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WO1998019162A1 - Identification de medicaments au moyen de bibliotheques combinatoires complementaires - Google Patents

Identification de medicaments au moyen de bibliotheques combinatoires complementaires Download PDF

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Publication number
WO1998019162A1
WO1998019162A1 PCT/US1997/019638 US9719638W WO9819162A1 WO 1998019162 A1 WO1998019162 A1 WO 1998019162A1 US 9719638 W US9719638 W US 9719638W WO 9819162 A1 WO9819162 A1 WO 9819162A1
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WIPO (PCT)
Prior art keywords
library
binding
peptide
protein
peptides
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PCT/US1997/019638
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English (en)
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WO1998019162A9 (fr
Inventor
Dana M. Fowlkes
Brian K. Kay
Jeffrey A. Frelinger
Robin Parish Hyde-Deruyscher
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Novalon Pharmaceutical Corporation
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Application filed by Novalon Pharmaceutical Corporation filed Critical Novalon Pharmaceutical Corporation
Priority to EP97948137A priority Critical patent/EP0937253A1/fr
Priority to CA002270394A priority patent/CA2270394A1/fr
Priority to JP52072798A priority patent/JP2001510453A/ja
Priority to AU54268/98A priority patent/AU744444B2/en
Priority to US09/069,827 priority patent/US6617114B1/en
Publication of WO1998019162A1 publication Critical patent/WO1998019162A1/fr
Publication of WO1998019162A9 publication Critical patent/WO1998019162A9/fr
Priority to US10/656,250 priority patent/US20050069951A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • This invention relates to methods of identifying drugs which can mediate the biological activity of a target protein.
  • binding partners ligands
  • ligands proteins
  • binding partner of a protein it is relatively straightforward to study how the interaction of the binding protein and its binding partner affects biological activity.
  • inhibitors are likely to affect the biological activity of the protein, at least if they can be delivered in vivo to the site of the interaction.
  • the binding protein is a receptor, and the binding partner an effector of the biological activity, then the inhibitor will antagonize the biological activity. If the binding partner is one which, through binding, blocks a biological activity, then an inhibitor of that interaction will, in effect, be an agonist.
  • the binding sites are typically relatively small surface patches. The binding characteristics of the protein may often be altered by local modifications at these sites, without denaturing the protein.
  • Peptides have been found to bind proteins at the same sites as those by which the proteins interact with other proteins, macromolecules and biologically significant substances e.g. nucleic acids, lipids and enzyme substrates.
  • the first examples of this property were in the publications of several groups who showed that there is a single peptide binding site on the biotin binding protein streptavidin. This is the same site responsible for biotin binding and these peptides compete with biotin for binding to this site
  • SAR structure/activity relationships
  • Another disadvantage to whole animal, organ and cell based screening is that certain limitations may prevent an active compound from being scored as such. For instance, an inability to pass through the cellular membrane may prevent a potent inhibitor, within a tested compound library, from acting on the activated oncogene ras and giving a spurious negative score in a cell proliferation assay. However, if it were possible to test ras in an isolated system, that potent inhibitor would be scored as a positive compound and contribute to the establishment of a relevant SAR. Subsequent, chemical modifications could then be carried out to optimize the compound structure for membrane permeability.
  • the overwhelming disadvantage to the receptor based methods for screening compounds is that they require a priori knowledge about the activity of receptor and its biological ligand. If through genetic mapping of a disease loci one determines that a particular gene product is responsible for the disease; and one lacks knowledge about the gene biochemical function because it is not a previously known receptor or enzyme, then it is very difficult to establish an assay with the methods previously known.
  • the present invention circumvents all these problems.
  • peptide libraries are quite similar to many of the natural macromolecules involved in biological processes and thus these libraries are rich in structures that mimic the natural ones which interact with the target protein.
  • the variants are composed of linear polymers such that each actually represents a sliding window of many differing chemical constituents. For instance, if a given macromolecular interaction is based on the side chains of four amino acids within a binding peptide, then a 13 amino acid peptide has 10 potential combinations of residues which may bind; therefore a library of 10 8 members has about 10 9 4-mer permutations. This, combined with ease of producing and screening exceptionally large and diverse peptide libraries, provides the incentive to use peptide combinatorial libraries for the initial identification and probing of protein functional domains.
  • peptides per se have limited utility for use as therapeutic entities. They are costly to synthesize, unstable in the presence of proteases and in general do not transit cellular membranes. Other classes of compounds have better properties for drug candidates.
  • acquiring chemical compound libraries has been a barrier to the entry of smaller firms into the drug discovery arena. Due to the large quantity of chemical required for testing on whole animals and even on cells in culture, it was a given that whenever a compound was synthesized it should be done in fairly large quantity. Thus, there was a synthesis and purification throughput of less than 50 compounds per chemist per year. Large companies maintained their enormous collections as trade barriers.
  • the present invention relates to a method of identifying drugs which can mediate the biological activity of a target protein via inhibition of binding of the target protein to a binding partner. Unlike prior methods, it does not require that the natural binding partner be used as a reagent, or even that it have been characterized. The need for the natural binding partner is obviated by the use of complementary combinatorial libraries.
  • library generally refers to a collection of chemical or biological entities which can be screened simultaneously for a property of interest. (They may be screened sequentially, if desired, but simultaneous screening is more efficient.) Typically, they are related in origin, structure, and/or function.
  • combinatorial library refers to a library in which the individual members are either systematic or random combinations of a limited set of basic elements, the properties of each member being dependent on the choice and location of the elements incorporated into it.
  • the members of the library are at least capable of being screened simultaneously. Randomization may be complete or partial; some positions may be randomized and others predetermined, and at random positions, the choices may be limited in a predetermined manner.
  • the members of a combinatorial library may be oligomers or polymers of some kind, in which the variation occurs through the choice of monomeric building block at one or more positions of the oligomer or polymer, and possibly in terms of the connecting linkage, or the length of the oligomer or polymer, too.
  • the members may be nonoligomeric molecules with a standard core structure, like the 1, 4-benzodiazepine structure, with the variation being introduced by the choice of substituents at particular variable sites on the core structure .
  • a “simple combinatorial library” In a “simple combinatorial library”, all of the members belong to the same class of compounds (e.g., peptides) and can be synthesized simultaneously.
  • a “composite combinatorial library” is a mixture of two or more simple libraries, e,g., DNAs and peptides. The number of component simple libraries in a composite library will, of course, normally be smaller than the average number of members in each simple library, as otherwise the advantage of a library over individual synthesis is small .
  • a biased combinatorial library is one in which, at one or more positions in the library member, only one of the possible basic elements is allowed for all members of the library, i.e., the biased positions are invariant.
  • amplifiable combinatorial library refers to a library in which the individual members, after found to bind to a target, may be amplified in vivo or in vitro, using elements already present in the library as starting materials.
  • nucleic acids may be amplified in vivo through natural replicative processes, or in vitro through techniques such as polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • peptides when presented on phage, or otherwise associated with an encoding nucleic acid, may be amplified indirectly by in vivo or in vitro amplification of the associated nucleic acid encoding the peptide, the amplified nucleic acid being expressed to produce the peptide.
  • biopolymeric library refers to a library composed of peptides (together with peptoids) , nucleic acids, and/or oligosaccharides . (It is not necessary that they be composed of naturally occurring amino acids, bases, or sugars, respectively.) However, because of the greater complexity of carbohydrate synthesis, peptides and nucleic acids are of greater interest.
  • a “panel of combinatorial libraries” is a collection of different (although possibly overlapping) and separately screenable simple or composite combinatorial libraries.
  • a “panel” differs from a composite library in that the component simple libraries have not been mixed together, that is, they may still be screened separately.
  • a "structural panel” is a panel as defined above where there is some structural relationship between the member libraries. For example, one could have a panel of 20 different biased peptide libraries where, in each library, the middle residue is held constant as a given amino acid, but, in each library the constant residue is different, so, collectively, all 20 possible genetically encoded amino acids are explored by the patent .
  • a “scanning residue library” refers to the preparation of panel of biased combinatorial peptide libraries such that the position of the constant residue shifts from one library to the next. For example, in library 1, residue 1 is held constant as a particular residue AA, in library, residue 2 is, and so forth through two or more (usually all) positions of the peptide .
