WO1999024065A1 - COMPOSES INHIBANT L'INTERACTION CD4-gp120 ET LEURS UTILISATIONS - Google Patents
COMPOSES INHIBANT L'INTERACTION CD4-gp120 ET LEURS UTILISATIONS Download PDFInfo
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- WO1999024065A1 WO1999024065A1 PCT/US1998/023906 US9823906W WO9924065A1 WO 1999024065 A1 WO1999024065 A1 WO 1999024065A1 US 9823906 W US9823906 W US 9823906W WO 9924065 A1 WO9924065 A1 WO 9924065A1
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- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002922 simulated annealing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
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- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000004305 thiazinyl group Chemical group S1NC(=CC=C1)* 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- HIV Human Immunodeficiency Virus
- gpl20 Human Immunodeficiency Virus -1 envelope glycoprotein
- AIDS acquired immunodeficiency syndrome
- CD4 two cellular receptors of the human host
- chemokine receptor primarily CXCR-4 or CCR-5, depending on viral strain
- the gpl20 protein has been an obvious target for structural investigation, and quantities of pure soluble protein have been available for several years, a byproduct in part from vaccine trials. Nevertheless, despite considerable effort, it has resisted crystallographic analysis for more than a decade.
- the mature gpl20 glycoproteins of different HIV-1 strains have approximately 470-490 amino acids (18) .
- Extensive N-linked glycosylation at approximately 20-25 sites accounts for roughly half its mass (18,19) .
- Sequences from many different viral isolates show that it contains five conserved regions (C1-C5) and five variable regions (V1-V5) (18, 20) and nine conserved disulfide bridges (19) .
- proteolytic digestion does not reveal a sub-domain structure. Indeed, even after extensive proteolytic cleavage, the unreduced protein runs near its native molecular weight on SDS-PAGE (Peter D. Kwong : unpublished data) .
- variable regions the V3 loop in particular, appear to be conformationally variable. Conformational change is also evidenced by shedding, the CD4-induced dissociation of gpl20 from the surface of the virus, and by ligand- induced variations in monoclonal antibody binding (21,22) . These changes may be related to the functional role of gpl20 in virus entry .
- This invention provides a method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammal in need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupt two or more of the following interactions:
- an alkyl group, R aromatic or heteraromatic group, Het, that binds to the side chain isobutyl (isopropyl or methyl group) of gpl20 isoleucine (valine or alanine) 371 or disrupts the hydrophobic interaction between the side chain isobutyl (isopropyl or methyl group) of gpl20 isoleucine (valine, or alanine) 371 and CD4 phenylalanine 43;
- Het an aromatic group or heteroaromatic group, Het, that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43, wherein Het is phenyl, Bn,
- X that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43, wherein X is hydroxyalkyl, hydroxyaryl , alkylamide, or arylamide;
- an aromatic group or heteroaromatic group, Het that binds to the side chain indole group of gpl20 tryptophan 427 or disrupts the hydrophobic interaction between the side chain indole group of gpl20 tryptophan 427 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- an alkyl group, R that binds to the beta or gamma carbons of the side chain propionate of gpl20 gluta ic acid 370 or disrupts the hydrophobic interaction between the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43, wherein
- R is alkyl, cycloalkyl, or haloalkyl
- Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- Y is alkylammonium, d i a 1 ky 1 ammo n i um , a r y 1 amm o n i urn , arylalkylammonium, alkylguanidiniu , piperidinium, pyrollidinium, or pyridinium;
- k an alkyl group, R, aromatic or heteroaromatic group, Het, that binds to the side chain isopropyl (or methyl) group of gpl20 valine (or alanine) 430 or disrupts the hydrophobic interaction between the side chain isopropyl
- valine (or alanine) 430 and the side ' chain guanidinium group of CD4 arginine 59;
- a group, Z that binds to the side chain propylalcohol group of gpl20 threonine 123 or disrupts the side chain propylalcohol group of threonine 123 and the hydrogen bond interaction between the alpha carbonyl group of CD4 Arg 59, wherein Z is alkoxyalkyl , aryloxyalkyl , alkoxyaryl , haloalkyl , haloaryl , alkylamide, arylamide, alkylcarboxylate, arylcarboxylate, arylalkyl ester, dialky ester, or alkylarl ester.
- This invention also provides a method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammalin need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupt two or more of the following interactions;
- an aromatic group of heteroaromatic group, Het that binds to the alpha, beta or gamma carbons fo the side chain propionate of gpl20 glutamic acid 370 or disrupts the hydrophobic interaction between the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine
- Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- Y that binds to the side chain carboxyl group of gpl20 aspartic acid 368 or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59, wherein Y is alkylammonium, d i a 1 k y 1 a mm o n i u m , a r y 1 a mmo n i u , arylalkylammonium , alkylguanidinium , piperidinium, pyrollidinium, or pyridinium;
- a group, X that binds to the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 with the side chain amide group of CD4 glutamine 40;
- a group, Z that binds tot he alpha amino group of gpl20 glycine (alanine, or glutamic acid) 472 at a distance of 3.6 ang . , or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (alanine, or glutamic acid) 473 with the side chain propionamide group of CD4 glutamine 40;
- m. a group, Z that binds to the alpha amino group of gpl20 aspartic acid (or asparagine) 474 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 aspartic acid (or asparagine) 474 with the side chain propionamide group of CD4 glutamine 40;
- a group, X that binds to the alpha carbonyl group of gpl20 methionine (or serine) 426 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 methionine (or serine) 426 and the side chain hydroxyl group of CD4 serine 42;
- X a group, X, that binds to the alpha carbonyl group of gpl20 tryptophan 427 or disrupts the hydrogen bond interaction between the alpha carbonyl of gpl20 tryptophan 427 and the alpha amino group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 valine (or alanine) 430 or disrupts the hydrogen bond between the alpha amino group of gpl20 valine (or alanine) 430 and the side chain hydroxyl group of CD4 serine 42;
- an alkyl group, R that binds to the isobutyl (or isopropyl) group of gpl20 isoleucine (or valine) 271 or disurpts the hydrophobic interaction between the isobutyl group of gpl20 isoleucine (or valine) 271 and the side chain hydroxypropl group of CD4 threonine 45;
- al . a group, X that binds to the alpha amino group of gpl20 clycine 367 or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine 367 and the alpha carbonyl group of CD4 lysine 46;
- Y that binds to the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 at a distance of 3.3 ang., or disrupts the hydrogen bond interaction between the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or seine) 280 and the side chain butylammoniu group of CD4 lysine 29;
- dl . a group, Z that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.4 ang., or disrupts the hydrogen bond interaction betweeen the alpha amino group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- a group, X that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the alpha carbonyl group of CD4 glutamine 33;
- a group, X that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the side chain amide group of CD4 glutamine 33;
- gl . a group, X that binds to the alpha amino group of gpl20 glycine (or valine) 459 or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (or valine) 459 with the side chain propiona ido group of CD4 glutamine 33;
- a group, X that binds to the alpha carbonyl group of gpl20 serine (or alanine) 365 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 serine (or alanine) 365 with the side chain amide of CD4 asparagine 52; and/or
- This invention also provides a Method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammal in need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupts two or more of the following interactions:
- a an aromatic group or heteroaromatic group, Het, that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43 ;
- an aromatic group or heteroaromatic group, Het that binds to the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 or disrupts the hydrophobic interaction between the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the side chain isobutyl (or isopropyl group) of gpl20 isoleucine (or valine) 371 or disrupts the hydrophobic interaction between the side chain isobutyl
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- Y a group, Y, that binds to the side chain carboxyl group of gpl20 aspartic acid 368 or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59, wherein Y is alkylammonium, d i a 1 k y 1 a mm o n i u m , a r y 1 a mm o n i u , arylalkylammonium , alkylguanidinium, piperidinium, pyrollidinium, or pyridinium.
- a group, X that binds to the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 with the side chain amide group of CD4 glutamine 40;
- a group, Z that binds tot healpha amino group of gpl20 glycine (alanine, or glutamic acid) 472 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (alanine, or glutamic acid) 473 with the side chain propionamide group of CD4 glutamine 40;
- m. a group, Z that binds to the alpha amino group of gpl20 aspartic acid (or asparagine) 474 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 aspartic acid (or asparagine) 474 with the side chain propionamide group of CD4 glutamine 40;
- n. a group, Z, that binds to the alpha amino group of gpl20 aspartic acid (or asparagine) 474 at a distance of 3.4 ang., or disrupts the hydrogen bond betweenthe alpha amino group of gpl20 aspartic acid (or asparagine) 474 with the alpha carbonyl group of CD4 glutamine 40;
- a group, X that binds to the alpha carbonyl group of gpl20 methionine (or serine) 426 or disrupts the hydrogen bond betweenthe alpha carbonyl group of gpl20 methionine (or serine) 426 and the side chain hydroxyl group of CD4 serine 42;
- X a group, X, that binds to the alpha carbonyl group of gpl20 tryptophan 427 or disrupts the hydrogen bond interaction between the alpha carbonyl of gpl20 tryptophan 427 and the alpha amino group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 lysine 429 or disrupts the hydrogen bond interaction betweeen the alpha amino group of gpl20 lysine (threonine or asparagine) 429 and the side chain hydroxyl group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 valine (or alanine) 430 or disrupts the hydrogen bond between the alpha amimo group of gpl20 valine (or alanine) 430 and the side chain hydroxyl group of CD4 serine 42;
- al . a group, X that binds to the alpha amino group of gpl20 glycine 367 or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine 367 and the alpha carbonyl group of CD4 lysine 46;
- Y that binds to the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 at a distance of 3.3 ang., or disrupts the hydrogen bond interaction between the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 and the side chain butylammonium group of CD4 lysine 29;
- cl . a group, Q that binds to the alpha methylene (or methine) group of gpl20 glycine (or valine) 459 at a distance of 3.1 ang., or disrupts the dipolar interaction between the alpha methylene (or methine) group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- dl . a group, Z that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.4 ang or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- a group, X that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the alpha carbonyl group of CD4 glutamine 33;
- gl . a group, X that binds to the alpha amino group of gpl20 glycine (or valine) 459 or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (or valine) 459 with the side chain propionamido group of CD4 glutamine 33;
- a group, X that binds to the alpha carbonyl group of gpl20 serine (or alanine) 365 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 serine (or ' alanine) 365 with the side chain amide of CD4 asparagine 52; and/or
- a group, X that binds to the side chain isopropylalcohol group of gpl20 threonine 123 at a distance of 3.8 ang., or disrupts the hydrogen bond between the side chain isopropyl alcohol group of gpl20 threonine 123 with the side chain ethyl alcohol group of CD4 serine 50.
- kl an alkyl group, R, or an aromatic or heteroaromatic group that binds to the side chain phenyl group of gpl20 phenylalanine 382 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine
- an aromatic or heteroaromatic group that binds to the side chain phenolic group of gpl20 tyrosine 384 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- ml an alkyl group, R, that binds to the side chain alkyl group of gpl20 valine (isoleucine, or glutamine) 255 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- Y that binds to the side chain carboxyl group of gpl20 glutamic acid 370 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine
- CD4 is in the top left
- gpl20 is toward the right
- Fab 17b is in the bottom left of the figure.
- Photomicrographs of crystals containing HIV-1 gpl20 Crystal types A-F are shown and correspond to the crystal types described in the text and Tables 3 and 4.
- the photomicrograph in A is at twice the magnification.
- the bar in A corresponds to 25 ⁇ m (50 ⁇ m for B-F) .
- Lane 1 (Pharmacia Phast system) .
- Lane 1 2.5 ug of ternary complex purified by gel filtration.
- the top band is the deglycosylated ⁇ 82 ⁇ Vl/2* ⁇ V3 ⁇ C5 gpl20, the next two bands are the alkylated and reduced heavy and light chains respectively of the Fab 17b, and the bottom band is the two-domain sCD4 (D1D2).
- Lane 2 standards: 94, 67, 43 (diffuse), 30, 20, and 14.
- Lane 3 supernant from the crystallization droplet.
- Lane 4 last wash of crystals.
- Lane 5 dissolved crystals.
- the gel is silver stained.
- Figure 5 Figure 5
- CD4 is toward the bottom and gpl20 is toward the top.
- FIGS 6A and 6B The HIV-1 entry process.
- the trimeric HIV-1 envelope glycoproteins anchored in the viral membrane, are depicted, with gpl20 in the lower right and gp41 in the upper right.
- the gpl20 variable loops are not shown, but would extend over the outer surface of the envelope glycoprotein complex.
- the receptors on the target cell, CD4 and chemokine receptor are also shown.
- the structures of gpl20, gp41, and CD4 are adapted from available X-ray crystallographic studies (5,20,21), whereas the chemokine receptor model is hypothetical.
- the molecular surface of the HIV-1 gpl20 core (20) is shown, with the arrow pointing towards the viral membrane.
- the inner domain, believed to interact with gp41, and the outer domain, which is probably exposed on the assembled trimer, are on the left and right, respectively.
- the gpl20 surface occluded by CD4 is shown and the gpl20 region thought to be involved in chemokine receptor binding (27) is also shown.
- the location of the base of the V3 loop is shown.
- Figure 7B conserveed gpl20 neutralization epitopes are shown on the gpl20 core, which is oriented identically to that in Figure 7A. The location of the epitopes was deduced from mutagenic analysis (45,46,48) .
- V2, V3 , and V4 The approximate location of gpl20 structures (20) that contribute to protection from antibody responses is shown.
- the relationship of different surfaces of the gpl20 core to the antibody response generated by the gpl20 glycoprotein is depicted.
- the surface of gpl20 that interacts with neutralizing antibodies (32) is shown, spans the inner and outer domains, and includes the V2 and V3 variable loops (not shown) .
- the surface of gpl20 that interacts with non-neutralizing antibodies is located on the inner domain, and includes gp41- interactive N- and C-terminal gpl20 regions (not shown) .
- the heavily glycosylated surface of the gpl20 outer domain, which appears to be minimally immunogenic, is also shown.
- the ribbon diagram shows gpl20, the N-terminal two domains of CD4 , and the Fab 17b (light chain) and (heavy chain) .
- the sidechain of Phe 43 on CD4 is also shown.
- the prominent CDR3 loop of the 17b heavy chain is evident in this orientation.