  • the middle residue AA X may be the same for all libraries, but the libraries also have a constant residue AA 2 which is scanned through all other residue positions.
  • a library screening program is a program in which one or more libraries (e.g., a structured panel of biased peptide libraries) are screened for activity.
  • the libraries may be screened in parallel, in series, or both. In serial screening, the results of one screening may be used to guide the design of a subsequent library in the series.
  • Applicants screen a first combinatorial library for binding to the target protein.
  • Applicants then screen a second
  • (complementary) library (preferably combinatorial in nature) for the ability to inhibit the binding of one or more of the target binding ligands in the first library to the target protein.
  • the successful inhibitors are candidate antagonists of one or more of the biological activities of the target protein.
  • the target-binding members of the library in question can be used as surrogates for an unknown or unavailable natural binding partner in screening a second combinatorial library (the "complementary library”) , which need not be a biopolymeric library, for members which can inhibit the complexing of target protein to its natural binding partner.
  • the surrogate library is preferably a peptide or nucleic acid library.
  • the size of a library is the total number of molecules in it, whether they be the same or different.
  • the diversity of a library as the number of different molecules in it .
  • "Diversity" does not measure how different the structures of the library; the degree of difference between two structures is referred to here as “disparity” or “dispersion” .
  • the "disparity” is quantifiable in some respects, e.g., size, hydrophilicity, polarity, thermostability, etc.
  • the average sampling frequency of a library is the ratio of size to diversity. The sampling frequency should be over the detection limit of the assay in order to assure that all members are screened.
  • the combinatorial libraries usually will have a diversity of at least 10 3 different structures.
  • the initial, surrogate-generating library is of high diversity, e.g., preferably at least about 10 6 , more preferably at least about 10 9 different members.
  • a peptide library is preferred, a library composed of a different class of compounds (e.g., peptoids or nucleic acids) is acceptable if there would be a detectable preference for binding the activity-mediating binding sites of the target protein.
  • the complementary library need not be, and preferably is not, a peptide library and it may be of lower overall diversity. It may be screened against all of the surrogate peptides; or only against selected ones. The screenings may be individual or collective. Often, the members of the complementary library will be less specific in their binding to the paratopes of the target protein than are the members of the first library, possibly because their surface area is smaller and offers fewer opportunities for favorable . (or unfavorable) interactions with other molecules.
  • a preferred complementary library is a benzodiazepine library.
  • the degree of complex- inhibitory activity of the members of the complementary library may be quantified by means of a labeled surrogate peptide and an insolubilized target protein. Either the amount of labeled surrogate peptide is fixed, and the amount of complementary compound varied, or, more preferably, the amount of labeled surrogate peptide is varied and the amount of complementary compound held constant. The greater the activity of the complementary compound, the less labeled surrogate peptide will be in the solid phase (i.e., complexed to the target protein) and the more will be in the liquid phase (i.e., uncomplexed) . The amount of label in either phase is then measured and correlated with the amount of the variable component. Conventional method of screening libraries for binding molecules do not lend themselves to quantification of the degree of affinity.
  • target protein binding members of the first library will not bind the target protein at the site bound by the natural binding partner which mediates the biological activity of interest, or bind it that site but still do not have an effect similar to that of the natural binding partner, i.e., that these nominal surrogates are not true surrogates for the natural binding partner.
  • these nominal surrogates are not true surrogates for the natural binding partner.
  • one or more of the identified members are true surrogates, if all of the nominal surrogates are used in screening the complementary library, then one necessarily will screen for inhibitors of the binding of the true surrogates to the target protein, too.
  • false hits i.e., compounds which inhibit the binding of a false surrogate to the target protein, or which inhibit binding of a true surrogate but at the wrong site
  • inhibitory compounds which bind the target protein from those which bind the surrogate peptide by use of either the target protein or surrogate peptide alone, in labeled or immobilized form, as an assay or affinity separation reagent.
  • Figure 1 Benzodiazepine scaffold used to create a combinatorial library, and a synthetic pathway leading to that structure.
  • FIG. 1 Vector pFLAG-ATS-BAP for expression of peptide ligands fused to bacterial alkaline phosphatase. This vector has been used to successfully express both large and small domains of signaling proteins. In most instances the protein is secreted and one simply concentrates the fusion protein by ammonium sulfated precipitation from the media. In rare instances the protein is not secreted, but accumulates within the cells. In this case, we wash the cells in tris buffered saline then sonicate to release active fusion protein. In either instance, sufficient amounts of the material are obtained from 100ml cultures. The FLAG epitope is disclosed, as a tag for a fusion protein, in Grihalde, et al . , Gene, 166:187-95 (1995) . Figure 3. Occurrence of Amino Acids (AA) in Binding Peptides Identified by screening from Phage Display Libraries.
  • AA Amino Acids
  • the sequences of the binding petpides identified by screening phage display libraries were analyzed.
  • a consensus sequence was determined, and from this, a core binding region.
  • the amino acids in the core region of all the peptides binding that target were tallied, and the tallies were divided by the number of peptides in question, to obtain a subtotal for each target.
  • the target subtotals were then added and divided by the number of targets. The final totals were converted into percentages. If all of the residues were represented equally their values would be 5%.
  • Figure 4 Occurrence of amino acid residues in binding peptides identified by screening phage display libraries, after correcting for codon usage. The numbers in figure 3 were divided by the number of codons available for each residue using an NNK coding scheme and the resulting numbers normalized to 100%.
  • FIG. 5 Enrichment of Phage Binding to CMV UL44.
  • the proportion of phage binding to UL44 was monitored after 2 and three rounds of selection as described in the text.
  • the ratio represents the number of blue plaques/# of white plaques.
  • the library letters represent the residue held constant in each library.
  • FIG. 6 Individual phage ELISAs for CMV UL44. Individual clones were picked after three rounds of selection on UL44 and tested for binding in a phage ELISA format. Letters under the graph represent the libraries that individual clones were isolated from. Letters A-H in the legend represent the row designation on the microtiter plate.
  • FIG. 7a glutathione
  • Fig. 7b DNA
  • Square boxes represent phage which bind to the GST portion of the fusion
  • diamonds represent phage which bind to the UL44 portion of the fusion protein.
  • Figure 8 ELS Assay of UL44 BioKey (surrogate ligand) . The peptide isolated from affinity selection against UL44. was synthesized and tested for specific binding as described in the text.
  • ELS Enzyme Linked Spectrophometric
  • Figure 9 Time Course of 50 pmol UL44 BioKey (surrogate ligand) Binding to GST-UL44.
  • the binding of the UL44 Surrogate ligand was monitored as a function of time. The signal increases in a linear fashion for at least 4 hours.
  • biotinylated peptide was monitored as a function of target concentration immobilized on the plate (from 0.125 migrograms to 2 micrograms) and as a function of peptide concentration (from 0.1 to 0.5 micrograms).
  • FIG. 12 Titration of PKC binding phage. A serial dilution of phage were incubated with immobilized PKC and the amount of phage binding monitored using a phage ELISA.
  • FIG. 13a Specific Binding of the ProRS BioKey (surrogate ligand) .
  • the peptide isolated from affinity selection against ProRS was synthesized and tested for specific binding as described in the text.
  • the targets tested were ProRS, GST- src SH3, GST, GST-UL44, TyrRS and GST-MDM2. The amount of biotinylated peptide binding was monitored using a streptavidin-HRP conjugate.
  • Surrogate ligands for src SH3 , UL44, and MDM2 were tested for binding to ProRS as described in the text.
  • FIG. 14 Self competition for ProRs BioKey. Self- Competition between biotinylated ProRS surrogate ligand and non-biotinylated surrogate ligand was determined. Competitor is the concentration on non-biotinylated ProRS peptide added.
  • BioKey concentration is the concentration of biotinylated ProRS surrogate ligand used.
  • the signals are presented as a percent of binding compared to no inhibitor added and are graphed in two formats: (a) linear (Fig. 14a) and (b) log (Fig. 14b) scales of the competitor concentrations.
  • FIG. 15 Concentration and time dependence of ProRS surrogate ligand binding.
  • the binding of biotinylated peptide was monitored as a function of peptide concentration (from 0.1 to 0.5 micrograms) (Fig. 15a) .
  • the binding of biotinylated peptide was monitored as a function of time (Fig .
  • TyrRS was synthesized and tested for specific binding as described in the text.
  • the targets tested were TyrRS, GST-src
  • FIG. 1 Concentration dependent binding of the TyrRS BioKey (surrogate ligand) .
  • the binding of biotinylated peptide was monitored as a function of peptide concentration (from 0.1 to
  • the present invention is directed to the identification of compounds in a compound library which can mediate the biological activity of a target receptor protein, even when the ligands which mediate that activity through binding to that receptor are not already known. Such compounds can then be used as "drug leads", i.e., used as a starting point for the design of analogues which can in turn be tested for activity.
  • the method of the invention comprises three steps:
  • a complementary library preferably a combinatorial library, (which is not limited to, and may not even include, peptides, or nucleic acids and hence is referred to on occasion as a "compound library") for compounds which inhibit the binding of one or more surrogates (peptides or nucleic acid which bind TP to TP .
  • a combinatorial library which is not limited to, and may not even include, peptides, or nucleic acids and hence is referred to on occasion as a "compound library” for compounds which inhibit the binding of one or more surrogates (peptides or nucleic acid which bind TP to TP .
  • a library screening program may include multiple rounds of surrogate library screening, with the first library sampling a broad "sequence space", and later libraries focusing on sequences related to those previously found to bind the target.
  • the different libraries may sample different sequence spaces, such as peptides and nucleic acids, or peptides of different length or composition. Similarly, it may include multiple rounds of- screening of complementary library. The later rounds may sample the same class of compounds, a different class of compounds, or a subclass of those previously screened. Different rounds may use different surrogates in the screen. The screenings may occur in any rational order, e.g., surrogate /complementary/surrogate /complementary , or surrogate/surrogate/complementary/complementary. The compounds of the complementary library may be screened simultaneously or sequentially.
  • the peptide library "samples" the surface of the TP for sites having a high interaction potential. It is likely that the TP evolved to offer one or more such sites and that such a site evolved to permit binding by the natural ligand (or that the natural ligand evolved to bind such a site on the TP) .
  • the peptide library is a biased library as hereafter defined.
  • TP-binding peptides may be screened for ability to act as a surrogate for the natural ligand, i.e., to mediate the biological activity of the TP . If so, then only the TP-binding peptides with this mediative property need be used in the second step.
  • a compound library is screened for the ability of the compounds to inhibit the binding of the TP- binding peptides of step (1) with the TP .
  • the compounds need not be peptides, and may be screened sequentially, or simultaneously.
  • the affinity range of the TP-binding (surrogate) peptides for the TP must be such that binding is detectable, and that inhibition of such binding by the compounds sought to be found is also detectable. Initially, the compounds screened are likely to have low inhibitory activity. As lead optimization progresses, compounds of higher inhibitory activity are more likely to be present. Different surrogate peptides may be preferred for initial and subsequent rounds of library construction and screening. Generally speaking, affinity in the range of 10-5 to 10 "11 M is desirable. The affinity of the surrogate ligand for the TP and the affinity range of compounds sought for in the first round of will determine the concentrations of surrogate ligand and drugs used in the screen.
  • the surrogate ligand concentration below its binding constant and the drug lead concentration above its binding constant. For example, if a peptide surrogate ligand has a binding constant of 1 x 10 "7 M, it should be used at concentrations of less than this in the assay. Conversely, if one wished to find compounds which bound to the TP with affinities around 1 x 10 "6 M, then the compounds should be present at higher concentrations than this in the assay. This does place limits on useful ligands and compounds. The ligand must be used at a concentration where its binding can still be detected.
  • the compounds may inhibit the binding of the TP-binding peptides to the TP either by binding to the peptide, or to the TP. Those that actually bind to the TP are more likely to mediate the biological activity of the TP . Those that bind to the TP-binding peptide alone are less likely to be useful, unless the TP-binding peptide is a true surrogate for .the natural ligand, i.e., the compound cross-inhibits the natural ligand.
  • Another advantage this method provides is focusing drug leads to sites on the TP which are biologically relevant. Assays which only require the compound binding to the target do not select for compounds which affect the activity of the TP, e.g., an interaction (e.g., enzymatic) of the TP with a biological ligand (for example another protein) .
  • a biological ligand for example another protein
  • the present invention is used to identify the biological activity of a target protein whose biological function is not known and perhaps cannot be determined directly.
  • compounds of known (or determinable) biological activity are screened for their ability to inhibit the binding of a peptide which binds the target protein. If such a compound inhibits such binding, it is hypothesized that the target protein mediates one or more of the biological activities of the compound.
  • the complementary library need not be a combinatorial library, provided that it is a library of substantial (e.g., 100 compound) structural diversity, e.g., a library of isolated natural products from various plant or animal sources, or a library of analogues previously made in various drug development programs.
  • the target protein may be a naturally occurring protein, or a subunit or domain thereof, from any natural source, including a virus, a microorganism (including bacterial , fungi, algae, and protozoa), an invertebrate (including insects and worms) , or the normal or cancerous cells of a vertebrate (especially a mammal, bird or fish and, among mammals, particularly humans, apes, monkeys, cows, pigs, goats, llamas, sheep, rats, mice, rabbits, guinea pigs, cats and dogs) .
  • the target protein may be a mutant of a natural protein.
  • the target protein may be, inter alia, a glyco-, lipo-, phospho-, or metalloprotein. It may be a nuclear, cytoplasmic, membrane, or secreted protein. It may, but need not, be an enzyme.
  • the known binding partners (if any) of the target protein may be, inter alia, other proteins, oligo- or polypeptides, nucleic acids, carbohydrates, lipids, or small organic or inorganic molecules or ions.
  • the biological activity or function of the target protein may be, but is not limited to, being a kinase protein kinase tyrosine kinase Threonine kinase
  • the binding protein may have more than one paratope and they may be the same or different. Different paratopes may interact with epitopes of different binding partners. An individual paratope may be specific to a particular binding partner, or it may interact with several different binding partners. A protein can bind a particular binding partner through several different binding sites. The binding sites may be continuous or discontinuous (vis-a-vis the primary sequence of the protein) .
  • a peptide library is a combinatorial library, at least some of whose members are peptides having three or more amino acids connected via peptide bonds.
  • the peptides may be linear, branched, or cyclic, and may include nonpeptidyl moieties.
  • the amino acids are not limited to the naturally occurring amino acids .
  • a biased peptide library is one in which one or more (but not all) residues of the peptides are constant residues.
  • the individual members are referred to as peptide ligands (PL) .
  • an internal residue is constant, so that the peptide sequence may be written as
  • Xaa is either any naturally occurring amino acid, or any amino acid except cysteine
  • m and n are chosen independently from the range of 2 to 20
  • the Xaa may be the same or different
  • AA X is the same naturally occurring amino acid for all peptides in the library but may be any amino acid.
  • m and n are chosen independently from the range of 4 to 9.
  • AA X is located at or near the center of the peptide. More preferably, AA X is either (a) at least five residues from both ends of the peptide, or (b) is in the middle 50% of the peptide. More preferably, that m and n are not different by more than 2; most preferably m and n are equal. Even if the chosen AA X is required (or at least permissive) of the TP binding activity, one may need particular flanking residues to assure that it is properly positioned. If AA X is more or less centrally located, the library presents numerous alternative choices for the flanking residues. If AAj is at an end, this flexibility is diminished.
  • the most preferred libraries are those in which AA- L is tryptophan, proline or tyrosine. Second most preferred are those in which AA X is phenylalanine, histidine, arginine, aspartate, leucine or isoleucine . Third most preferred are those in which AA-, ⁇ is asparagine, serine, alanine or methionine. The least preferred choices are cysteine and glycine. These preferences are based on evaluation of the results of screening random peptide libraries for binding to many different TPs . The effect of fixing one position in a library is to increase the occurrence of that particular residue from 1 in 20 to 20 in 20, an increase of 20 fold.
  • Ligands that bind to functional domains tend to have both constant as well as unique features. Therefore, by using "biased" peptide libraries, one can ease the burden of finding ligands .