- the complete N- and C- termini of gpl20 are missing, the positions of the gpl20 termini are consistent with the proposal that gp41, and hence the viral membrane, is located towards the top of the diagram. This would position the target membrane at the diagram base.
- the vertical dimension of gpl20 in this orientation is roughly 50 A.
- Precisely perpendicular views of gpl20 are shown in Figures 9 and 11. Drawn -with RIBBONS 49 .
- core gpl20 Structure of core gpl20.
- the orientation of gpl20 in each of the panels shown in this figure is related to Figure 8 by a 90° rotation about a vertical axis.
- the viral membrane would be oriented above, the target membrane below, and the C-terminal tail of CD4 coming out of the page.
- the left portion of core gpl20 as the "inner” domain
- the right portion as the "outer” domain
- the 4-stranded sheet at the bottom left of gpl20 as the "bridging sheet.”
- the bridging sheet ( ⁇ 3, ⁇ 2, ⁇ 21, ⁇ 20) can be seen packing primarily over the inner domain, although some surface residues of the outer domain, e.g. Phe 382, reach in to form part of its hydrophobic core.
- Ribbon diagram Helices and ⁇ -strands are depicted.
- strand ⁇ l5 makes an antiparallel ⁇ -sheet alignment with strand C' of CD4.
- the dashed line to the right of the diagram represents the disordered V4 loop. Selected parts of the structure are labeled.
- Helices are shown as corkscrews and labeled ⁇ l-o-5.
- ⁇ -strands are shown as arrows: black and labeled represent the 25 ⁇ -strands of core gpl20; gray and unlabeled represent the continuation of hydrogen bonding across a sheet; white and labeled represents the C' strand of CD4. Spatial proximity between neighboring strands implies mainchain hydrogen bonding.
- Loops are labeled ⁇ A- ⁇ F and V1-V5. The labels of loops with high sequence variability are circled.
- Solvent accessibility is indicated for each residue by an open circle if the fractional solvent accessibility is greater than 0.4, a half-closed circle if 0.1 to 0.4, and a closed circle if less than 0.1. Sequence variability observed among primate immunodeficiency viruses is indicated below the solvent accessibility by the number of horizontal hash marks: 1 mark, residues conserved among all primate immunodeficiency viruses; 2 marks, conserved among all HIV-1 isolates; 3 marks, exhibits moderate variation among HIV-1 isolates; and 4 marks, exhibits significant variability among HIV-1 isolates. In accessing conservation, all single atom changes were permitted as well as larger substitutions if the character of the sidechain was conserved (e.g. K to R or F to L) .
- N-linked glycosylation is indicated by "m” for the high mannose additions and "c” for the complex additions observed in mammalian cells (6) .
- Residues of gpl20 in direct contact with CD4 are indicated by "*".
- Direct contact is a more restrictive criterion of interaction than the often used loss of solvent accessible surface; residues of gpl20 which show loss of solvent accessible surface but are not in direct contact are 123, 124, 126, 257, 278, 282, 364, 471, 475, 476 and 477. Parts (a) and (b) were drawn with MOLSCRIPT (P. J. Kraulis) .
- FIG. 10B Electron density in the Phe 43 cavity.
- the 2Fo-Fc electron density map at 2.5A, 1. l ⁇ contour, is shown.
- the orientation is the same as in (a) .
- the foreground has been clipped for clarity removing the overlying ⁇ 24- ⁇ 5 connection.
- In the upper middle of the picture is the central unidentified density.
- Phe 43 of CD4 can be seen reaching up to contact the cavity.
- the gpl20 residues are Trp 427 (with its indole ring partially clipped by foreground slabbing), Trp 112, Val 255, Thr 257, Glu 370 (packing under the Phe 43 ring), lie 371, and Glu 368 (partially clipped in the bottom right corner) . Hydrophobic residues lining the back of the cavity can be partially glimpsed around the central unidentified density.
- Electrostatic surface of CD4 and gpl20 The electrostatic potential is displayed at the solvent accessible surface, which is shaded according to the local electrostatic potential. The slight "puffiness" of the surface arises from the enlarged nature of the solvent accessible surface relative to the standard molecular surface.
- the gpl20 surface is shown in an orientation similar to that of Figures 9A and 9C, but rotated ⁇ 20° around a vertical axis to depict the recessed binding pocket more clearly.
- a thin yellow Ca worm of CD4 is shown to aid in orientation.
- the CD4 surface is shown, rotated relative to the gpl20 panel by an exact 180° rotation about the vertical axis shown.
- a thin red C ⁇ worm of gpl20 is shown.
- CD4-gpl20 contact surface On the right, the gpl20 surface is shown with the surface within 3.5 A of CD4 (surface-to-atom center distance). This effectively creates an "imprint" of CD4 on the displayed gpl20 surface. On the left (180° rotation), the corresponding CD4 surface and gpl20 "imprint" is also shown.
- the surface of gpl20 is shown with the surface of gpl20 residues shown by substitution to affect CD4 binding highlighted: substantial effect -- residues 257, 368, 370 and 427; moderate effect -- residue 457.
- substantial effect -- residues 257, 368, 370 and 427 is also depicted.
- residues important for gpl20 binding are shown on the CD4 surface: substantial effect -- residues 43 and 59; moderate effect — residues 29, 35, 44, 46, 47.
- Sequence variability mapped to the gpl20 surface The sequence variability observed among primate immunodeficiency viruses ( Figure 9D) is depicted mapped onto the gpl20 surface. Also shown is the carbohydrate: N-acetylglucosamine and fucose residues present in the structure; Asn-proxi al N-acetylglucosamines modeled at residues 88, 230, 241, 356, 397, 406, 462. Much of the carbohydrate (22 residues) is hidden on the back side of the outer domain. Figure 10H
- Phe 43 cavity The surface of the Phe 43 cavity is shown, buried in the heart of gpl20.
- a worm representation of gpl20 shows the three stretches that are incorrectly predicted by secondary structure prediction: the ⁇ B loop, bending around the top of the cavity, strands ⁇ 20- ⁇ 21 just below the cavity, and strand ⁇ l5, slightly more distal to the cavity right.
- the orientation shown here is the same as for the gpl20 surfaces in Figure 10C-10G.
- Electrostatic surface The electrostatic potential is displayed at the solvent accessible surface, which is shaded according to the local electrostatic potential.
- the electrostatic shading is the same scale as that shown in Figure IOC.
- the surface that corresponds to the 17b epitope is the most electropositive region of the molecule.
- the V3 loop is truncated here, but sequence analysis shows that it is generally quite positively charged.
- FIG HE Worm diagram of gpl20 The gpl20 is shown shaded according to the same scheme given in Figure HA. The orientation is the same as in Figures HC and 11D, that is, 90° from Figure HA.
- This conformational change strains the interactions at the N- and C- termini of gpl20 with the rest of the oligomer, priming the CD4-bound gpl20 core.
- the chemokine receptor binds to the bridging sheet and the V3 loop (at the bottom left and right, respectively, of gpl20), causing an orientational shift of core gpl20 relative to the oligomer. This triggers further steps, which ultimately lead to the fusion of the viral and target membranes.
- FIG. 13 Structure of HIV-1 gpl20 with neutralizing antibody and human receptor CD .
- Figure 14B View of the molecular surface of the gpl20 outer domain, from the perspective indicated in Figure 14A.
- the molecular surface in the figure on the left is shaded according to the variability observed in gpl20 residues among primate immunodeficiency viruses.
- the variability of the gpl20 surface shown is underestimated since the V4 variable loop, which is not resolved in the structure, contributes to this surface.
- the position of the V5 region is shown.
- the highly conserved glycosylation site (asparagine 356 and threonine/serine 358) within the L e loop, between the V5 and V4 regions.
- the V4 loop and the carbohydrates are modeled, as described in Materials and Methods .
- Figure 14D View of the molecular surface of the gpl20 core inner domain.
- variability is indicated by the shading scheme used in Figure 14B .
- the CD4-binding site is to the right of the figure, and the protruding V1/V2 stem is indicated.
- the conserved molecular surface, which is associated with the inner domain of the gpl20 core, is devoid of know N- linked glycosylation. These are modeled in the figure on the right, which is shaded as described in Figure 14B .
- FIG 15A The molecular surface of the gpl20 core is shown, from the same perspective as that in Figure 14A.
- the modeled N-terminal gpl20 core residues, V4 loop and carbohydrate structures are included.
- the variability of the molecular surface is indicated, using the shading scheme described in Figure 14B .
- the approximate locations of the V2 and V3 variable loops are indicated. Note the well-conserved surfaces near the "Phe 43" cavity and the chemokine receptor- binding site (see Figure 14A) .
- FIG. 14A A Co. tracing of the gpl20 core, oriented similarly to Figure 14A.
- the gpl20 residues within Figure 17A of the 17b CD4i antibody are shown.
- the residues implicated in the binding of CD4BS antibodyies (20) are shown. Changes in these residues significantly affect the binding of at least 25 percent of the CD4BS antibodies listed in the table from the fourth series of experiments.
- the residues implicated in 2G12 binding (19) are shown.
- the V4 variable loop, which contributes to the 2G12 epitope, (19) is indicated by dotted lines (see figure 14A) .
- the neutralizing face of the complete gpl20 glycoprotein includes the V2 and V3 loops, which reside adjacent to the surface shown (see Figure 15A) .
- the approximate location of the gpl20 face that is poorly accessible on the assembled envelope glycoprotein trimer and therefore elicits only non-neutralizing antibodies (5 , 6) is shown.
- the approximate location of an immulogically "silent" face of gpl20, which roughly corresponds to the highly glycosylated outer domain surface, is also shown.
- a likely arragement of the HIV-1 gpl20 glycoproteins in a trimeric complex The gpl20 core was organized into a trimeric array, based on the criteria discussed in the text.
- the perspective if from the target cell membrane, similar to that shown in Figure 14C.
- the CD4 binding pockets are indicated by black arrows, and the chemokine receptor-binding regions are darkly shaded.
- the lightly shaded areas indicate the more variable, glycosylated surface of the gpl20 core.
- the approximate locations of the 2G12 epitopes are indicated by open arrows. The approximate locations for the V3 loops and V4 regions are shown.
- the positions of the V5 regions and some complex carbohydrate addition sites are shown.
- the approximate locations of the large V1/V2 loops, centered on the known positions of the VI/V2 stems, are indicated.
- On one of the gpl20 subunits the positions of the L D and L E loops are indicated.
- the distance of each of the gpl20 monomers from the 3 -fold symmetry axis is arbitrary.
- the HIV gpl20 derivative used in the binding assay The wild-type gpl20 and gp41 envelope glycoproteins are shown in the upper figure. conserveed (black) and variable (white) regions (25) are indicated.
- the N-terminal and V1/V2 deletions correspond to those previously described for the HXBc2 gpl20 mutants ⁇ 82 and ⁇ 128-194, respectively (8,9).
- SIG signal peptide.
- Figure 18 The gpl20-CCR5 binding assay.
- FIG 18A The radiolabeled wt ⁇ protein was incubated either with the parental Ll .2 cells or with the L1.2-CCR5 cells. Incubations were carried out either in the absence or presence of sCD4 (lOOnM) . The wt ⁇ protein bound to the cells is shown. The two bands represent different glycoforms of gpl20.
- the wt ⁇ protein was incubated with both sCD4 and 17b antibody at the indicated concentrations prior to adition to the L1.2-CCR5 cells.
- the L1.2-CCR5 cells were incubated with 2D7 anti-CCR5 antibody or MIP-13 at the indicated concentrations prior to incubation with wt ⁇ -sCD4 complexes.
- the wt ⁇ protein bound to the cells is shown.
- FIG. 19 Structure of the HIV-1 gpl20 region implicated in CCR5 binding .
- a ribbon drawing of the HIV-1 gpl20 glycoprotein (6) complexed with CD4 is shown. The perspective is that from the target cell membrane. The two amino-terminal domains of CD4 are shown. The gpl20 inner domain is shown, the outer domain is shown and the "bridging sheet" is shown. The gpl20 residues in which changes resulted in a >90% decrease in CCR5 binding are labeled. The V1/V2 stem and base of the V3 loop (strands l2 and j ⁇ l3 and the associated turn) are indicated.
- Figure 19B A ribbon drawing of the HIV-1 gpl20 glycoprotein (6) complexed with CD4 is shown. The perspective is that from the target cell membrane. The two amino-terminal domains of CD4 are shown. The gpl20 inner domain is shown, the outer domain is shown and the "bridging sheet" is shown. The gpl20 residues in which changes resulted in a >90% decrease in CCR5 binding are labeled. The V1/V
- a molecular surface of the gpl20 glycoprotein from the same perspective as that of Figure 19A is shown. Shaded surfaces are associated with gpl20 residues in which changes resulted in either a ⁇ 75% decrease, a > 90% decrease or a ⁇ 50% increase in CCR5 binding, when CD4 binding was at least 50% of that seen for the wt ⁇ protein.
- the surface depicted in Figure 19B is shaded according to the degree of conservation observed among primate immunodeficiency viruses (25) .
- the molecular surface of the gpl20 glycoprotein is shown, indicating residues in which changes resulted in a > 70% decease in 17b antibody binding, in the absence of sCD4.
- the invention relates to a crystals of gpl20 suitable for x-ray diffraction.
- the three dimensional structure of gpl20 provides information which has a number of uses; principally related to the development of pharmaceutical compositions which mimic the action of gpl20.
- the essence of the invention resides in the obtaining of crystals of gpl20 of sufficient quality to determine the three dimensional (tertiary) structure of the protein by x-ray diffraction methods.
- This invention provides crystals of sufficient quality to obtain a determination of the three-dimensional structure of gpl20 to high resolution, preferably to the resolution of 2.5 angstroms.
- the value of crystals of gpl20 extends beyond merely being able to obtain a structure for gpl20.
- the knowledge of the structure of gpl20 provides a means of investigating the mechanism of action of these proteins in the body. For example, binding of these proteins to various receptor molecules can be predicted by various computer models. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows chemists to design and attempt to synthesize molecules which mimic the binding of gpl20 to its receptors. This is the method of "rational" drug design.
- One skilled in the art may use one of several methods to screen chemical entities for their ability to associate with gpl20. This process may begin by visual inspection of, for example, the active site on the computer screen based on the gpl20 coordinates. Docking may be accomplished using software such as Quanta and Sybyl , followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
- Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:
- GRID [P.J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules” , J. Med. Chem. 28:849-857 (1985)]. GRID is available from Oxford Universit, Oxford, UK.