  • HPQ occurs in most streptavidin-binding peptides, which bind with the HPQ side chains oriented inward so as to interact with the biotin-binding site of the TP streptavidin. Some of the residues that participate in binding biotin also interact with the peptides; however, the peptides adopt an alternate method of utilizing binding determinants
  • the example above showed a biased library with one residue held constant. The net effect of this is to increase the number of peptides with the constant residue in that position. If this residue at this position is helpful for binding, then the number of individuals per library that will bind to the target protein will be increased. If all the amino acids are represented equally, then the number of potential binding peptides is increased 20 fold in a library made up of the 20 naturally occurring amino acids. Libraries using different ratios of amino acids will be enriched according to the proportion of each residue in the starting library.
  • Trp Trp
  • Glu Glu
  • NNK coding scheme to represent all 20 amino acids. Residues L, R, and S are overrepresented 3 fold in this scheme while V, T, A, G, and P are overrepresented 2 fold. These residues are present in higher amounts using the NNK coding scheme and the increase in the number of surrogate ligands isolated by enriching for these residues will be less than those residues that are coded for only once. The degree of overrepresentation/underrepresentation may be reduced by using non-equimolar mixtures of bases at each position. This problem could be avoided altogether if the nucleic acid were synthesized triplet by triplet, rather than base by base, so at each step one added one out of 20 possible trinucleotides, each encoding a different amino acid.
  • residues that are important for protein-peptide interactions contain side chains that can interact with other amino acids and are less likely to pack tightly, allowing a greater degree of freedom for interaction with other ligands.
  • a study of residues at protein binding sites showed an overrepresentation of R, H, W, and Y (Villar and Kauvar, FEBS Letters 349: 125-130 (1994) Amino acid preferences at protein binding sites) .
  • a compilation of peptide sequences derived from the phage display against a series of proteins reveals that the amino acids are not found in equal amounts, that is to say that some amino acids appear in peptides that bind to various targets more frequently than other amino acids.
  • FIG. 3 A graph which shows the raw incidence of residue occurrence in peptides binding to any of 16 proteins is shown in figure 3; figure 4 shows the effect of correcting for codon usage.
  • figure 4 shows the effect of correcting for codon usage.
  • aromatic residues proline, cysteine and aspartic acid.
  • Biased libraries with these residues fixed or scanning through the displayed peptide are preferred, whereas biased libraries with residues that are under represented (such as alanine, methionine, and lysine) are less preferred with libraries containing the remaining residues fixed or scanning of intermediate usefulness.
  • this data set should be updated and reevaluated, however, the trends are quite clear.
  • An empirical way of determining which residues are preferred would be to take a representative mixture of proteins and bind to them a random synthetic peptide library. After washing away the peptides that did not bind, the remaining peptides could be eluted and the molar ratio of residues remaining bound could be determined. The profile should tell which residues result in peptides which would bind to the original mixture of proteins. This approach would also work on an individual target, providing initial information on residues important for binding.
  • An alternative method for determining which residues are preferred would be to take the mixture of proteins and use a set of phage display libraries in which one residue of the displayed peptide is fixed to select for binding phage. After several rounds of affinity selection, the libraries with the greatest number of binding phage should be those where the fixed residue is contributing to the binding of the displayed peptides.
  • the number of libraries can be reduced greatly if the central residue that is held constant is from group 1 through 7 and 13.
  • the C residue is a special case where the C held constant will be placed at the end of the peptide, not in the middle.
  • there are special uses for libraries having cysteine contrained loops see examples 1, 2, and 4) and this method would be especially useful to increase the number of binding peptides from these libraries.
  • These 8 constant residues can be combined with residues from groups 1-7, giving a structured panel of 56 libraries, a practical number to construct and manipulate.
  • the present invention is not limited to any particular method of synthesizing a combinatorial peptide library with one or more predetermined positions held constant, or with a particular mixture of amino acids at a given position.
  • Another way of thinking about the biased libraries is that they represent a more efficient way of generating complexity. If the structural properties of the binding site on the TP is such that only PLs with a certain residue, say W can bind, then a library with a single fixed, centrally positioned W will have more potential binding motifs than will a totally random library with the same numbers of unique members.
  • a TP might have two very similar FDs (such as two SH3 domains within the adaptor protein Grb-2) and thus the PLs may be of a single class even though the TP has two FDs.
  • Ser, Thr hyroxylated species especially those aliphatic in nature. Also thiolated species .
  • Glu carboxylated species especially those aliphatic in nature. Esters and alcohols.
  • Lys, Arg aminated species derivatives of the delta-guanido group.
  • Tyr as for Phe, but also hydroxylated species.
  • Trp aromatic structures especially with two or three fused rings; the rings may include heterocyclic nitrogen; indole derivatives are of particular interest. Cys thiolated and hydroxylated species.
  • Met thiolated species sulfonium salts; sulfoxides; sulfones.
  • peptide ligand If more than one peptide ligand is identified, they can be tested against each other. Competitive inhibition implies that they bind to the same site; its absence, that they bind to different sites. If the functional domains of the TP are known, these domains can be used separately as screening targets to identify which ligands bind which domains.
  • a peptide library may be prepared by biological or nonbiological synthesis methods.
  • a gene encoding the peptides of interest is expressed in a host cell so that the peptides are displayed either on the surface of the cell or on the outer coat of phage produced by the cell.
  • the gene must be randomized at those codons corresponding to variable residues of the peptide. It thus is not a single DNA, but rather a DNA mixture, which is introduced into the host cell culture, so that each cell has the potential, depending on which DNA it receives, of expressing any of the many possible peptide sequences of the library.
  • the gene may be randomized by, in the course of synthesis, using a mixture of nucleotides rather than a pure nucleotide during appropriate synthetic cycles.
  • the synthesis cycles may add one base at a time, or an entire codon.
  • the peptide library may also be prepared nonbiologically by stepwise addition of amino acids.
  • the activated AA is chosen randomly from an AA mixture.
  • the synthesis is carried out on a solid surface, such as a pin or bead. See (Proc Natl Acad Sci U S A 81: 3998-4002 (1984) [84248046], Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid, H. M. Geysen, R. H. Meloen & S. J.
  • the tags are such that only a minimal number of steps are necessary and that special handling such as that required for using radioisotopes can be minimized.
  • Suitable epitopes e.g. cMyc or influenza hemagglutinin, or enzymes such as beta-galactosidase, luciferase, or glutathione-S-transferase (GST) , or fluorochromatic proteins (e.g., the green fluorescent protein of algae) , are incorporated into the primary structure of the TP using recombinant DNA skills.
  • Such epitopes are conveniently detected by use of the appropriate enzyme
  • tags that can be incorporated via recombinant techniques include substrate sites for enzymes such as protein kinase A which allows for the rapid and efficient labeling of the TP with 32 P. Less desirable, but still feasible, is the radio labeling of the recombinant protein, e.g., in vivo with 14 C or 3 H labeled amino acids or in vi tro with 125 I.
  • the TP may be immobilized, and the library screened by the method of Cantley, infra .
  • the target may be immobilized, on chromatographic media either directly, e.g., using Affigel (BioRad), or indirectly.
  • the TP is noncovalently conjugated to the support by means of an affinity reagent.
  • target protein tagged with six histidines may be immobilized on Qiagen nickel binding resin, or a GST (glutathione S-transferase) tagged target immobilized on glutathione Sepharose (Pharmacia) , or a maltose binding protein/target protein fusion immobilized on maltose (New England Biolabs) or dextran media. Subsequently, one uses the immobilized target to separate out peptides with desired activity by the method of Cantley et . al . (Trends Biochem. Sci. 20: 470-475 (1995) [96108162] Recognition and specificity in protein tyrosine kinase-mediated signalling . S .
  • SH2 domain specificity determination using oriented phosphopeptide library S. Zhou & L. C. Cantley and Cell 72: 767-78 (1993) [93201599] SH2 domains recognize specific phosphopeptide sequences. S. Zhou, S. E. Shoelson, M. Chaudhuri , G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider &). In this method a mixture of peptides are eluted from the TP and the whole mixture sequenced by automated techniques.
  • phage display libraries The advantage of identifying peptides using the chemical synthesis-on-a-support (e.g., pins or beads) approach over peptide from phage display libraries are mostly due to the ease of identification of D-amino acid containing peptides which gives one a significant additional source of diversity. Conversely, the advantage of utilizing phage display libraries is that one can screen greater numbers of phage (perhaps 10 9 or 10 10 ) than pins (perhaps 10 4 or 10 5 ) or even possibly beads (perhaps 10 6 ) .
  • Peptoids are oligomers, similar to peptides, in which the peptide bond (-NHCO-) is replaced by an analogous linkage.
  • the -NH- may be replaced by -NR- , where R is a functional group other than H. It may be, for example, an alkyl or aryl group.
  • the -CO- may be replaced by, e.g., -SiO-, -CS-, -S0 2 -, -PO(OH)-, or -COR- .
  • a peptoid may include one or more peptidyl bonds, provided that at least one of the main chain linkages between units is not a peptidyl bond. It is likely that peptoids will enjoy advantages similar to those of peptides for screening purposes, except that peptoid libraries cannot be prepared biologically.
  • the compound library is a combinatorial library whose members are suitable for use as drugs if, indeed, they have the ability to mediate a biological activity of the target protein.
  • Peptides have certain disadvantages as drugs. These include susceptibility to degradation by serum proteases, and difficulty in penetrating cell membranes. Preferably, all or most of the compounds of the compound library avoid, or at least do not suffer to the same degree, one or more of the pharmaceutical disadvantages of peptides.
  • disjunction in which a lead drug is simplified to identify its component pharmacophoric moieties
  • conjunction in which two or more known pharmacophoric moieties, which may be the same or different, are associated, covalently or noncovalently, to form a new drug
  • alteration in which one moiety is replaced by another which may be similar or different, but which is not in effect a disjunction or conjunction.
  • disjunction in which a lead drug is simplified to identify its component pharmacophoric moieties
  • conjunction in which two or more known pharmacophoric moieties, which may be the same or different, are associated, covalently or noncovalently, to form a new drug
  • alteration in which one moiety is replaced by another which may be similar or different, but which is not in effect a disjunction or conjunction.
  • the use of the terms "disjunction”, “conjunction” and “alteration” is intended only to connote the structural relationship of the end product to the original leads, and not how the new drugs are actually synth
  • Alterations may modify the size, polarity, or electron distribution of an original moiety. Alterations include ring closing or opening, formation of lower or higher homologues, introduction or saturation of double bands, introduction of optically active centers, introduction, removal or replacement of bulky groups, isosteric or bioisosteric substitution, changes in the position or orientation of a group, introduction of alkylating groups, and introduction, removal or replacement of groups with a view toward inhibiting or promoting inductive (electrostatic or conjugative (resonance) effects.
  • the substituents may include electron acceptors and/or electron donors.
  • Typical electron donors (+1) include - CH 3 , -CH 2 R, -CHR 2 , -CR 3 and -COO " .
  • the substituents may also include those which increase or decrease electronic density in conjugated systems.
  • the former (+R) groups include -CH 3 , -CR 3 , -F, -Cl, -Br, -I, -OH, -OR, - OCOR, -SH, -SR, -NH 2 , -NR 2 , and -NHCOR.
  • the later (-R) groups include -N0 2 , -CN, -CHC, -COR, -COOH, -COOR, -CONH 2 , -S0 2 R and - CF 3 .
  • the modifications may be achieved by a variety of unit processes, including nucleophilic and electrophilic substitution, reduction and oxidation, addition elimination, double band cleavage, and cyclization.
  • a compound, or a family of compounds, having one or more pharmacological activities may be disjoined into two or more known or potential pharmacophoric moieties.
  • Analogues of each of these moieties may be identified, and mixtures of these analogues reacted so as to reassemble compounds which have some similarity to the original lead compound. It is not necessary that all members of the library possess moieties analogous to all of the moieties of the lead compound.
  • benzodiazepines The design of a library may be illustrated by the example of the benzodiazepines.
  • Benzodiazepine drugs including chlordiazepoxide, diazepam and oxazepam, have been used on anti-anxiety drugs.
  • Derivatives of benzodiazepines have widespread biological activities; derivatives have been reported to act not only as anxiolytics, but also as anticonvalsants, cholecystokin (CCK) receptor subtype A or B, kappa opioid receptor, platelet activating factor, and HIV transactivator Tat antagonists, and GPIIblla, reverse transcriptase and ras farnesyltransferase inhibitors.
  • CCK cholecystokin
  • the benzodiazepine structure has been disjoined into a 2- aminobenzophenone, an amino acid, and an alkylating agent. See Bunin, et al . , Proc. Nat. Acad. Sci. USA, 91:4708 (1994). Since only a few 2-aminobenzophenone derivatives are commercially available, it was later disjoined into 2- aminoarylstannane, an acid chloride, an amino acid, and an alkylating agent. Bunin, et al . , Meth. Enzymol., 267:448
  • the arylstannane may be considered the core structure upon which the other moieties are substituted, or all four may be considered equals which are conjoined to make each library member.
  • the basic library synthesis plan and member structure is shown in Figure 1.
  • the acid chloride building block introduces variability at the R 1 site.
  • the R 2 site is introduced by the amino acid, and the R 3 site by the alkylating agent.
  • the R 4 site is inherent in the arylstannane.
  • Bunin, et al . generated a 1, 4 -benzodiazepine library of 11,200 different derivatives prepared from 20 acid chlorides, 35 amino acids, and 16 alkylating agents.
  • variable elements included both aliphatic and aromatic groups.
  • aliphatic groups both acyclic and cyclic
  • the aromatic groups featured either single and multiple rings, fused or not, substituted or not, and with heteroatoms or not.
  • the secondary substitutents included -NH 2; -OH, -OMe, -CN, -Cl, -F, and - COOH. While not used, spacer moieties, such as -O- , -S-, -00-, -CS-, -NH- , and -NR- , could have been incorporated.
  • Bunin et al suggest that instead of using a 1, 4- benzodiazepine as a core structure, one may instead use a 1, 4-benzodiazepine-2 , 5-dione structure. As noted by Bunin et al . , it is advantageous, although not necessary, to use a linkage strategy which leaves no trace of the linking functionality, as this permits construction of a more diverse library.
  • DeWitt, et al . , Proc. Nat. Acad. Sci. (USA), 90:6909-13 (1993) describes the simultaneous but separate, synthesis of 40 discrete hydantoins and 40 discrete benzodiazepines. They carry out their synthesis on a solid support (inside a gas dispersion tube), in an array format, as opposed to other conventional simultaneous synthesis techniques (e.g., in a well, or on a pin) .
  • the hydantoins were synthesized by first simultaneously deprotecting and then treating each of five amino acid resins with each of eight isocyanates.
  • the benzodiazepines were synthesized by treating each of five deprotected amino acid resins with each of eight 2 -amino benzophenone imines.
  • candidate simple libraries which might be evaluated include derivatives of the following:
  • Heteronitrogen pyrroles pentasubstituted pyrroles pyrrolidines pyrrolines prolines indoles beta-carbolines pyridines dihydropyridines
  • one or more compounds of the chemical structures listed below have been found to exhibit the indicated pharmacological activity, and these structures, or derivatives, may be used as design elements in screening for further compounds of the same or different activity.
  • one or more lead drugs of the class are indicated.
  • hypnotics higher alcohols clomethiazole
  • aldehydes chloral hydrate
  • carbamates meprobamate
  • acetylcarbromal barbiturates
  • barbital benzodiazepine
  • narcotic analgesics morphines phenylpiperidines (meperidine) diphenylpropylamines (methadone) phenothiazihes (methotrimeprazine)
  • analgesics analgesics, antipyretics, antirheumatics salicylates (acetylsalicylic acid) p-aminophenol (acetaminophen)
  • anxiolytics propandiol carbamates meprobamate
  • benzodiazepines chlordiazepoxide, diazepam, oxazepam
  • CNS stimulants xanthines (caffeine, theophylline)
  • phenylalkylamines (amphetamine)
  • adrenergics aromatic amines epinephrine, isoproterenol, phenylephrine
  • alicyclic amines cyclopentamine
  • aliphatic amines methylhexaneamine
  • imidazolines naphazoline
  • anti -adrenergics mdolethylamine alkaloids (dihydroergotamine) imidazoles (tolazoline) benzodioxans (piperoxan) beta-haloalkylamines (phenoxybenzamine) dibenzazepines ' (azapetine) hydrazinophthalazines (hydralazine)
  • antihistamines ethanolamines (diphenhydramine) ethylenediamines (tripelennomine) alkylamines (chlorpheniramine) piperazines (cyclizine) phenothiazines (promethazine)
  • vasodilators polyol nitrates (nitroglycerin)
  • antibiotics penicillins cephalosporins octahydronapthacenes (tetracycline) sulfonamides nitrofurans cyclic amines naphthyridines xylenols
  • antitumor alkylating agents nitrogen mustards aziridines methanesulfonate esters epoxides amino acid antagonists folic acid antagonists pyrimidine antagonists purine antagonists antiviral adamantanes nucleosides thiosemicarbazones inosines amidines and guanidines isoquinolines benzimidazoles piperazines
  • pharmacological classes see, e.g., Goth, Medical Pharmacology: Principles and Concepts (C.V. Mosby Co. : 8th ed. 1976) ; Korolkovas and Burckhalter, Essentials of Medicinal Chemistry (John Wiley & Sons, Inc.: 1976) .