- MCSS [A. Miranker and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method", Proteins: Structure, Function and Genetics, 11:29-34 (1991)]. MCSS is available from Molecular Systems, Burlington, MA.
- AUTODOCK [D.S. Goodsell and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins, Structure, Function, and Genetics, 195-202 (1990)] AUTODOCK is available from Scripps Research Institute, La Jolla, CA.
- Assembly may be proceeded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of gpl20. This would be followed by manual model building using software as Quanta or Sybyl.
- CAVEAT [P.A. Bartell et al . , "CAVEAT: A Program of Facilitate the Structure-Derived Design of Biologically Active Molecules” , in Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc . 78, pp. 182-196 (1989)]. CAVEAT is available from the University of California, Berkeley, CA.
- 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, CA) . This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35:2145-2154 (1992).
- inhibitory or other type of binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion (s) of a known inhibitor (s) .
- LUDI [H.-J. Bohm "The Computer Program LUDI : A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec . Design, 6:61-78 (1992)]. LUDI is available from Biosym Technologies, San Diego, CA.
- LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)]. LENGEND is available from Molecular Simulations, Burlington, MA.
- the gpl20 or CD4 antagonist may be tested for bioactivity using standard techniques.
- structure of the invention may be used in binding assays using conventional formats to screen inhibitors .
- Suitable assays for use herein include, but are not limited to, the enzyme-linked immunosorben assay (ELISA) , or a fluoresence quench assay.
- ELISA enzyme-linked immunosorben assay
- fluoresence quench assay Other assay formats may be used; these assay formats are not a limitation on the present invention.
- the gpl20 structure of the invention permit the design and identification of synthetic compounds and/or other molecules which have a shape complimentary to the conformation of the gpl20 active site of the invention.
- the coordinates of the gpl20 structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the invention. Subsequently, suitable candidates identified as above may be screened for the desired gpl20 inhibitory bioactivity, stability, and the like.
- inhibitors may be used therapeutically or prophylactically to block gpl20 activity.
- this invention also provides material which is the basis for the rational design of drugs which mimic the action of gpl20.
- the subject invention provides a crystal suitable for X- ray diffraction comprising a polypeptide having an amino acid sequence of a portion of a Human Immunodeficiency Virus envelope glycoprotein gpl20.
- the subject invention also provides the above-described crystals, which effectively diffract X-rays for determination of the atomic coordinates of the polypeptide to a resolution of 4 angstroms or better than 4 angstroms .
- the subject invention also provides the above-described crystals, which effectively diffract X-rays for determination of the atomic coordinates of the polypeptide to a resolution of 2.5 angstroms or better than 2.5 angstroms .
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a CD4 binding site .
- the subject invention further provides the above- described crystals, further comprising a compound bound to the CD4 site.
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a chemokine receptor binding site.
- the subject invention also provides the above-described crystals, further comprising a compound bound to the chemokine receptor binding site.
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a CD4 binding site and a chemokine receptor binding site.
- the subject invention also provides the above-described crystals, further comprising of a first compound bound to the CD4 binding site of the polypeptide and a second compound bound to the chemokine receptor binding site of the polypeptide.
- the subject invention also provides the above-described crystals, wherein the first compound is the second compound.
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 lacking the VI, V2 , V3 , and C5 regions.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the conserved stem of the V1/V2 stem- loop structure.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the base of the V3 loop.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the C5 region.
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 with 5% by weight of the carbohydrate residues linked to the gpl20 in substantially the same manner as they are linked to gpl20 in unmodified gpl20.
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 with 15% by weight of the carbohydrate residues linked to the gpl20 polypeptide in substantially the same manner as they are linked to gpl20 in unmodified gpl20.
- the subject invention also provides the above-described crystals, further comprising a Fab, a CD4 , a polypeptide having amino acid sequence of a portion of CD4 , or a combination thereof, bound to the gpl20.
- the subject invention also provides the above-described crystals, wherein the Fab is produced from an antibody to a discontinuous epitope.
- the subject invention also provides the above-described crystals, wherein the monoclonal antibody is designated 17b.
- the subject invention ' additionally provides a method for producing a crystal suitable for X-ray diffraction comprising: (a) deglycosylating a polypeptide having amino acid sequence of a portion of a gpl20 wherein said portion is produced by deleting or replacing part of the gpl20 to reduce the surface loop flexibility; (b) contacting the polypeptide with a ligand so as to form a complex which exhibits restricted conformational mobility; and (c) obtaining crystal from the complex so formed to produce a crystal suitable for X-ray diffraction.
- the subject invention also provides the above-described methods, wherein the VI, V2 , or V3 loop of the gpl20 contained in the polypeptide are partially truncated, deleted or replaced.
- the subject invention also provides the above-described methods, wherein the polypeptide lacks the VI, V2 , V3 and C5 loop of the gpl20.
- the subject invention also provides the above-described methods, wherein the polypeptide also lacks up to fifty N-terminal amino acids of the gpl20 or up to fifty C- terminal amino acid of gpl20.
- the subject invention also provides the above-described methods, wherein the ligand is a Fab, a CD4 , or a polypeptide having amino acid sequence of a portion of CD4.
- the subject invention also provides the above-described methods, wherein the resulting polypeptide after the deglycosylation contains at least 5% of the carbohydrate.
- the subject invention also provides the crystal produced by the above-described methods.
- the subject invention also provides a method for identifying a compound capable of binding to a portion of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining a binding site on the portion of gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising the portion of gpl20; and (b) determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the gpl20.
- the subject invention also provides a method for designing a compound capable of binding to a portion of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining a binding site on the portion of gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising the portion of gpl20; and (b) designing a compound to fit the binding site.
- the subject invention also provides the above-described methods, wherein the fitting is determined by shape complementarity or by estimated interaction energy.
- the subject invention also provides the above-described methods, wherein the X-ray diffraction data are set forth in Table A.
- the subject invention also provides the above-described methods, wherein the atomic coordinates are set forth in Table B.
- the subject invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the compound identified by the above-described methods and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers means any of the standard pharmaceutical carriers.
- suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.
- Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.
- Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- Such carriers may also include flavor and color additives or other ingredients.
- Compositions comprising such carriers are formulated by well known conventional methods.
- the subject invention also provides the above-described methods, wherein the compound is not previously known.
- the subject invention also provides the compounds identified by the above-described methods.
- the subject invention also provides the compound designed by the above-described methods.
- the subject invention also provides a composition comprising the above-described compounds and a suitable carrier.
- This invention provides a method of inhibiting the interaction of HIV-gpl20 with CD4 which comprises administering to a mammal in need thereof a compound, with the proviso that the compound is not CD4 , which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupts two or more of the following interactions: (a) a benzyl group that binds to the side chain isobutyl (or isopropyl group) of gpl20 isoleucine (or valine 371 at a distance of 3.4 ang.
- a phenyl group tht binds to the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 at a distance of 3.4-3.5 ang.
- said distance is the distance between nearest interacting heavy atoms in said groups of gpl20 and CD4 in the crystal structure. Said distances to comparable groups in other gpl20 isolates (shown in parentheses) have not been measured. Side chains do not include alpha carbons or alpha substituents.
- a method of inhibiting the interaction of HIV-gpl20 with CD4 which comprises administering to a mammal in need thereof a compound, with the proviso that the compound is not CD4 , which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupts two or more of the following interactions:
- a benzyl group that binds to the side chain isobutyl (or isopropyl group) of gpl20 isoleucine (or valine) 371 at a distance of 3.4 ang. or otherwise or disrupts the hydrophobic interaction between the side chain isobutyl (or isopropyl group) og gpl20 isoleucine (or valine) 371 and CD4 phenylalanine 43;
- a benzyl group that binds to the side chain carboxylate group of gpl20 aspartic acid 368 at a distance of 3.1 ang. or otherwise or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine
- a phenyl group that binds to the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 at a distance of 3.4-3.5 ang. or otherwise or disrupts the hydrophobic interactio nbetween the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43;
- a phenyl group that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 at a distance of 3.1 ang. or otherwise or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- a propylguanidinium group that binds to the side chain carboxyl group of gpl20 aspartic acid 368 at a distance of 1.7 ang. or otherwise or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59;
- a propylguanidinium group that binds to the side chain isopropyl (or methyl) group of gpl20 valine (or alanine) 430 at a distance of 3.3 ang. or otherwise or disrupts the ionic interaction between the side chain isopropyl (or methyl) group of valine (or alanine) 430 and the side chain guanidinium group of CD4 arginine 59; j . an amide group that binds to the side chain propylalcohol group of gpl20 threonine 123 at a distance of 4.6 ang. or otherwise or disrupts the hydrogen bond interaction between the side chain propylalcohol group of threonine 123 and the alpha carbonyl group of CD4 Arg 59;
- a propionamide group that binds to the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 at a distance of 3.6 ang. or otherwise or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 with the side chain propionamid group of CD4 glutamine 40;
- a propionamide group that binds to the alpha amino group of phl20 aspartic acid (or asparagine) 474 at a distance of 3.6 ang. or otherwise or disrupts the hydrogen bond between the alpha amino group of gpl20 aspartic acid (or asparagine) 474 with the side chain propionamide group of CD4 glutamine 40;
- n. a methyl alcohol group that binds to the alpha amino group of gpl20 lysine 429 at a distance of 3.2 ang. or otherwise or disrupts the hydrogen bond interaction betweenthe alpha amino group of gpl20 lysine (threonine or asparagine) 429 and the side chain hydroxyl group of CD4 serine 42;
- a methyl alcohol group that binds to the alpha carbonyl group of gpl20 lysine 429 at a distance of 3.2 ang. or otherwise or disrupts the hydrogen bond interaction betweenthe alpha carbonyl group of gpl20 lysine (threonine or asparagine) 429 and the side chain hydroxyl group of CD4 serine 42;
- p. a methyl alcohol group that binds to the alpha carbonyl group of gpl20 tryptophan 427 at a distance of 3.2 ang. or otherwise or disrupts the hydrogen bond interaction between the alpha carbonyl of gpl20 trptophan 427 and the side chain hydroxyl group of CD4 serine 42;
- a methyl alcohol group that binds to the alpha amino group of gpl20 valine (or alanine) 430 at a distance of 3.4 ang. or otherwise or disrupts the hydrogen bond between the alpha amino group of gpl20 valine (or alanine) 430 and the sidechain hydroxyl group of CD4 serine 42;
- r. a methyl alcohol group that binds to the alpha carbonyl group of gpl20 methionine (or serine 426 at a distance of 3.7 ang. or otherwise or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 methionine (or serine) 426 and the sidechain hydroxyl group of CD4 serine 42;
- a butylammonium group that binds to the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 at a distance of 3.3 ang. or otherwise or disrupts the hydrogen bond interaction between the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 and the side chain butylammonium group of CD4 lysine 29;
- cl an amido group that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.4 ang. or otherwise or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- dl an amido group that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 at a distance of 2.6 ang. or otherwise or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the alpha carbonyl group of CD4 glutamine 33; el . a propionamido group that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 at a distance of 4.2 ang. or otherwise or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the sidechain amide group of CD4 glutamine 33;
- a propionamido group that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.9 ang. or otherwise or disrupts the hydrogen bond between the alpha amino group of gpl20 alycine (or valine) 459 with the sidechain propionamido group of CD4 glutamine 33; and/or
- This invention also provides a method of inhibiting the interaction of HIV-gpl20 with CD4 which comprises administering to a mammal in need thereof a compound, with the proviso that the compound is not CD4 , capable of disrupting two or more of the contacts between gpl20 and CD4 as set forth in table C.
- This invention also provides a method for identifying a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD4 ; and (b) determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the CD4 binding site of the gpl20.
- This invention also provides a method for designing a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD4 ; and (b) designing a compound to fit the CD4 binding site.
- This invention also provides the above-described methods, wherein the crystal further comprising a CD4 , a second polypeptide having amino acid sequence of a portion of CD4 , or a compound known to be able to bind to the CD4 site of the gpl20, bound to the polypeptide.
- This invention also provides the above-described methods, wherein the fitting is determined by shape complementarity or by estimated interaction energy.
- This invention also provides the above-described methods, wherein the X-ray diffraction data are set forth in Table A.
- This invention also provides the above-described methods, wherein the atomic coordinates are set forth in Table B.
- This invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the compound identified the by above-described methods and a pharmaceutically acceptable carrier.
- This invention also provides the above-described methods, wherein the compound is not previously known.
- This invention also provides the compound identified by the above-described methods.
- This invention also provides the compound designed by the above-described methods.
- This invention also provides a composition comprising the above-described compounds and a suitable carrier.
- This invention also provides a method of inhibiting Human Immunodeficiency Virus infection in a subject comprising adminstering effective of amount of the above-described composition to the subject.
- the above-described compounds are nonpeptidyl.
- This invention provides a method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammal in need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupt two or more of the following interactions:
- an alkyl group, R aromatic or heteraromatic group, Het, that binds to the side chain isobutyl (isopropyl or methyl group) of gpl20 isoleucine (valine or alanine) 371 or disrupts the hydrophobic interaction between the side chain isobutyl (isopropyl or methyl group) of gpl20 isoleucine (valine, or alanine) 371 and CD4 phenylalanine 43;
- Het an aromatic group or heteroaromatic group, Het, that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43, wherein Het is phenyl, Bn, EtPh, or heteroarylalkyl
- X that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43, wherein X is hydroxyalkyl, hydroxyaryl, alkylamide, or arylamide;
- an aromatic group or heteroaromatic group, Het that binds to the side chain indole group of gpl20 tryptophan 427 or disrupts the hydrophobic interaction between the side chain indole group of gpl20 tryptophan 427 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43 ;
- an alkyl group, R that binds to the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 or disrupts the hydrophobic interaction between the beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43, wherein R is alkyl, cycloalkyl, or haloalkyl;
- an aromatic group of heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- Y that binds to the side chain carboxyl group of gpl20 aspartic acid 368 or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59, wherein Y is alkylammonium, d i a 1 ky 1 ammo n i urn , a r y 1 a mm o n i urn , aryl alkyl ammonium , alkylguanidinium , piperidinium, pyrollidinium, or pyridinium; k. an alkyl group, R, aromatic or heteroaromatic group, Het, that binds to the side chain isopropyl (or methyl) group of gpl20 valine
- valine (or alanine) 430 and the side chain guanidinium group of CD4 arginine 59;
- a group, Z that binds to the side chain propylalcohol group of gpl20 threonine 123 or disrupts the side chain propylalcohol group of threonine 123 and the hydrogen bond interaction between the alpha carbonyl group of CD4 Arg 59, wherein Z is alkoxyalkyl, aryloxyalkyl , alkoxyaryl, haloalkyl, haloaryl, alkylamide, arylamide, alkylcarboxylate, arylcarboxylate, arylalkyl ester, dialky ester, or alkylarl ester.