  • For synthetic methods see, e.g., Warren, Organic Synthesis: The Disconnection Approach (John Wiley & Sons, Ltd. : 1982) ; Fuson,
  • the library is preferably synthesized so that the individual members remain identifiable so that, if a member is shown to be active, it is not necessary to analyze it.
  • Several methods of identification have been proposed, including:
  • each member is synthesized only at a particular coordinate on or in a matrix. This might be, for example, the location of a particular pin, or a particular well on a microtiter plate.
  • the present invention is not limited to any particular form of identification.
  • Solid phase synthesis permits greater control over which derivatives are formed. However, the solid phase could interfere with activity. To overcome this problem, some or all of the molecules of each member could be liberated, after synthesis but before screening.
  • assays for soluble compounds are composed of 1) an immobilized target and 2) a soluble, labeled ligand.
  • the design of the assay is such that one measures the formation of PL/TP complex.
  • Compounds that bind to the PL's cognate TP binding site i.e. the FD
  • Such interactions generally follow first order kinetics, that is, the amount of complex formed decreases with increasing amounts of compound.
  • Compounds with potential utility shall display elements of "specificity. This means that useful compounds will inhibit the formation of one or only some PL/TP complexes, but not all. It is necessary to establish assay kits which contain controls for the inhibition of the readout per se .
  • the target is the TP of interest and its cognate PL.
  • the PL is a peptide
  • One very convenient means of labeling the peptide is to have it chemically synthesized with a biotin moiety attached to its amino or carboxy terminus.
  • the biotin moiety is separated from the peptide sequence identified above by the addition of one or more residues common in protein loops (reverse turns) , or in interdomain spacers in natural multidomain proteins, e.g., glycine and serine residues.
  • many permutations work well e.g. two glycines, G-S-G, S-G-S etc.
  • the purpose is to have the biotin flexibly linked some distance away from the residues on the peptide that bind to the FD within the TP .
  • the PL/TP complex can be detected by a procedure using commercially available alkaline phosphatase-conjugated streptavidin to form a tertiary complex which is then detected through the use of soluble colorimetric substrates for alkaline phosphatase. Since the amount of substrate hydrolyzed per unit of time is a direct reflection on the amount of initial PL/TP complex present one can conveniently use a microtiter plate reader with kinetic capacity to estimate the inhibition of complex formation by a given compound without the requirement for testing multiple dilutions of a compound in the initial screen.
  • the peptide can be complexed to dextran polymers (DEX) which have been pre-labeled with biotin as described by Dwyer (Nature Biotechnology, 14:348-351, Detection of Low Affinity Interactions between Peptides and Heat Shock Proteins by Chemiluminescence of Enhanced Avidity reactions (CLEAR) , Leslie D. Causey and Donard S. Dwyer) . This very nicely circumvents any need to laboriously seek out PL of high affinity for the TP .
  • DEX dextran polymers
  • the dextran/PL complex is premixed with the labeled PT which are together brought into contact with the immobilized compound.
  • the presence of the dextran per se minimizes the formation of non-specific complexes with the compound's plastic support and the dextran/PL complex modulates specific, high avidity binding of the labeled TP to the immobilized compound.
  • Phage Libraries have been used as a means of displaying large collections of peptides while at the same time carrying the genetic information which encode them. This is a powerful tool for the discovery of peptide ligands for various molecules, however, it does have some limitations. At the present time it is possible to make libraries with a complexity on the order of 1-5X10 9 clones . It is technically feasible to make libraries with random stretches of 25 residues, however, in order to represent all possible combinations of amino acids, the length of the displayed peptide is limited to about 7 amino acids (20 7 or 1.28X10 9 possibilities). This may be sufficient for some interactions, however, some protein:peptide interactions may require more that a 7 amino acid peptide to accurately imitate the interaction of two proteins.
  • the consensus sequences for ligands have at least one amino acid residue which is highly conserved.
  • Purpose built libraries i.e. a X-X-X-P-P-X-X-P-X-X library (Yu H, Chen JK, Feng S, Dalgarno DC, Brauer AW and Schreiber SL (1994) Cell 76:933- 945) ) based on consensus ligand sequences have been successfully used to isolate large numbers of phage displaying binding peptides using proteins in the same family.
  • the four different biased libraries used contained a fixed proline, arginine, aspartate or phenylalanme residue.
  • the predicted effect of the fixed proline was to be advantageous, the arginine and aspartate residues neutral, and the phenylalanme deleterious.
  • the results indicate that the biased proline library was much more efficient than the random library and was almost as good as the purpose built library for isolating phage that bind specifically.
  • the biased arginine library was more efficient that the random library and the biased aspartate and phenylalanme libraries were less efficient than the random library.
  • Phage were panned. Briefly, Src and Abl GST fusion proteins were immobilized on ELISA plates and blocked with BSA. Approximately 2X10 11 phage from each library was added to each well an allowed to bind. After washing the phage were eluted and amplified overnight in E. coli DH5 ⁇ F' . The amplified phage were then put through 2 additional rounds of binding and elution without any amplification. Titers were approximated by spotting serial dilutions on a lawn of DH5 ⁇ F' . Individual dilutions were then plated and clonal phage isolated by picking well separated plaques. Specificity was established using an anti-phage ELISA as previously described. Plates were coated with specific fusion protein or GST alone and phage bound as above. Bound phage were detected using an anti-phage antibody conjugated to horseradish peroxidase .
  • the library that was most effective for isolating phage that bound specifically was the Class I SH3 purpose built : 10 out of 12 specific and of those 8 gave very strong signals indicating that the affinity is likely to be very high.
  • the proline biased library was almost as effective with 7 out of 12 specific, all of them with very strong signals.
  • the arginine biased library gave 6 out of 12 specific signals, however only two of those were of moderate strength.
  • the aspartate biased library gave only one signal which was of moderate strength.
  • the phenylalanme library gave signals which did not differ appreciably from background.
  • the random library gave 12 out of 12 signals which were of low strength, indicating that they are likely not of very high affinity.
  • Table A-l Phage isolated with Abl SH3 domain data
  • single residue biased libraries greatly enhances the isolation of phage displayed peptides which have desirable binding characteristics.
  • single residue biased libraries yielded a larger number of high affinity binders than the random library. This enhancement is both in the number of phage isolated and in the affinity that the phage have for the target.
  • the biased libraries were as good as the so-called purpose built library.
  • the purpose built library was made using a priori knowledge of the residues necessary for ligand affinity of the SH3 domain containing target .
  • Human cytomegalovirus disease associa tions .
  • Human cytomegalovirus (HCMV) is a ubiquitous human pathogen (for recent reviews see Huang and Kowalik, 1993; Britt and Alford, 1996) .
  • HCMV is highly species specific. Humans are the only reservoir for the virus, and transmission occurs by direct or indirect contact among individuals. HCMV infections are generally asymptomatic in healthy children and adults. However, HCMV is responsible for about 8% of mononucleosis cases (Klemola et al . , 1970; Horwitz et al . , 1979) and for transfusion disease (Reyman, 1966) in some individuals that receive blood products contaminated with the virus . HCMV can cause serious disease in unborn children and in immunocompromised people.