- This invention also provides a method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammalin need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupt two or more of the following interactions;
- Het that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group of heteroaromatic group, Het that binds to the alpha, beta or gamma carbons fo the side chain propionate of gpl20 glutamic acid 370 or disrupts the hydrophobic interaction between the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43 ;
- Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- Y that binds to the side chain carboxyl group of gpl20 aspartic acid 368 or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59, wherein Y is alkylammonium, d i a 1 k y 1 a mm o n i u m , a r y 1 a mmo n i u m , arylalkylammonium , alkylguanidinium, piperidinium, pyrollidinium, or pyridinium;
- an alkyl group, R aromatic or heteroaromatic group, Het, that binds to the side chain isopropyl (or methyl) group of ghl20 valine (or alanine) 430 or disrupts eh hydrophobic interaction between the side chain isopropyl (or methyl) group of valine (or alanine) 430 and the side chain guanidinium group of CD4 arginine 59;
- a group, X that binds to the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 with the side chain amide group of CD4 glutamine 40;
- a group, Z that binds tot he alpha amino group of gpl20 glycine (alanine, or glutamic acid) 472 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (alanine, or glutamic acid) 473 with the side chain propionamide group of CD4 glutamine 40;
- m. a group, Z that binds to the alpha amino group of gpl20 aspartic acid (or asparagine) 474 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 aspartic acid (or asparagine) 474 with the side chain propionamide group of CD4 glutamine 40;
- a group, X that binds to the alpha carbonyl group of gpl20 methionine (or serine) 426 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 methionine (or serine) 426 and the side chain hydroxyl group of CD4 serine 42;
- p. a group, X that binds to the alpha carbonyl group of gpl20 tryptophan 427 or disrupts the hydrogen bond interaction between the alpha carbonyl of gpl20 tryptophan 427 and the alpha amino group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 valine (or alanine) 430 or disrupts the hydrogen bond between the alpha amino group of gpl20 valine (or alanine) 430 and the side chain hydroxyl group of CD4 serine 42;
- gpl20 isoleucine (or valine) 271 or disurpts the hydrophobic interaction between the isobutyl group of gpl20 isoleucine (or valine) 271 and the side chain hydroxypropl group of CD4 threonine 45;
- al . a group, X that binds to the alpha amino group of gpl20 clycine 367 or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine 367 and the alpha carbonyl group of CD4 lysine 46;
- Y that binds to the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 at a distance of 3.3 ang., or disrupts the hydrogen bond interaction between the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or seine) 280 and the side chain butylammonium group of CD4 lysine 29;
- dl . a group, Z that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.4 ang., or disrupts the hydrogen bond interaction betweeen the alpha amino group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- a group, X that binds to the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 or disrupts the hydrogen bond between the side chain amide (or hydroxyl) group of gpl20 asparagine (or serine) 280 with the side chain amide group of CD4 glutamine 33;
- gl . a group, X that binds to the alpha amino group of gpl20 glycine (or valine) 459 or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (or valine) 459 with the side chain propionamido group of CD4 glutamine 33;
- a group, X that binds to the alpha carbonyl group of gpl20 serine (or alanine) 365 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 serine (or alanine) 365 with the side chain amide of CD4 asparagine 52 ;
- This invention also provides a Method of inhibiting the interaction of HIV-gpl20 with leukocyte CD4 which comprises administering to a mammal in need thereof a compound which contains certain functional groups that interact with HIV-gpl20 in a manner that disrupts two or more of the following interactions: a. an aromatic group or heteroaromatic group, Het, that binds to the side chain carboxylate group of gpl20 aspartic acid 368 or disrupts the dipolar interaction between the side chain carboxylate group of gpl20 aspartic acid 368 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 or disrupts the hydrophobic interaction between the alpha, beta or gamma carbons of the side chain propionate of gpl20 glutamic acid 370 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha carbonyl group of gpl20 asparagine (or arginine) 425 or disrupts the CHO hydrogen bond interaction between the alpha carbonyl group of asparagine (or arginine) 425 and the side chain phenyl group of CD4 phenylalanine 43;
- an aromatic group or heteroaromatic group, Het that binds to the alpha methylene group of gpl20 glycine 473 or disrupts the hydrophobic interaction between the alpha methylene group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine
- Het that binds to the alpha carbonyl group of gpl20 glycine 473 or disrupts the dipolar interaction between the alpha carbonyl group of gpl20 glycine 473 and the side chain phenyl group of CD4 phenylalanine 43;
- Y a group, Y, that binds to the side chain carboxyl group of gpl20 aspartic acid 368 or disrupts the ionic interaction between the side chain carboxyl group of aspartic acid 368 and the side chain guanidinium group of CD4 arginine 59, wherein Y is alkylammonium, d i a 1 k y 1 a mm o n i u m , a r y 1 a mmo n i urn , aryl alkyl ammonium , alkylguanidinium , piperidinium, pyrollidinium, or pyridinium.
- an alkyl group, R aromatic or heteroaromatic group, Het, that binds to the side chain isopropyl (or methyl) group of gpl20 valine (or alanine) 430 or disrupts the hydrophobic interaction between the side chain isopropyl (or methyl) group of valine (or alanine) 430 and the side chain guanidinium group of CD4 arginine 59; j.
- a group, X that binds to the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 glycine (alanine, or glutamic acid) 472 with the side chain amide group of CD4 glutamine 40;
- a group, Z that binds tot healpha amino group of gpl20 glycine (alanine, or glutamic acid) 472 at a distance of 3.6 ang., or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (alanine, or glutamic acid) 473 with the side chain propionamide group of CD4 glutamine 40;
- X a group, X, that binds to the alpha carbonyl group of gpl20 tryptophan 427 or disrupts the hydrogen bond interaction between the alpha carbonyl of gpl20 tryptophan 427 and the alpha amino group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 lysine 429 or disrupts the hydrogen bond interaction betweeen the alpha amino group of gpl20 lysine (threonine or asparagine) 429 and the side chain hydroxyl group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 valine (or alanine) 430 or disrupts the hydrogen bond between the alpha amimo group of gpl20 valine (or alanine) 430 and the side chain hydroxyl group of CD4 serine 42;
- X that binds to the alpha amino group of gpl20 aspartic acid 368 or disrupts the hydrogen bond between the alpha amino group of gpl20 aspartic acid 368 and the alpha carbonyl group of CD4 leucine 44;
- al . a group, X that binds to the alpha amino group of gpl20 glycine 367 or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine 367 and the alpha carbonyl group of CD4 lysine 46;
- Y that binds to the side chain acetamide (or methyl alcohol) group of gpl20 asparagine (or serine) 280 at a distance of 3.3 ang., or disrupts the hydrogen bond interaction between the side chain acetamide
- dl . a group, Z that binds to the alpha amino group of gpl20 glycine (or valine) 459 at a distance of 3.4 ang or disrupts the hydrogen bond interaction between the alpha amino group of gpl20 glycine (or valine) 459 and the alpha carbonyl group of CD4 asparagine 32;
- gl . a group, X that binds to the alpha amino group of gpl20 glycine (or valine) 459 or disrupts the hydrogen bond between the alpha amino group of gpl20 glycine (or valine) 459 with the side chain propionamido group of CD4 glutamine 33;
- a group, X that binds to the alpha carbonyl group of gpl20 serine (or alanine) 365 or disrupts the hydrogen bond between the alpha carbonyl group of gpl20 serine (or alanine) 365 with the side chain amide of CD4 asparagine 52; and/or
- j 1. an alkyl group, R, or an aromatic or heteroaromatic group that binds to the side chain indole group of gpl20 tryptophan (or phenylalanine) 112 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- kl an alkyl group, R, or an aromatic or heteroaromatic group that binds to the side chain phenyl group of gpl20 phenylalanine 382 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- an aromatic or heteroaromatic group that binds to the side chain phenolic group of gpl20 tyrosine 384 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- Y that binds to the side chain carboxyl group of gpl20 glutamic acid 370 and/or disrupts the aforementioned interactions of gpl20 with CD4 phenylalanine 43;
- rl an aromatic or heteroaromatic group that binds to the side chain phenolic group of gpl20 tyrosine 435 and/or disrupts the afroementioned inteactins of gpl20 with CD4 phenylalanine 43.
- This invention also provides
- This invention comprises compounds of formula (I) of formula (II) :
- X is a group that is designed to mimic Arg- 59 in CD4 ;
- Y is a group that is designed to mimic Phe-43 in CD4;
- Z is -NRC(R (R 2 )C0-;
- Q is -OH, -OR, -NR 1# R 2 or -NH-Lys-OR;
- W is either or W is NR X R 2
- R, R lr and R 2 are the same or different and are hydrogen, alkyl, Bn, EtPh, (cycloalkyl) alkyl , arylalkyl, heteroarylalkyl , haloalkyl, hydroxyalkyl, aminoalkyl, amino acid sidechain.
- Y is Aryl(CH 2 )n-; Cyclohexyl (CH 2 ) n- . Unless otherwise indicated, the terms are defined as follows :
- alkyl is used herein at all occurrences to mean a straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl , isobutyl, tert-butyl, and the like.
- halo or halogen are used interchangeably herein at all occurrences to mean radicals derived from the elements chlorine, fluorine, iodine and bromine.
- aryl or “heteroaryl” are used herein at all occurrences to mean substituted and unsubstituted aromatic ring(s) or ring systems which may include bi-or tri-cyclic systems and heteroaryl moieties, which may include, but are not limited to, heteroatoms selected from 0, N, or S.
- Representative examples include, but are not limited to, phenyl, benzyl, naphthyl, pyridyl, quinolinyl, thiazinyl, and furanyl .
- Ph is used herein at all occurrences to mean phenyl .
- moiety Btd. is synthesized by the procedure described in Ngai et al., Tetrahedron, 1993, 49, 3577-3592, (incorporated herein by reference) .
- This invention also provides the above-described vaccine, wherein the 6 or more amino acids are identical to the amino acids of naturally occurring gpl20.
- This invention further provides the above-described vaccines, wherein the amino acids are within 1 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the amino acids are within 3 angstroms of their distances in naturally occurring gpl20.
- This invention provides the above-described vaccines, wherein the amino acids are within 5 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the above-described vaccines, further comprising a carrier.
- This invention also provides the above-described vaccines, further comprising an adjuvant.
- This invention provides a vaccine comprising a polypeptide having 6 or more continuous amino acids from the Phe 43 cavity of gpl20.
- This invention provides the above-described vaccines, wherein the polypeptide is or is part of an epitope a conserved neutralization epitope.
- This invention also provides the above-described vaccines, further comprising a carrier.
- This invention further provides the above-described vaccines, further comprising an adjuvant.
- This invention further provides a vaccine comprising a polypeptide having 6 or more amino acids in the same spatial proximity to each other as the surface accessible amino acids adjacent to the Phe 43 cavity of naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the 6 or more amino acids are identical to the amino acids of naturally occurring gpl20.
- This invention provides the above-described vaccines, wherein the amino acids are within 1 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the amino acids are within 3 angstroms of their distances in naturally occurring gpl20.
- This invention further provides the above-described vaccines, wherein the amino acids are within 5 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the above -described vaccines, further comprising a carrier.
- This invention also provides the above-described vaccines, further comprising an adjuvant.
- This invention also provides the above-described vaccines, wherein the surface accessible amino acids comprise Lysine 432, Proline 369, and Threonine 373.
- This invention further provides a vaccine comprising a polypeptide having 6 or more continuous surface accessible amino acids adjacent to the Phe 43 cavity of gpl20.
- This invention also provides the above -described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the above-described vaccines, further comprising a carrier.
- This invention also provides the above-described vaccines, further comprising an adjuvant.
- This invention further provides a method of inhibiting cell entry by HIV, comprising blocking or inhibiting the residues from 2 or more the sets of the CCR5 -binding residues set forth above, thereby inhibiting or preventing gpl20 from binding to CCR5 and thereby inhibiting cell entry by HIV.
- This invention also provides the above described method wherein 3 or more the sets of the CCR5-binding residues set forth above are blocked or inhibited from interacting with CCR5.
- This invention also provides the above described methods, wherein the blocking or inhibiting comprises contacting the CCR5-binding residues with an antibody.
- Crystalline order is explicitly dependent on lattice homogeneity. Reducing heterogeneity can be thought of as increasing the proportion of surface area available for formation of lattice contacts, increasing the probability of crystallization.
- the probability that a single lattice contact between two molecules is homogeneous is in part related to the fraction of surface that is homogeneous on one molecule multiplied by the fraction homogeneous on the other, that is, to:
- the overall crystallization probability is related to: ⁇ (H- ⁇ ) 2*c , where the sum is over all possible lattices, "H” is the fraction of the surface which may form lattice contacts, “ ⁇ ” is a function of the size of the lattice contact and the degree of surface homogeneity - related to the occlusion of available surface area upon formation of each lattice contact as well as the spatial distribution of homogenous surface over the molecular surface; and "C” is the number of unique contacts required to make a set of symmetry-related molecules into a crystal lattice
- the use of multiple variants of the same protein also increases the probability of crystal formation.
- the overall probability of crystallization is exponentially related to the number of variants. Assuming independence of variants (a reasonable assumption with different protein ligands; not as valid with minor changes) with n variants and a probability of crystallization for each variant of P, the overall probability P ⁇ is:
- the overall probability is 1 - (1-0.25) n ; with 15 variants, the probability increases to almost 99%.
- the enhancement in overall probability is given by the ratio of (P ⁇ / P) - 1. If one tries many variants, and (1-P) n is much smaller than 1, then
- the enhancement is related to the initial probability of crystallizing a single variant.
- the more difficult a protein is to crystallize the more it benefits from this multiple variant strategy.
- Gpl20 constructs The various recombinant gpl20 glycoporteins used for crystallization trials were produced in stable Drosophila Schneider 2 producer lines under the control of an inducible promoter as previously described (20) (Table 1
- Sequence numbers refer to the translated gpl60, with the mature gpl20 beginning at +31. N-terminal sequencing showed that all constructs contained 4 additional amino acids, Gly-Ala-Arg-Ser, an artifact of the signal peptide cleavage. GAG here refers to the tripeptide, Gly-Ala-Gly, which was substituted for the removed amino acids.
- the N-terminal two domains of CD4 (D1D2), residues 1-182, were produced in Chinese hamster ovary (CHO) cells and purified as described previously (21) .
- Secreted gpl20 from Drosophila cells was purified by F105-Protein A affinity chromatography which used a glycine pH 2.8 elution step followed by immediate Tris base neutralization.
- Fabs were produced by papain digestion of monoclonal antibodies. Briefly, the antibody was reduced in 100 mM DTT, 100 mM NaCl, 50 mM
- PBS phosphate-buffered saline
- alkylating solution PBS titrated to pH 7.5 with 2 mM iodoacetamide, 48hr
- the reduced and alkylated antibody was concentrated to at least 2 mg/ml and digested with papain using the commercial protocol (Pierce) .
- An additional gel filtration chromatographic step on a Superdex S-200 column (Pharmacia, FPLC) was added to ensure oligomeric homogeneity.
- the gpl20 proteins were subject to protease digestion, papain, elastase, and subtilisin (Boehringer Manneheim) to assay for proteolytic susceptibility.
- the gpl20 concentration was kept constant and the protease diluted serially (3.3x) from a ratio of 1:10 to 1:1000.
- the digestion mix was incubated for 1 hr at 37° C and quenched by addition of 1% SDS (1:10 ratio) with immediate heating in boiling water for 2 minutes. Digestion products were analyzed with SDS- polyacrylamide gel electrophoresis (PAGE) with and without DTT reduction.
- Carboxypeptidase Y digestion was used to analyze the C- terminus of gpl20. A 1:10 ratio of carboxylpeptidase Y (Boehringer Manneheim) to gpl20 was incubated for 1 hr at 37° C, pH 7.0. Even though digestion could not be easily seen by SDS-PAGE, the C-terminus of gpl20, HXBc2 strain, contains a number of positively charged amino acids, and the extent of the reaction could be monitored by native-PAGE.
- Drosop ila-produced gpl20 proteins were deglycosylated enzymatically . Briefly, 0.5 mg/ml of gpl20 was incubated with various deglycosylating enzymes (singly or in combination) in 0.5 M NaCl, 100 mM Na Acetate, pH 5.7, for 10 hr at 37° C. Endoglycosidase D was used at a concentration of 0.1 U/ml, Endoglycosidase F at 0.25 U/ml, Endoglycosidase H at 0.25 U/ml, and Glycopeptidase F at 0.1 U/ml (all from Boehringer Manneheim) .
- Monoclonal antibody binding assay The various gpl20 glycoproteins were assessed for recognition by a variety of monoclonal antibodies directed against both linear and discontinuous gpl20 epitopes by either immunoprecipitation (31) or by ELISA (32) .
- the ELISA was performed with both fully glycosylated and deglycosylated ⁇ V1/2 ⁇ V3 glycoproteins immobilized on ELISA plates using a capture antibody specific for the gpl20 carboxyl-terminus, 6205 (International Enzymes) (32) .
- Crystallization The vapor diffusion hanging droplet technique was used for all crystallizations. Small volumes, 0.5 ⁇ l protein solution + 0.5 ⁇ l reservoir solution, were used for virtually all crystallizations, screenings as well as final optimizations.
- Crystal Screen I The Crystal Screen I (Hampton Research) was used, augmented by roughly 20 conditions which tested high protein concentrations (vapor diffusion concentration of the protein at various pHs) as well as mixtures of organic additives (2-5% MPD, PEG 400, or PEG 4000) combined with high ionic strength (2-4 M NaCl, Am 2 S0 4 or Na/K ⁇ O ) at pH 5.5-9.5.
- high protein concentrations vapor diffusion concentration of the protein at various pHs
- mixtures of organic additives 2-5% MPD, PEG 400, or PEG 4000
- high ionic strength 2-4 M NaCl, Am 2 S0 4 or Na/K ⁇ O
- Type E crystals were grown from the following conditions: Protein ( ⁇ 82 ⁇ V1/2* ⁇ V3 ⁇ C5 gpl20, two-domain CD4 (D1D2) , Fab 17b purified as a ternary complex on the Superdex S-200) ; Droplet (0.5 ⁇ l protein solution consisting of -10 mg/ml protein in gel filtration buffer + 0.4 ⁇ l droplet mix containing 0.1 M NaCitrate, 0.02 M NaHepes, 10% isopropanol, 8% PEG 5000 (Fluka) , 0.0075% SeaPrep Agarose (FMC BioProducts) , pH 6.4; Reservoir: (0.35 M NaCl, 0.1 M NaCitrate, 0.02 M Hepes, 10% isopropanol, 8% PEG 5000, pH 6.4) .
- the droplet 0.5 ⁇ l protein solution consisting of -10 mg/ml protein in gel filtration buffer + 0.4 ⁇ l droplet mix containing 0.1 M Na
- Lattice contacts are made solely at the molecular surface . Unlike small molecules, macromolecules have interiors -considerable surface, and hence crystallization, variability is tolerated while maintaining the same basic fold or even enzymatic abilities.
- a prescient example that pre-dates the powerful methods of modern molecular biology was John Kendrew' s screening of myoglobins from many different organisms until he found one, from sperm whale, that crystallized well (37) . Indeed, human myoglobin requires a Lys to Arg mutation in order to produce crystals suitable for structural analysis (38) .
- crambin is actually a mixture of two isoforms with sequence variation at internal residues (39) .
- the molecular weight for the glycosylated gpl20 is approximately 90kDa; the deglycosylated gpl20, 60 kDa; and the deglycosylated ⁇ V1/2 ⁇ V3 gpl20, 47 kDa.
- the ⁇ -terminus is resistant to proteolysis from +39 to +82, and thus probably adopts an ordered conformation. This number was calculated assuming only the C-terminal 19 and the ⁇ -terminal 8 amino acids were disordered.
- Variants of gpl20 were developed through an iterative cycle which strove to eliminate heterogeneity.
- the cycle involved recombinant production of gpl20 variants, deglycosylation, and then assessment of heterogeneity and flexibility by examination of glycosylation status, monoclonal antibody binding, and protease sensitivity, leading to the design of new constructs. For example. at the gpl20 C-terminus, protease digestion and native PAGE detected variability, and carboxyl peptidase Y digestion generated a 15-20 amino acid deletion which retained CD4 binding activity.
- variable loops VI, V2 , and V3 were replaced. Little effect was found on CD4 binding (32,40,41).
- Three constructs were made which contained deletions of the VI, V2 , and V3 loops (Table 1) . With ⁇ V1/2 ⁇ V3, the entire base and stem of the variable loops VI, V2 and V3 were excised. With ⁇ V1/2* ⁇ V3, the conserved stem of the V1/V2 stem-loop structure was retained, restoring the CD4-induced antibody epitopes in the presence of soluble CD4. With ⁇ Vl/2* ⁇ V3* the base of the V3 loop was retained as well, fully restoring CD4-induced antibody epitopes, even in the absence of soluble CD4.
- Endoglycosidase H which has specificity for oligosaccharides with 5-9 mannoses, removed roughly 60% of the carbohydrate, and addition of Endoglycosidase D, which cleaves oligosaccharides with 3 or 4 mannoses, removed up to 90% of the carbohydrate .
- CD4 Protein ligands, CD4 and the Fabs of monoclonal antibodies, were used in an attempt to reduce overall surface, and hence potential crystal lattice, mobility. This was complicated by the internal mobility of these ligands: CD4 has a flexible juncture between the second and third extracellular domains (42) , and Fabs have a conformationally mobile "elbow bend" between their variable and constant domains (43) .
- CD4 we used a construct containing only the N-terminal two domains (1- 182) , for which there was previous success in structure determination (14) .
- monoclonal antibodies we limited the crystallization screens to using only one Fab at a time, even though combinations with multiple Fabs were possible.
- Fab F105 no crystals to the percent of N- linked sites cleaved by endoglycosidase D or H.
- the "fully deglycosylated" protein still contains N-acetyl glucosamine and fucose moieties.
- D1D2 sCD4 refers to two-domain soluble CD4. Antibody epitopes are described in the text.
- small volume droplets were used, typically 0.5 ⁇ l of protein per crystallization trial. With small volumes, only 1-2 mg of protein were sufficient to evaluate each gpl20 crystallization variant. Smaller volumes were found to be more efficient at nucleation than larger droplets, perhaps due to higher surface tension effects resulting in greater variations in precipitant concentration, thereby permitting each droplet to sample a wider range of precipitant concentrations. Indeed, droplets that were "spread-out" also showed enhanced nucleation. This explanation may also account for the well-known observation that crystals frequently nucleate from the edges of crystallizaton droplets.
- the initial crystallization screens produced six different types of crystals (Table 4) For crystal types A-D, extensive optimization was unable to produce single crystals large enough to be characterized.
- crystal types E and F single crystals of needle morphology could be grown. With type E crystals, the needle axis was coincident with the a axis, with the cross-section perpendicular to the needle axis a rhombehedron bounded by faces of the form (0 1 1) and (0-1 1) . These could be distinguished from type F crystals, where the cross- section was hexagonal.
- Single crystals of type E and F were analyzed for diffraction in capillary mounts. Only type E crystals showed diffraction. Gel electrophoresis of these crystals demonstrated that they contained gpl20, D1D2 and Fab 17b ( Figure 4) .
- D1D2 sCD4 refers to the two domain soluble CD4. ** The protein concentration is given as the absorbance (280 nm) of the complex per ml of solution. *** Most of the reservoirs are conditions from Crystal Screen 1 (Hampton Research) ; the reagent numbers given here refer to the crystallization reagent from this commercial kit. Hanging droplets were 0.5 ⁇ l protein (in 0.35 M NaCl, 5 mM Tris pH 7.0, 0.02% NaN 3 ) + 0.5 ⁇ l reservoir, except for crystal type B, which used 0.5 ul of 3 -fold diluted reservoir.
- Crystallization reservoirs were 500 ⁇ l; an additional 35 ul of 5 M NaCl was added after the droplet was mixed to compensate for the NaCl in the protein solution. All dilutions used H 2 0, except for crystal type F, where 22.5% isopropanol was used. Crystallizations were setup at room temperature and incubated at 20 °C.
- Table 6 which follows, shows the critical residues in gpl20 for interactions with CD4. Table 6. Critical Residues in GP120 for Interactions with CD4
- Kelders H. A., Kalk, K. H. , Gros, P., and . G., H.
- the human immunodeficiency viruses (HIV-1 and HiV-2) and simian immunodeficiency viruses (SIV) are the etiologic agents of acquired immunodeficiency syndrome (AIDS) in their respective human and simian host (1) .
- infection with primate immunodeficiency viruses is characterized by an initial phase of high-level viremia, followed by a long period of persistent virus replication at a lower level (2) .
- Viral persistence occurs despite specific antiviral immune responses, which include the generation of neutralizing antibodies.
- the primate immunodeficiency viruses like all retroviruses, are surrounded by an envelope consisting of a host cell-derived lipid bilayer and virus-encoded envelope glycoproteins (3) .
- the viral membrane To enter host cells, the viral membrane must be fused with the plasma membrane of the cell, a process mediated by the envelope glycoproteins.
- the exposed location of these proteins on the virus allows them to carry out their function but also renders them uniquely accessible to neutralizing antibodies.
- dual selective forces, virus replication and immune pressure have shaped the evolution of the envelope glycoproteins and continue to do so within each infected host.
- the envelope glycoproteins are synthesized as approximately 845-870 amino acid precursor in the rough endoplasmic reticulum. N- linked, high- mannose sugar chains are added to form the gpl60 glycoprotein, which assembles into oligomers (4-6) . The preponderance of evidence suggests that these oligomeric complexes are trimers (4,5) .
- the gpl60 trimers are transported to the Golgi apparatus, where cleavage by a cellular protease generates mature envelope glycoproteins: gpl20, the exterior envelope glycoprotein, and gp41, the transmembrane glycoprotein (3) .
- the gp41 glycoprotein possesses an ectodomain that is largely responsible for trimerization (7) , a membrane-spanning anchor, and a long cytoplasmic tail. Most of the surface-exposed elements of the mature, oligomeric envelope glycoprotein complex are contained on the gpl20 glycoprotein. Selected, presumably well-exposed, carbohydrates on the gpl20 glycoprotein are modified in the Golgi apparatus by the addition of complex sugar (6) . The gpl20 and gp41 glycoproteins are maintained in the assembled trimer by non-covalent , somewhat labile interactions between the gp41 ectodomain and discontinuous structures composed of N- and C-terminal gpl20 sequences (8) .
- Virus attachment also involves the interaction of the gpl20 envelope glycoproteins with specific receptors, the CD4 glycoprotein (11) and members of the chemokine receptor family (12, 13) (Fig. 6) .
- the CD4 glycoprotein is expressed on the surface of T lymphocytes, monocytes, dendritic cells, and brain microglia, the main target cells for primate immunodeficiency virus in vivo. The requirement for CD4 binding exhibited by most primate immunodeficiency viruses for efficient entry is consistent with this observed in vivo tropism.
- CD4 binding A major function of CD4 binding is to induce conformational changes in the gpl20 glycoprotein that contribute to the formation and/or exposure of the binding site for the chemokine receptor (13, 14).
- the use of CD4 as a receptor may have evolved subsequently, allowing the high-affinity chemokine receptor-binding site of primate immunodeficiency viruses to be sequestered from host immune surveillance.
- the more conserved regions fold into a gpl20 core which has been recently crystallized in a complex with fragments of CD4 and a neutralizing antibody (20) .
- the gpl20 core is composed of two domains, an inner domain and an outer domain (Fig. 7a) . These names reflect the likely orientation of gpl20 in the assembled envelope glycoprotein trimer: the inner domain faces the tri er axis and, presumably, gp41, while the outer domain is mostly exposed on the surface of the trimer. Elements of both domains contribute to CD4 binding.