  • HCMV congenital HCMV
  • HCMV congenital HCMV
  • Infected HCMV HCMV is the most common congenital infection in humans (Britt and Alford, 1996); about 40,000 infected children are born each year in the United States. Given estimates that about 10-15% of these infected infants exhibit long-term neurological pathology, then HCMV is the leading infectious cause of central nervous system maldevelopment in newborn children (Fowler et al . , 1992) . Damage to perceptual organs is the most common outcome of intrauterine infection, and congenital HCMV infection appears to be the most common non- genetic cause of childhood hearing loss in the United States (Hicks et al . , 1993) .
  • HCMV disease is a common posttransplantation complication in solid organ allograft recipients.
  • the degree of immunosuppression in the allograft recipient correlates with the probability of clinically significant HCMV disease. More than 60% of heart, kidney and liver allograft recipients develop active HCMV infections (reviewed in Pollard, 1988; Britt and Alford, 1996) .
  • Solid organ allograft recipients exhibit a range of clinical syndromes resulting from HCMV infection, such as prolonged fever, leukopenia, thrombocytopenia, atypical lymphocytosis and elevated hepatic transaminases (Hofflin et al . , 1987; Singh et al . , 1988; Smyth et al .
  • HCMV infection is seen in about 40-50% of allogeneic bone marrow transplant recipients, and pneumonia is the most common clinical syndrome that results (reviewed in Wingard et al . , 1990) . Even with the availability of gangcyclovir, the mortality rate following bone marrow transplant remains at 10- 20% for patients with HCMV pneumonia (Goodrich et al . , 1991; Schmidt et al . , 1991; Yau et al . , 1991; Enright et al . , 1993; Winston et al . , 1993).
  • HCMV might be a cofactor in the pathogenesis of HIV.
  • Epidemiological studies have suggested that HCMV infection is associated with increased risk for the development of AIDS in HIV- infected individuals (Webster et al . , 1989; Webster, 1991; Webster et al . , 1992) .
  • HCMV can influence HIV gene expression and growth in cultured cells (Barry et al . , 1990; Rando et al . , 1990; Biegalke et al . , 1991; Koval et al . , 1991; Peterson et al . , 1992).
  • HCMV ulcerative colitis
  • HCMV disease in AIDS has been reported to affect almost every organ system, clinically significant HCMV infections have been reported most frequently in the central nervous system (principally retinitis which is found in 20-25% of long lived AIDS patients) , the gastro-intestinal system and the lung (Britt and Alford, 1996) .
  • Ganciclovir a congener of acyclovir, and foscarnet both exhibit potent anti-HCMV activity in cell culture assays.
  • Ganciclovir is preferentially phosphorylated in HCMV- infected cells by a viral gene product, UL97, and its subsequent incorporation into growing DNA chains by viral DNA polymerase encourages chain termination (Frank et a 1 . , 1984; Reid et al . , 1988) .
  • Mutations in the viral polymerase or in UL97 confer resistance to ganciclovir (Sullivan et al . , 1993; Baldanti, et al . , 1995; Hanson, et al . 1995).
  • Foscarnet inhibits the HCMV DNA polymerase directly (Snoeck et al . , 1993). Numerous studies have demonstrated the efficacy of ganciclovir and foscarnet in the control of HCMV disease in both allograft recipients and AIDS patients (reviewed in Britt and Alford, 1996) , although the utility of foscarnet is limited since its chronic use often leads to renal toxicity (Chrisp and Clissold, 1991; Reusser et al . , 1992).
  • anti-HCMV drug therapy In the case of AIDS, anti-HCMV drug therapy often must be continued on a long term basis since viral replication resumes shortly after withdrawal of the drug, and, as a consequence, viral variants that are resistant to one or both drugs are becoming increasingly common (Drew et al . , 1991; Balfour, 1992), underscoring the need for additional drugs with anti-HCMV activity. Recently, a new drug with anti-HCMV activity was approved by the FDA for the treatment of retinitis in patients with AIDS. Vistide is a nucleotide analog which has performed well in clinical trials, although it can cause renal impairment .
  • HCMV gene products that might be targets for the development of antiviral drugs.
  • two HCMV proteins are most often cited as potential targets.
  • the first is a proteinase encoded by the HCMV UL80 open reading frame and termed assemblin (Welch et al . , 1991a and b) . This proteinase functions during assembly of the virion particle.
  • a temperature-sensitive allele of the homologous herpes simplex virus gene is defective for virus assembly at the nonpermissive temperature (Preston et al . , 1983) , predicting that a drug that interfered with the activity of assemblin, would block viral assembly and spread.
  • the second target is the UL44-coded polymerase accessory protein. This protein, together with the UL54-coded polymerase, is the topic of this example.
  • HCMV DNA replication requires both a cis-acting DNA origin of replication, termed oriLyt (Hamzeh et al . , 1990; Anders et al . , 1992; Masse et al . , 1992) , and a set of trans-acting viral proteins.
  • trans- acting viral replication proteins have been identified (Pari and Anders, 1993; Pari et al . , 1993) using a transient replication assay originally developed to study herpes simplex virus DNA replication (Challberg, 1986) .
  • a transient replication assay originally developed to study herpes simplex virus DNA replication (Challberg, 1986) .
  • cells are co-transfected with a reporter plasmid containing the oriLyt sequence plus combinations of plasmids or cosmids that supply required viral trans-acting replication proteins.
  • Replicated reporter plasmid DNA that is resistant to Dpn I is then monitored by DNA blot assay.
  • the eleven HCMV products required for replication of the reporter are listed in Table 101.
  • HCMV replication genes Five of the HCMV replication genes were originally identified on the basis of their sequence homology to known herpes simplex virus type 1 (HSV-1) replication proteins and on the basis of biochemical studies that confirmed predicted functions. These include a DNA polymerase, encoded by the UL54 HCMV open reading frame (Huang, 1975; Ye and Huang, 1993), and a polymerase accessory protein, UL44 (Ertl et al . , 1991; Ertl and Powell, 1992) ,- a single-stranded DNA-binding protein, UL57 (Anders et al . , 1986; Kemble et al .
  • HSV-1 herpes simplex virus type 1
  • HCMV UL102 was located on the viral chromosome relative to other viral genes at a similar position as the HSV-1 UL8 gene encoding a primase associated factor (Chee et al . , 1990), i.e., these genes were positional homologs. These proteins probably act directly at the replication fork to mediate HCMV DNA replication. Candidate homologs of this set of proteins have been found in all herpes viruses sequenced to date (EBV: Baer et al .
  • the IE1/IE2, UL36-38, and IRS1-TRS1 HCMV genes encode immediate early gene products have been shown to regulate the expression of viral and cellular genes at the level of transcription (e.g. Pizzorno et al . , 1988; Cherrington and Mocarski, 1989; Depto and Stenberg, 1989; Stenberg et al . , 1990; Colberg-Poley et al . , 1992; Stasiak and Mocarski, 1992) .
  • the UL84 and 112-113 genes encode early viral proteins with unknown functions (Wright et al . , 1988; He et al . , 1992).
  • HSV-1 UL9 protein together with the six HSV proteins identified in Table 101 are necessary and sufficient to reconstitute HSV-1 DNA replication in the transfection assay (Challberg, 1986) described above.
  • HSV proteins that participate directly in viral replication, with the exception of an origin-binding protein, have been identified. If HCMV utilizes an origin-binding protein, it presumably is one of the 11 proteins identified in Table 101.
  • the HCMV UL44 protein is generally referred to as the DNA polymerase accessory protein.
  • the HCMV UL44 protein has some homology to the HSV-1 UL42 protein (data not shown) . Both of these proteins exist in a 1:1 complex with viral DNA polymerase (UL42 : Powell and Purifoy, 1977; Gallo et al . , 1988, Crute and Lehman, 1989; UL44 : Huang, E.-S., 1975; Ertl and Powell, 1992) .
  • the HSV-1 UL42 protein has been shown to be essential for viral DNA replication by analysis of viral mutants (Johnson et al . , 1991; Digard et al .
  • the goal of this example is the development of a high throughput screen for the identification of small molecule anti-HCMV drug candidates that target the viral UL44 processivity factor.
  • This drug discovery system is especially useful .for proteins that are not readily placed into biochemical assays, e.g. regulatory factors.