- CD4 binds in a recessed pocket on gpl20, making extensive contact over approximately 800 A° 2 of the gpl20 surface.
- a shallow cavity is filled with water molecules, while a deep cavity extends 10-15 A° into the interior of gpl20.
- the opening of this deep cavity is occupied by phenylalanine 43 of CD4 , which has been shown by mutagenic analysis to be critical for gpl20 binding (21) .
- Most of the gpl20 residues previously identified as important for CD4 binding (22,23) surround the opening of the deep cavity and contribute to interactions with phenylalanine 43 of CD4.
- aspartic acid 368 of gpl20 forms a salt bridge with arginine 59 of CD4 , also shown by mutagenesis to be important for gpl20 binding (21) .
- mainchain atoms on gpl20 and CD4 form hydrogen bonds bridging the two proteins.
- the formation of the deep cavity in gpl20 likely contributes to the transmission of CD4-induced conformational changes to gpl20 elements involved in the interaction with chemokine receptors and/or gp41.
- the deep cavity may be a useful target for intervention by small molecular weight compounds .
- CCR5 chemokine receptors
- V3-deleted versions of gpl20 do not bind CCR5, even though CD4 binding occurs at wild-type levels (14) .
- Antibodies against the V3 loop interfere with gpl20-CCR5 binding (14) .
- These results support an involvement of the V3 loop in chemokine receptor binding.
- Other, conserved gpl20 structures also appear to play an important role in chemokine receptor binding.
- Antibodies that recognize conserved, discontinuous gpl20 epitopes that are more exposed after CD4 binding are potent inhibitors of gpl20-CCR5 interaction (14) . These CD4-induced (CD4i) epitopes are discussed further below.
- Recent mutagenic and structural analysis have revealed the existence of a highly conserved gpl20 structure that is important for CCR5 binding (20,27) (Fig. 7, a and b) . This structure is adjacent to the V3 loop and the CD4i epitopes, and is oriented to face the target cell upon gpl20-CD4 binding.
- the gp41 ectodomain structures reveal an extended, trimeric coiled coil that could potentially bridge the viral and target cell membranes (5) .
- Interactions of other gp41 helical segments near the membrane-spanning region with the interhelical grooves of the internal coiled coil are important for fusion-related conformational changes in gp41. This interaction can be inhibited by helical peptides that mimic either of the involved gp41 helices
- HIV-1 envelope glycoproteins as antigens.
- the success of these viruses in achieving persistent infections implies that the viral envelope glycoproteins have evolved to be less-than-ideal immunogens and antigens.
- Structures on the viral envelope glycoproteins that are conserved among diverse viral strains are, in general, poorly exposed to the humoral immune system.
- the moieties involved in gpl20-gp41 association are buried in the interior of the functional envelope glycoprotein spike (18, 31, 32) .
- the CD4 binding sites is recessed, flanked by variable regions exhibiting considerable glycosylation
- HIV-1 viruses that have been passaged in immortalized cell lines are typically more sensitive to neutralization by antibodies or soluble CD4 than are primary, clinical isolates (34) .
- a major determinant is the structure of the gpl20 major variable loops, V1/V2 and V3 (35) .
- V1/V2 and V3 variable loops of a laboratory-adapted virus with those of a neutralization-resistant primary isolate creates a virus similar to the parental primary virus (35) .
- the basis for the decreased sensitivity of primary HIV-1 isolates to neutralization appears to involve a decreased exposure of the relevant gpl20 epitopes to soluble CD4 or antibody.
- the temporal pattern of the antibody response to HIV-1 infection The noncovalent nature of the association between gpl20 and gp41 contributes to the lability of the functional envelope glycoprotein trimer (8,9).
- the interactive regions of gpl20 and gp41 are particular immunogenic (37) .
- the cognate antibodies cannot bind the assembled, functional envelope glycoprotein complex, they do not exhibit neutralizing activity.
- antibodies against the envelope glycoproteins typically can be detected in the sera of HIV-1-infected individuals by two-three weeks after infection, most of these antibodies lack the ability to inhibit virus infection. By the time that neutralization antibodies are efficiently elicited, HIV-1 is firmly established in the host.
- neutralizing antibodies can be detected in the sera of infected animals or humans (38) . These antibodies neutralize the infecting virus but often exhibit little of no activity against other stains of virus. A subset of these strain- restricted antibodies recognize the HIV-1 V3 loop (38) . These antibodies can block chemokine receptor binding
- variable gpl20 elements can contribute to the epitopes recognized by the strain-restricted neutralizing antibodies. It is known, for example, that antibodies directed against the gpl20 V2 loop can also exhibit neutralizing activity (39) .
- the V2 loop- associated neutralization epitopes are typically conformation-dependent.
- the ability of some V2-or V3- directed antibodies to recognize more than one HIV-1 strain (39,40) suggests that these major variable loops assume a finite number of conformations. This is consistent with the functional consequences on virus entry of some changes in these variable structures (41) , and with the observation that amino acid substitutions in the variable loops are not random (42) .
- the requirement for chemokine receptor binding probably constrains V3 loop variation.
- the V2 loop although dispensible for the replication of some HIV-1 viruses in culture (33) , helps protect the V3 loop and the conserved epitopes near the chemokine receptor binding site from neutralizing antibodies.
- the V2 and V3 loops reside proximal to the chemokine receptor binding site (Fig. 7) , masking more conserved gpl20 elements and presenting potentially variable epitopes to the immune system.
- the gpl20 residues important for antibody binding are all located within the CD4 -binding pocket on gpl20 (Fig. 7b) , and several of the most important residues are near the opening of the deep cavity (20) . Therefore, some broadly neutralizing antibodies can apparently access the more recessed elements of the CD4 binding pocket. This is consistent with the observation that the gpl20-CD4 interface is as large as that of a typical antibody- antigen complex (20) .
- CD4i CD4-induced epitopes
- the CD4i epitopes are located near conserved gpl20 structures important for chemokine receptor interaction (14) (Fig. 7b) .
- CD4 binding has been shown to cause a change in the V2 loop conformation that allows better CD4i epitope exposure (33) .
- the antibodies recognizing the CD4i epitopes must bypass the overlapping V2 and V3 loops (33) . Indeed, as is evident in the current crystal structure (20) , this is accomplished by the protrusion of the CDR3 loop of the antibody heavy chain.
- Antibodies against CD4i epitopes need to bind viruses before CD4 binding occurs to achieve neutralization (47) .
- the reason is that once the envelope glycoprotein complex binds cell surface CD4 , there are severe steric constraints on the binding of an antibody to the gpl20 surface facing the target cell (Fig. 6) .
- Another fairly conserved gpl20 neutralization epitope is recognized by the 2G12 antibody (48) .
- the 2G12 antibody Unlike the other characterized HIV-1 neutralizing antibodies, which recognize gpl20 structures near or within the receptor- binding sites, the 2G12 antibody apparently binds an epitope in the outer domain (Fig. 7b) . Given the variability in this outer domain, the ability of the 2G12 antibody to neutralize a fair number of HIV-1 strains (48) seems paradoxical.
- the marked sensitivity of 2G12 antibody may recognize more conserved carbohydrate structures formed as a result of the heavy concentration of N-linked glycosylation in the gpl20 outer domain.
- the apparent rarity with which 2G12-like antibodies are elicited attests to the success of the viral strategy of employing a heavily glycosylated outer domain surface in immune evasion.
- the HIV-1 envelope glycoproteins as vaccine components. That the human and simian immunodeficiency virus envelope glycoproteins are not ideal immunogens is an expected consequence of the immunological selective forces that drove the evolution of these viruses.
- the same features of the envelope glycoproteins that dictate poor immunogenicity in natural infections have hampered vaccine development.
- the lability of envelope glycoprotein complex has frustrated attempts to present oligomers mimicking the functional spike to the immune system.
- the disintegration of envelope glycoprotein oligomers contributes to the preferential elicitation of non-neutralizing antibodies by the newly exposed gpl20 N- and C-termini.
- variable loops elicit the majority of neutralizing antibodies, probably due to the exposed nature of these epitopes. It is still unclear whether conserved features in the V2 and V3 variable loops exist that can be exploited in vaccine design, or whether all possible functional configurations of these variable structures need to be represented in a cocktail of immunogens .
- the discontinuous gpl20 structures surrounding the receptor binding sites exhibit a relatively high degree of conservation (20), in keeping with the minimal polymorphism in the host cell receptors.
- the CD4 binding site contributes a particularly attractive target. It appears to be accessible to antibodies, more so than the conserved elements of the chemokine receptor-binding region. A large fraction of the broadly neutralizing antibodies that eventually appear in HIV-1-infected individuals is directed against the CD4 binding site (43), indicating that ability of the human immune system to recognize this gpl20 region and to generate an appropriate response. Nonetheless, these antibodies have been difficult to elicit in animals and vaccinated humans
- HIV-1 envelope glycoproteins have evolved to be inefficient at eliciting effective antiviral antibody responses.
- the availability of structural information on the conserved HIV-1 gpl20 neutralization epitopes should facilitate the modification of this important antigen and allow the rational testing of hypotheses regarding its poor immunogenic properties. These efforts should complement ongoing efforts to improve antigen presentation to the immune system and to create suitable animal models for the screening of vaccine candidates .
- HIV human immunodeficiency virus
- gpl20 The entry of human immunodeficiency virus (HIV) into cells requires sequential interactions of the viral exterior envelope glycoprotein, gpl20, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes.
- gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system.
- the structure reveals a cavity-laden CD4-gpl20 interface, a conserved binding site for the chemokine receptor, evidence for conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion.
- Our results provide a framework for understanding the complex biology of HIV entry into cells and will guide efforts to intervene.
- HIV-1 and HIV-2 and the related simian immunodeficiency viruses (SIV) cause the destruction of CD4+ lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndrome (AIDS) (1, 2) .
- AIDS acquired immunodeficiency syndrome
- the entry of HIV into host cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric, spikes displayed sparsely on the surface of the virion. These envelope complexes are anchored in the viral membrane by the gp4l transmembrane envelope glycoprotein.
- the surface of the spike is composed primarily of the exterior envelope glycoprotein, gpl20, associated by noncovalent interactions with each subunit of the trimeric gp41 glycoprotein complex (3, 4.)
- V1-V5 variable regions
- the first four variable regions form surface-exposed loops that contain disulfide bonds at their bases6.
- the conserved gpl20 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gpl20 regions are extensively glycosylated6.
- the variability and glycosylation of the gpl20 surface likely modulate the immunogenicity and antigenicity of the gpl20 glycoprotein, which is the major target for neutralizing antibodies elicited during natural infection (7) .
- gpl20 envelope glycoprotein binds to the CD4 glycoprotein, which serves as the primary receptor.
- the gpl20 glycoprotein binds to the most amino-terminal of the four immunoglobulin- like domains of CD4. Structures of both the N-terminal two domains (8, 9) and the entire extracellular portion of CD410 have been determined, and mutagenesis studies indicate that the CD4 structure analogous to the second complementarity-determining region (CDR2) of immunoglobulins is critical for gpl20 bindingll, 12. conserveed gpl20 residues important for CD4 binding have likewise been identified by mutagenesis (3, 13, 14) .
- CD4 binding induces conformational changes in the gpl20 glycoprotein, some of which involve the exposure and/or formation of a binding site for specific chemokine receptors.
- chemokine receptors mainly CCR5 and CXCR4 for HIV, serve as obligate second receptors for virus entry (15, 16.)
- the gpl20 third variable (V3) loop is the major determinant of chemokine receptor specificity (17) .
- V3 loop is the major determinant of chemokine receptor specificity (17) .
- other more conserved gpl20 structures that are exposed upon engagement of CD4 also appear to be involved in chemokine-receptor binding.
- CD4-induced exposure is indicated by the enhanced binding of several gpl20 antibodies (18, 19) which, like V3-loop antibodies, efficiently block the binding of gpl20-CD4 complexes to the chemokine receptor (20) . These are called the CD4-induced (CD4i) antibodies.
- CD4 binding may trigger additional conformational changes in the envelope glycoproteins. For example, the binding of CD4 to the envelope glycoproteins of some HIV-1 isolates induces the release or "shedding" of the gpl20 protein from the complex (21) , although the relevance of this process to HIV entry is uncertain.
- HIV and related retroviruses belong to a class of enveloped fusogenic viruses that includes corona-, paramyxo- and orthomyxoviruses (e.g. influenza virus), all of which require post-translational cleavage for activation.
- the transmembrane coat proteins of these viruses (gp41 equivalents) share sequence resemblance, particularly in their N-terminal fusion peptides, and they participate directly in membrane fusion.
- the ectodomain of gp41 can form a coiled coil resembling that of influenza hemagglutinin HA (23, 4, 22,) supporting the notion that this class of viruses may share some common aspects with respect to virus entry. In other respects, enveloped viruses tend to be distinctive.
- CD4i epitope A companion report relates this structure to the antigenic properties of the gpl20 envelope proteins (24) .
- the crystallized gpl20 is from the HXBc2 strain of HIV-1. It has deletions of 52 and 19 residues from the N- and C- termini, respectively; Gly-Ala-Gly tripeptide substitutions for 67 Vl/V2-loop residues and 32 V3-loop residues; and the removal of all sugar groups beyond the linkages between the two core N-acetylglucosamine residues.
- This deglycosylated core gpl20 eliminates over 90% of the carbohydrate but retains , over 80% of the non-variable-loop protein. Its capacity to interact with CD4 and relevant antibodies is preserved at or near wild-type levels26.
- the final model, composed of 7877 atoms comprises residues 90-396 and 410-492 of gpl20 (excepting loop substitutions), residues 1-181 of CD4 , and residues 1-213 of the light chain and 1-229 of the heavy chain of the 17b monoclonal antibody.
- 11 N-acetylglucosamine and 4 fucose residues, and 602 water molecules have been placed.
- the overall structure of the complex of gpl20 with D1D2 of CD4 and Fab 17b is as depicted in Fig. 8.
- the deglycosylated core of gpl20 as dissected from the ternary complex approximates a prolate ellipsoid with dimensions of 50 x 50 x 25 ⁇ , although its overall profile is more heart-shaped than circular.
- Its backbone structure is shown in Figs. 9a & c in an orientation precisely perpendicular to that in Fig. 8 (Fig. He gives a mutually perpendicular view) .
- This core gpl20 comprises 25 b strands, 5 a helices and 10 defined loop segments, all organized with the topology shown in Fig. 9b. Specific spans of structural elements are given in Fig. 9d.
- the structure confirms the chemically determined disulfide bridge assignments (6; Fig. 9c) .
- the polypeptide chain of gpl20 is folded into two major domains plus certain excursions that emanate from this body.
- the inner domain (inner with respect to the N- and C-termini) features a two-helix, two-strand bundle with a small five-stranded b sandwich at its termini-proximal end and a projection at the distal end from which the V1/V2 stem emanates.
- the outer domain is a stacked double barrel that lies alongside the inner domain such that the outer barrel and inner bundle axes are approximately parallel.
- the proximal barrel of the outer-domain stack is composed from a 6-stranded, mixed-directional b sheet that is twisted to embrace helix a2 as a 7th barrel stave.
- the distal barrel of the stack is a 7-stranded antiparallel b barrel.
- the two barrels share one contiguous hydrophobic core, and the staves also continue from one barrel to the next except at the domain interface.
- This interruption is centered at a side between barrels where the chain enters the outer domain with loop LB insinuated as a tongue between strands bl6 and b23.
- the extended segment just preceding LB is like an 8th stave of the distal barrel, but it is slightly out of reach for hydrogen bonding with its bl6 and b9 neighbors.
- the chain returns to complete the inner domain after b24.
- the proximal end of the outer domain includes variable loops V4 and V5 and loops LD and LE, which are variable in sequence as well.
- Loop LC is also at this end, close in space to loop LA of the inner domain, although by topology it is at the other end of this domain.
- the distal end does include the stem of the excised variable loop V3 and also an excursion via loop LF into a b hairpin, b20-b21, which in turn hydrogen bonds with the VI/V2 stem emanating from the inner domain.
- This bridging sheet also participates in the separated interactions of gpl20 with both CD4 and the 17b antibody (Fig. 8 and below) .
- One further excursion from the body of the outer domain produces strand bl5 and helix a3 , which are also important in CD4 binding.
- This structure of core gpl20 should be a prototype for the class.
- FIG. 9d shows that an HIV-2 sequence is 35% identical with that of the HXBc2 strain expressed in this crystallized construct, and the identity level rises to 77% and 51%, respectively, for the more closely related HIV-1 clade C and clade O representatives.
- the inner domain is appreciably more conserved than the outer domain with 86%, 72% and 45% identity for the respective C, O and HIV-2 comparisons. Variability correlates with the degree of solvent exposure of residues (Fig. 9d) , in keeping with the conservation of hydrophobic cores.
- the seven disulfide bridges retained in core gpl20 are absolutely conserved and mostly buried (Fig. 9c) .
- Glycosylation sites are all surface exposed and are conserved above average (Fig. 9d) .
- the previously identified HIV variable segments ⁇ are all on loops connecting elements of secondary structure, and loops LD and LE are also especially variable. Indeed, LE is more variable than V5 in light of current sequence data. These loops are also relatively mobile as reflected in high B factors or disorder, as in V4.
- variable segments in the outer domain, including the exposed face of a2 appear to arise from neutral mutation rather than selective pressure since they are on non-immunogenic surfaces, presumably masked by glycosylation.
- CD4 is bound into a depression formed at the interface of the outer domain with the inner domain and the bridging sheet of gpl20 (Figs. 10a) .
- This interaction buries a total of 742 A° 2 from CD4 and 802 A° 2 from gpl20.
- the surface areas that are actually in contact are considerably smaller (Fig. lOd) because an unusual mismatch in surface topography creates large cavities that are occluded in the interface, as described below. There is, however, a general complementarity in electrostatic potential at the surfaces of contact, although the match is imprecise in this respect as well.
- the focus of CD4 positivity is displaced from the center of greatest negativity on gpl20 (Fig. 10c) .
- the binding site is devoid of carbohydrate (Fig. lOg) .
- the structure of CD4 in this complex differs only locally from that in free D1D2 structures and at only a few places : residues 17-20 at the poorly ordered CDRl-like loop and residues 41,42,47,49 and 60, which are at or near the contact site and have low B factors in the gpl20-bound state.
- Direct interatomic contacts are made between 22 CD4 residues and 26 gpl20 amino-acid residues. These include 219 van der Waals contacts and 12 hydrogen bonds. Residues in contact are concentrated in the span from 25 to 64 of CD4 , but they are distributed over six segments of gpl20 (Figs. 9d & lOi) : 1 residue from the V1/V2 stem, loop LD, the beta-15-alpha-3 excursion, the beta-20-beta- 21 hairpin, strand beta-23 and the beta-24-alpha-5 connection. These interactions are compatible with previous analyses of mutational data on both CD411, 12, 29 and gpl203, 13, 14.
- Lys 29 makes a direct ionic hydrogen bond, and while Asp 457 of gpl20 is near to these electropositive groups (Figs. lOe & i) it does not make hydrogen bonds .
- gpl20 residues that are covered by CD4 are variable in sequence. This variation is accommodated in part by the large interfacial cavity (Fig. lOe) .
- the gpl20 residues in contact with this water-filled cavity are especially variable (Fig. lOg) .
- half of the gpl20 residues that make contacts with CD4 do so only through main-chain atoms (including Cb) of gpl20, and 60% of CD4 contacts are made by gpl20 main-chain atoms (Fig. lOf) . Included among these are 5 of the 12 hydrogen bonds in the interface.
- One such contributing element is an antiparallel b-sheet alignment of CD4 strand C" with gpl20 strand beta-15 (Figs. 10a & i) .
- Atomic details of the interaction are particularly intricate and unusual for the contacts made between gpl20 and the mutationally critical CD4 residues Phe 43 and Arg 59 (Fig. lOj).
- Arg 59 interacts with Asp 368 and Val 430.
- the carboxylate group of Asp 368 makes double hydrogen bonds with the guanidinium Nh atoms of Arg 59, but it also hydrogen bonds back to the backbone NH group of residue 44 and it appears to be optimally positioned to receive a CH...0 hydrogen bond (3.20 A°) from the Phe 43 ring.
- Phe 43 interacts with residues Glu 370, He 371, Asn 425, Met 426, Trp 427 and Gly 473 as well as Asp 368, but only the contacts with He 371 have a conventional hydrophobic character. Those to 425-427 and 473, including Trp 427, are only to backbone atoms. A surprisingly large fraction of the Phe 43 contacts (28%) are to polar groups. The phenyl group is stacked on the carboxylate group of Glu 370, and there are contacts with the carbonyl oxygen atoms of residues 425, 426 and 473 and the NH group of Trp 427.
- the larger cavity is lined by mostly hydrophilic residues, half derived from gpl20 and half from CD4. It is not deeply buried; while formally a cavity in the crystal structure, minor changes in sidechain orientation would make it solvent accessible.
- the observed electron density and predicted hydrogen bonding are consistent with at least 8 water molecules in the cavity.
- Residues from gpl20 that actually line the cavity include Ala 281, Ser 364, Ser 365, Thr 455, Arg 469) exhibit sequence variability, whereas surrounding this variable patch are conserved residues, the substitution of which affect CD4 binding. These include the critical contact residues Asp 368, Glu 370 and Trp 427, which flank one end of the cavity, and Asp 457 at the other end (Fig. lOe) .
- CD4 residues that line the cavity can be mutated with only moderate effect on gpl20 binding, whereas Arg 59 suffers less loss of solvent accessible surface upon gpl2_0 binding but is highly sensitive to mutation.
- This cavity thus serves as a water buffer between gpl20 and CD4 (Fig. lOe) .
- the tolerance for variation in the gpl20 surface associated with this cavity produces a variational island (Fig. lOg) , or "anti-hot spot", which is centrally located between regions required for CD4 binding, and may help the virus escape from antibodies directed against the CD4 binding site.
- the "Phe 43" cavity (Fig. 10b & h) is very different in character from the larger binding- interface cavity. It is roughly spherical, with a diameter of ⁇ 8 A° (atom center to atom center) across the center of the cavity. It is positioned just beyond Phe 43 of CD4 , at the intersection of the inner domain, the outer domain and the bridging sheet. It is relatively deeply buried, extending into the hydrophobic interior of gpl20. The phenyl ring of Phe 43 is the only non-gpl20 residue contacting this cavity, forming a lid which covers the bottom of the cavity (Fig. 10b) .
- Residues that line the Phe 43 cavity are primarily hydrophobic. They are also highly conserved, as much so as the buried gpl20 hydrophobic core. Despite a lack of steric hindrance, almost no substitutions to larger residues are found. Given the frequency of gpl20 sequence divergence, such conservation strongly implies functional significance. Indeed, although residues that line this cavity provide little direct contact to CD4 , they do nevertheless affect the gpl20-CD4 interaction. Thus, mutations at Thr 257 (no contacts) and Trp 427
- the 17b antibody is a broadly neutralizing human monoclonal isolated from the blood of an HIV-infected individual. It binds to a CD4-induced (CD4i) gpl20 epitope that overlaps the chemokine receptor-binding site20.
- the interface between Fab 17b and core gpl20 in the ternary complex involves a small area of interaction.
- the solvent accessible area excluded upon binding is only 455 A° 2 from gpl20 and 445 A° 2 from 17b, which is largely from the heavy chain (371A° 2 ) .
- the long (15 residue) complementarity-determining region 3 (CDR3) of the heavy chain dominates, but the heavy-chain CDR2 and the light -chain CDR3 also contribute.
- the 17b contact surface is very acidic (3 Asp, 3 Glu, no Arg or Lys) although hydrophobic contacts (notably a cis proline and tryptophan from the light chain) predominate at the center.
- the 17b epitope lies across the base of the four-stranded bridging sheet (Fig. He & e) . All four strands make substantial contact with 17b, suggesting that the integrity of the bridging sheet is necessary for 17b binding.
- the gpl20 surface that contacts 17b consists of a hydrophobic center surrounded by a highly basic periphery (3 Lys, 1 Arg, and no Asp or Glu) (Fig. lid) . Although this basic gpl20 surface complements the acidic 17b surface, only one salt bridge is observed (between Arg 419 of gpl20 and Glu 106 of the 17b heavy chain) . The rest of the specific contacts occur between hydrophobic and polar residues.
- the interaction between 17b and gpl20 involves a hydrophobic central region flanked on the periphery by charged regions, predominately acidic on 17b and basic on gpl20.
- CD4-17b contacts There are no direct CD4-17b contacts and none of the gpl20 residues contacts both 17b and CD4. Rather, CD4 binds on the opposite face of the bridging sheet, providing specific contacts that appear to stabilize its conformation (Fig. lOi and lOj) and may explain in part the CD4-induction of 17b binding.
- the 17b epitope is well conserved among HIV-1 isolates. Of the 18 residues that show loss in solvent accessible surface upon contact with 17b, 12 residues (67%) are conserved among all HIV-1 viruses. By contrast, only 19 of the 37 gpl20 residues (51%) that show loss of solvent accessible surface upon CD4 binding are similarly conserved.
- CD4i epitopes tend to be masked from immune surveillance by the adjacent V2 and V3 loops (see accompanying paper) . Indeed, in the complex structure, a large gap is seen between gpl20 and tips of the light-chain CDR1 and CDR2 loops. Pointing directly at this gap is the base of the V3 loop.
- variable loops may need to be bypassed for access to the conserved structures in the bridging sheet .
- the 17b epitope may be further protected from the immune system by a CD4- induced conformational change (see below) .
- the site of interaction with the chemokine receptor CCR5 overlaps with the 17b epitope30. Both are induced upon CD4 binding and both involve highly conserved residues.
- the basic and polar gpl20 residues (Lys 121, Arg 419, Lys 421, Gin 422) that contact the 17b heavy chain also are important for CCR5 interaction30.
- the hydrophobic and acidic surface of the 17b heavy chain may mimic the tyrosine-rich, acidic N-terminal region of CCR5 , which is important for gpl20 binding and HIV-1 entry (31, 32) .
- this site is directed at the cellular membrane when gpl20 is engaged by CD4. Electrostatic interactions between the basic surface of the bridging sheet and the acidic chemokine receptor (and possibly the acid headgroups in the target membrane) could drive conformational changes related to virus entry.
- gpl20 Although monomeric in isolation, gpl20 likely exists as a trimeric complex with gp41 on the virion surface.
- the large electroneutral surface on the inner domain (Fig. 10c) is the probable site of trimer packing based on its lack of glycosylation, its conservation in sequence, the location of CD4 and CCR5 binding sites, and the immune response to this region.
- the Phe 43 cavity (now a pocket) would present a perplexing structural dilemma.
- the cavity-lining residues have few structural restrictions, with ample room for larger substitutions into the cavity, yet these residues are highly conserved and inexplicably hydrophobic if exposed in a pocket .
- This pocket structure is in turn intimately connected to the bridging sheet, itself peculiar in absence of CD4.
- the backbone amide of bridging-sheet residue 425 is hydrogen-bonded to Glu 370, a critical CD4 contact residue (Fig.
- Trp 427 packs perpendicular to Trp 112, which lines the pocket from the inner domain (Fig. 10b) .
- NS of Trp 427 is delicately poised for hydrogen-bonding with the 7r-electrons of the indole ring of Trp 112. Structures such as these would necessarily be very sensitive to orientational shifts between the inner and outer domains .
- core gpl20 may differ in the absence of CD4 comes from comparison with theory.
- the evolutionary algorithm of PHD37 gives secondary-structure predictions with 90% estimated reliability for roughly 45% of the core gpl20 sequence. Compared to our structure, it is accurate except at three places where it is markedly wrong (four consecutive residues with reliability index greater than 90%) . All of these are at the Phe 43 cavity or in contacts with CD4 : loop LB, strand 315, and the segment of 320 into the turn to /321.
- Fig. 10c stabilizes a nascent complex state, and inserts the Phe 43 to induce formation of the Phe 43 cavity.
- the HIV surface proteins function to fuse the viral membrane with the target cell membrane.
- the gpl20 glycoprotein plays roles crucial to the control and initiation of fusion.
- One set of roles concerns positioning: locating a cell capable of productive viral infection, anchoring the virus to the cell surface, and orienting the viral spike next to the target membrane.
- Another set concerns timing: holding the gp41 in a metastable conformation and triggering the coordinate release of the three N-terminal fusion peptides of the trimeric gp41. While it is clear that this is a complex multi-conformational process, the simplicity of the system, composed only of two membranes, the viral oligomer, and two host receptors, raises the possibility that we may be able to understand the entire mechanism.
- Crystallography has now provided two snapshots : an intermediate state in which gpl20 is bound to CD4 , described herein; and a probably final, "fusion-active" state of the gp41 ectodomain (40,41) .
- gp41 ectodomain 40,41
- the entry process is initiated by the binding of HIV-1 to the cellular receptor CD4 (Fig. 12, step 1). Although the extracellular portion of CD4 has some segmental flexibility, this binding roughly orients the viral spike.
- This orientation can be simulated by an alignment of the D1D2 CD4 in the ternary complex with the previously solved structure of the four-domain, entire extracellular portion of CD4(10) . Such alignment orients the N- and C- termini of core gpl20 towards the viral membrane, while the 17b epitope/chemokine receptor-binding site on the gpl20 surface faces the target cell membrane.
- Such an orientation is consistent with the proposed oligomeric structure and gp41-interactive surfaces described above.
- CD4 binding also induces conformational changes in gpl20, which result in the creation of a metastable oligomer. Although some of the more flexible gpl20 regions and gp41 are missing, the structure of the core gpl20-CD4 complex presented here describes this state in atomic detail . CD4 binding results in movement of the V2 loop, which numerous experiments suggest partially occludes the V3 loop and CD4i epitopes (18, 36) . It also creates, or at least stabilizes, the bridging sheet on which these epitopes are located (described above for the core) .
- CD4 binding results in changes in the conformation of the V3 region, with the tip of the loop becoming more accessible, as judged by enhanced proteolytic susceptibility and altered exposure of V3 epitopes (19) .
- the V3 loop together with the uncovered epitopes comprise the chemokine-receptor binding site.
- CD4 binding not only orients the gpl20 surface implicated in chemokine receptor binding to face the target cell, but it also forms and exposes the site itself.
- these changes may all result from a single, concerted shift in the relative orientation of the inner and outer domains.
- This conformational shift may alter the orientation of the N- and C- termini, at the proximal end of the inner domain, perhaps partially destabilizing the oligomeric gpl20/gp41 interface (21) .
- Such a shift would also alter the relative placement of the V1/V2 stem (in the CD4i site) , which emanates from the inner domain, and the V3 loop, which emanates from the outer domain.
- mutations that permit an adaptation of HIV-1 to CD4-independent entry using CXCR4 involve sequence changes in both the VI/V2 stem and the V3 loop (42) .
- the next step in HIV-1 entry is the interaction of the gpl20-CD4 complex with the chemokine receptor (Fig. 12, step 2) .
- chemokine receptor Fig. 12, step 2
- interactions between CD4 and chemokine receptor may occur, mutagenic analyses (H. Choe and J. Sodroski, unpublished observations) and the known examples of CD4-independent virus entry or chemokine-receptor binding suggest that direct gpl20 contacts dominate in the interaction with the chemokine receptor. Since most of the chemokine receptor is encased in the host membrane, binding would necessarily move the gpl20 bridging sheet close to the target membrane. This movement requires CD4 flexibility since the initial HIV binding at the N-terminal DI domains probably occurs above the glycocalyx.
- the structure of the gpl20/CD4/l7b antibody ternary complex described here reveals some of the molecular aspects of HIV-1 entry, including the atomic structure of gpl20, the explicit interactions with CD4 , and the conserved site of binding for the chemokine receptor. Still unknown are details of the apo state of core gpl20, the oligomeric structure, the interaction with the chemokine receptor, the conformational changes that trigger the reorganization of the gp41 ectodomain and the structural basis for insertion of the fusion peptide of gp41 into the target membrane. Further understanding will require snapshots of other intermediates.
- the conformational complexity and observed intricate domain associations of gpl20 may reflect genome restrictions at the protein level akin to those that lead to overlapping reading frames at the transcription level. Multiply protected infection machinery is contained in these condensed intricacies. Its mechanisms frustrate host defenses; understanding them may inspire medical intervention.
- the two-domain CD4 (D1D2, residues 1-182) was produced in Chinese hamster ovarian cells (8), the monoclonal antibody 17b in an Epstein-Barr virus immortalized B-cell clone isolated from an HIV-1 infected individual and fused with a murine B-cell fusion partner(18), and the core gpl20 from Drosophila Schneider 2 lines under control of an inducible metallothionein promoter (20) .
- the various biochemical manipulations e.g. deglycosylation for the gpl20 and papain digestion to produced the Fab 17b
- protein purification e.g. ternary complex crystallization are described elsewhere (25) .
- the best crystals were small needles of cross-section only 30-40 ⁇ m. These were crosslinked with vapor diffusion glutaraldehyde treatment
- cryoprotectant containing stabilizer (10% ethylene glycol with 10.5% monomethyl-PEG 5,000, 10% isopropanol, 50 mM NaCl, 100 mM Citrate/HEPES buffer pH 6.3), transferred into immiscible oil (Paratone-N; Exxon) , suspended in a small ethylene loop at the end of a mounting pin, and flash-frozen in a cryostat nitrogen stream at 100 K .
- Diffraction data were collected at beamline X4A, Brookhaven National Laboratory, using phosphor image plates and a Fuji BAS2000 scanner. To avoid overlap problems from the relatively high mosaicity (-1.0°), oscillation data were collected using a rotation axis that was off-set at least 30° from the 197A c axis. Although crystals initially diffracted to Bragg spacing of greater than 2A, ⁇ axis mosaicity and substantial radiation damage despite cryogenic cooling reduced the overall resolution to 2.5A. Data processing and reduction were performed using DENZO and SCALEPACK (45) (Table 1) .
- each of the top 100 possible rotational solutions with each of three different CD4 models (lcdi, lcdh, 3cd4) , were searched for a distinctive translation solution (AMoRe; J. Navaza) .
- the translation searches used the rigid body refined Fab as a partial structure to help discriminate the correct solution.
- Two distinctive solutions were found: the 25th rotational solution of 3cd4 gave a translation correlation of 0.171 (verses 0.128 for the second highest translation solution) , and the 61st rotational solution of lcdh gave 0.149 (verses 0.140). These two solutions were virtually identical .
- Rigid body refinement in XPLOR(46) gave a Patterson correlation of 7.9% for the CD4 alone and 32.4% for the Fab and CD4. All molecular replacement and rigid body refinements used 8-4A data.
- crystals were soaked in over 20 different heavy atom solutions and screened for isomorphous replacement using the statistical ⁇ chi>2 test in SCALEPACK (45) .
- Derivatives were identified from two heavy atom compounds : 10 mM K3IrCl6 (10 hr equilibration in heavy atom containing cryoprotectant stabilizer; 2.8A) and 5 mM K20sCl6 (24 hr soak; 3.5A) .
- Isomorphism was found to be highest between these heavy atom data sets and a native data set collected at pH 7.0 (cryoprotectant stabilizer buffered with 50 mM BisTris pH 7.0) .
- K3IrCl6 derivative was modeled as 9 partially occupied sites; two sites of occupancy 0.158 and 0.142, and 7 of less than 0.07. While relatively isomorphous, poor data quality (Rsym of greater than 20% past 3.0A) combined with relatively small isomorphous differences (Riso of 12.0%) reduced the quality of phasing. In contrast, the K20sC16 derivative had an Riso of 15.6%, but was only isomorphous to roughly 5A. It was modeled as 4 sites of occupancy 0.321, 0.207, 0.194 and 0.128, with the highest site at the same position as the second highest site from K3IrC16.
- Deviations of the CD4 structure in the complex from the free state were measured by the procedure of Wu et al.10. Deviations were taken as significant when the root mean square (rms) residue deviation was greater than the overall value and also more than 0.5u greater than variation among the free structures .
- Interatomic contacts were defined as in Zhu et . al.48. Structural alignments were made by visual comparison of the SCOP databas, and automatic searches were performed with PrISM (A.-S. Yang and B. Honig) .
- HIV-1 entry co-factor functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272, 872-877 (1996) .
- chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids in the envelope V3 loop. J. Virol. 71, 7136-7139 (1997) .
- HIV-1 gpl20 glycoproteins with the chemokine receptor CCR-5 Nature 384, 179-183 (1996).
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Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
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US6511826B2 (en) | 1995-06-06 | 2003-01-28 | Human Genome Sciences, Inc. | Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10 |
US6743594B1 (en) | 1995-06-06 | 2004-06-01 | Human Genome Sciences, Inc. | Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5) |
US7175988B2 (en) | 2001-02-09 | 2007-02-13 | Human Genome Sciences, Inc. | Human G-protein Chemokine Receptor (CCR5) HDGNR10 |
US7393934B2 (en) | 2001-12-21 | 2008-07-01 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7501123B2 (en) | 2004-03-12 | 2009-03-10 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7807671B2 (en) | 2006-04-25 | 2010-10-05 | Bristol-Myers Squibb Company | Diketo-piperazine and piperidine derivatives as antiviral agents |
US7829711B2 (en) | 2004-11-09 | 2010-11-09 | Bristol-Myers Squibb Company | Crystalline materials of 1-(4-benzoyl-piperazin-1-yl)-2-[4-methoxy-7-(3-methyl-[1,2,4]triazol-1-yl)-1H-pyrrolo[2,3-C]pyridine-3-yl]-ethane-1,2-dione |
US7851476B2 (en) | 2005-12-14 | 2010-12-14 | Bristol-Myers Squibb Company | Crystalline forms of 1-benzoyl-4-[2-[4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-YL)-1-[(phosphonooxy)methyl]-1H-pyrrolo[2,3-C]pyridin-3-YL]-1,2-dioxoethyl]-piperazine |
WO2011024175A1 (fr) | 2009-08-28 | 2011-03-03 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Composés macrocycliques, compositions associées et méthodes de prévention ou de traitement d'une infection par le vih |
US8637036B2 (en) | 2009-09-25 | 2014-01-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to HIV-1 and their use |
US9341639B2 (en) | 2013-07-26 | 2016-05-17 | Industrial Technology Research Institute | Apparatus for microfluid detection |
US9403763B2 (en) | 2011-12-14 | 2016-08-02 | Dana-Farber Cancer Institute, Inc. | CD4-mimetic inhibitors of HIV-1 entry and methods of use thereof |
US9695230B2 (en) | 2011-12-08 | 2017-07-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Broadly neutralizing HIV-1 VRC07 antibodies that bind to the CD4-binding site of the envelope protein |
US9776963B2 (en) | 2008-11-10 | 2017-10-03 | The Trustees Of The University Of Pennsylvania | Small molecule CD4 mimetics and uses thereof |
US9975848B2 (en) | 2014-08-13 | 2018-05-22 | The Trustees Of The University Of Pennsylvania | Inhibitors of HIV-1 entry and methods of use thereof |
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US5109123A (en) * | 1988-06-14 | 1992-04-28 | Dana Farber Cancer Institute | Alteration of ability of soluble CD4 fragments to bind HIV |
US5614612A (en) * | 1990-03-09 | 1997-03-25 | Haigwood; Nancy L. | Purified gp120 compositions retaining natural conformation |
US5851529A (en) * | 1988-03-21 | 1998-12-22 | Guber; Harry E. | Recombinant retroviruses |
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1998
- 1998-11-10 WO PCT/US1998/023906 patent/WO1999024065A1/fr active Application Filing
- 1998-11-10 AU AU14545/99A patent/AU1454599A/en not_active Abandoned
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US5851529A (en) * | 1988-03-21 | 1998-12-22 | Guber; Harry E. | Recombinant retroviruses |
US5109123A (en) * | 1988-06-14 | 1992-04-28 | Dana Farber Cancer Institute | Alteration of ability of soluble CD4 fragments to bind HIV |
US5614612A (en) * | 1990-03-09 | 1997-03-25 | Haigwood; Nancy L. | Purified gp120 compositions retaining natural conformation |
Non-Patent Citations (1)
Title |
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CHEN S., ET AL.: "DESIGN AND SYNTHESIS OF A CD4 BETA-TURN MIMETIC THAT INHIBITS HUMAN IMMUNODEFICIENCY VIRUS ENVELOPE GLYCOPROTEIN GP120 BINDING AND INFECTION OF HUMAN LYMPHOCYTES.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 89., 1 July 1992 (1992-07-01), US, pages 5872 - 5876., XP002916732, ISSN: 0027-8424, DOI: 10.1073/pnas.89.13.5872 * |
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US6743594B1 (en) | 1995-06-06 | 2004-06-01 | Human Genome Sciences, Inc. | Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5) |
US6759519B2 (en) | 1995-06-06 | 2004-07-06 | Human Genome Sciences, Inc. | Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor) |
US6800729B2 (en) | 1995-06-06 | 2004-10-05 | Human Genome Sciences, Inc. | Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor) |
US7160546B2 (en) | 1995-06-06 | 2007-01-09 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7175988B2 (en) | 2001-02-09 | 2007-02-13 | Human Genome Sciences, Inc. | Human G-protein Chemokine Receptor (CCR5) HDGNR10 |
US7393934B2 (en) | 2001-12-21 | 2008-07-01 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7501123B2 (en) | 2004-03-12 | 2009-03-10 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7829711B2 (en) | 2004-11-09 | 2010-11-09 | Bristol-Myers Squibb Company | Crystalline materials of 1-(4-benzoyl-piperazin-1-yl)-2-[4-methoxy-7-(3-methyl-[1,2,4]triazol-1-yl)-1H-pyrrolo[2,3-C]pyridine-3-yl]-ethane-1,2-dione |
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US7807676B2 (en) | 2006-04-25 | 2010-10-05 | Bristol-Myers Squibb Company | Diketo-Piperazine and Piperidine derivatives as antiviral agents |
US9776963B2 (en) | 2008-11-10 | 2017-10-03 | The Trustees Of The University Of Pennsylvania | Small molecule CD4 mimetics and uses thereof |
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US10815295B2 (en) | 2011-12-08 | 2020-10-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Broadly neutralizing HIV-1 antibodies that bind to the CD4-binding site of the envelope protein |
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US9975848B2 (en) | 2014-08-13 | 2018-05-22 | The Trustees Of The University Of Pennsylvania | Inhibitors of HIV-1 entry and methods of use thereof |
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