  • We chose to use UL44 as a target for our combinatorial recognition system because it is known to be necessary for viral replication and in vi tro synthesis of CMV DNA. Thus, we can rapidly evaluate the action of any potential compounds that act on UL44 with both biochemical and viral replication assays.
  • the first step is production of recombinant protein to use as a molecular affinity selection tool.
  • GST glutathione-S-transferase
  • the mRNA encoding UL44 is not spliced. Therefore, it is possible to isolate the complete UL44 coding sequence as a functional unit from HCMV genomic DNA prepared from purified virions.
  • oligonucleotide primers and the PCR to amplify the coding region of UL44 from CMV DNA: 5' -CTGTGCGGATCCATGGATCGCAAGACG-3 ' and 5' -CTGTGCGAATTCCTAGCCGCACTTTTG-3 ' .
  • the resulting 1.3 kb product was purified using a Wizard PCR Preps clean up resin, blunted with T4 DNA Polymerase (NEB) , cut with BamHI repurified on an agarose gel and cloned into the vector pGex2T digested with BamHI and Smal .
  • NEB T4 DNA Polymerase
  • E. coli DH5aF' carrying the plasmid were induced with IPTG to produce the fusion protein and the product was purified by affinity chromatography using glutathione sepharose as recommended by the manufacturer (Pharmacia) .
  • the resulting protein was used as a fusion protein or was cleaved from the glutathione sepharose using the protease thrombin
  • Thrombin 50 Units of Thrombin (Pharmacia) for 2 hours at room temperature.
  • the resulting UL44 protein was analyzed by SDS gel electrophoresis and contained two major cleavage products, one of full length and the other roughly 5 kdaltons smaller.
  • Phage libraries were made using published protocols (Construction of Random Peptide Libraries in Bacteriophage M13 in Phage Display of Peptides and Proteins: A Laboratory Manual. Edited by B. Kay, J. Winter and J. McCafferty. Academic Press 1996.). Briefly, oligonucleotides which encoded the random peptide with one residue fixed were converted to double stranded DNA by extending a complementary primer using Sequenase (USB) . The resulting fragments were digested with Xhol and Xbal, gel purified and ligated into previously digested mBAX vector. The ligation was introduced into bacteria by ten successive electroporations and the transformed bacteria were amplified overnight.
  • USB Sequenase
  • the X 5 DX 5 library is referred to as a "D” library, X 5 FX 5 as an “F” library, etc.
  • the oligonucleotide sequence for each was 5'- GACTGTGCCTCGAGK(NNK) 5 xxx(NNK) 5 TCTAGACGTGTCAGT-3' where xxx is the codon shown above for each residue fixed.
  • a library with 10 random residues followed by a fixed C (TGC) was constructed with the same flanking sequences. This is referred to as the "X 10 C” library.
  • the oligonucleotide with the sequence of 5 ' -ACTGACACGTCTAGA-3 ' was used as the primer to convert the ssDNA to double stranded.
  • the Carolina Workshop Library (CWL) is a 12-a.a. "unbiased" (no constant residue) peptide phage library, with each amino acid encoded by NNK.
  • UL44 or GSTUL44 was immobilized on microtiter plates (Costar) by incubating 1 ⁇ g of protein in 200 ⁇ l of 0.1 M NaHC0 3 , pH 8.5 overnight at 4° C. The remaining protein binding sites on the plate were blocked by adding 150 ⁇ l of 1% BSA in 0.1 M NaHC0 3 and incubating the plate at room temperature for 1 hour. The plate was then washed 5 times with 300 ⁇ l of TBST (100 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1 % tween-20). Phage libraries were then added to the wells in 200 ⁇ l of TBST and allowed to incubate at room temperature for 5 hours.
  • TBST 100 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1 % tween-20
  • the wells were washed 5X with TBST and the phage were eluted by incubating with 200 ⁇ l of 50 mM Glycine, pH 2.0 for 10 minutes.
  • the eluant was removed to a tube at the pH neutralized with 200 ⁇ l of 200 mM NaHP04 buffer, pH 7.0.
  • the phage were then amplified by adding the eluted phage to 5 ml of 2XYT broth containing 1:100 dilution of an overnight culture of E. coli DH5 ⁇ F' .
  • the cultures were grown with agitation overnight at 37° C.
  • the next morning the bacteria were removed by centrifugation at 3000 xg for 10 minutes in a SS-34 rotor. 100 ⁇ l of the supernatant containing the amplified phage were then used in the next round of affinity purification.
  • Enrichment for phage that bind to UL44 were monitored by including a non-specific phage which formed white plaques on DH5 ⁇ F' in the affinity selection process starting at round 2.
  • the ratio of blue:white plaques was monitored by plating the phage in the presence of X-gal and IPTG. Phage from our libraries appear blue and the non-specific control phage appear as white plaques. If a particular library has been enriched for phage which bind to the target, then they should be selectively retained in higher numbers than the control phage and this is reflected in .
  • the ratio of blue to white plaques (Fig. 5) .
  • Phage specific for either the GST portion or the UL44 portion of the fusion were then added to separate wells and at the same time various concentrations of either GSH or DNA were added. The results are shown in Figure 7. It is important to keep in mind that these phage were isolated from the same affinity selection run. This clearly shows that phage specific for GST bind at the active site of the enzyme because the binding of phage is competed in a dose dependent fashion with GSH. The concentrations of GSH used here had no effect on the binding of the UL44 specific phage to the same GSTUL44 protein. Conversely, the phage specific for UL44 are binding in the DNA binding pocket of UL44 and are competed away by the addition of DNA in a dose dependent manner. The same concentrations of DNA have little effect on the binding of the GST specific phage to the same GSTUL44 fusion protein.
  • Enzyme Linked Spectrophotometric Assay for CMV UL44 Using Peptides derived from Phage Display Immulon 4 (cat.# 011-010-3855) 96-well plates were purchased from Dynatech. Bovine serum, albumin (BSA) (A2153),
  • PBS Phosphate buffered saline
  • Ultrapure glycerol #16374
  • Biotinylated surrogate ligands were prepared as a 1 mM stock solution in the appropriate solvent (H 2 0 or 10% acetonitrile) .
  • SA-AP was prepared as a 1 mg/ml stock solution in PBS containing 10% glycerol and stored in aliquots at -80 °C.
  • Target protein was immobilized in microtiter wells by incubating 0.5-2.0 ⁇ g per well in 100 ⁇ l of 0.1 M NaHC0 3 overnight at 4 °C. (Studies have indicated that maximal protein binding can be attained with 0.5 ⁇ g of target protein per well.) The target protein was removed and the wells were blocked with 200 ⁇ l of 1% BSA prepared in 0.1 M NaHC0 3 for 1 hour at room temperature. During the 1 hour blocking period, the SA-AP : surrogate ligand conjugate was prepared by mixing 2 ⁇ g SA-AP and 50 pmol biotinylated surrogate ligand for each well of target protein.
  • the surrogate ligand:SA-AP conjugate was then added (100 ⁇ l/well) and incubated at room temperature for 2 hours. The wells were then washed 5 times with 300 ⁇ l TBST using a BioRad 1575 Immunowash plate washer. The assay was developed by adding 100 ⁇ l of pNPP and incubating at room temperature for 5-10 minutes. The absorbance was read at 405 nm.
  • Figure 8 shows that a peptide derived from phage display binds specifically to UL44. The only significant binding is to GST-UL44 and not to streptavidin, GST or a GST-SrcSH3 fusion protein, indicating that the structure recognized by this peptide is UL44 and not the GST tag.
  • This binding is time and dosage dependent ( Figures 9 and 10) .
  • the binding is dependent on the concentration of the surrogate ligand and the concentration of target protein on the plate. Saturation of the target protein binding to the plate occurs at about 0.5 ⁇ g of protein per well.
  • the binding of this peptide to UL44 can be competed specifically with an identical peptide that is not biotinylated ( Figure 11) .
  • phage displaying peptides from biased peptide libraries which bind specifically to a GSTUL44 fusion protein. These phage bind to one of two domains: the GSH binding site on the GST portion of the fusion or the DNA binding site on the UL44 fusion. The phage are specific for each site and they can be competed for binding with their respective ligands. A peptide of the same sequence as the phage which binds to the DNA binding domain of UL44 also binds to UL44 specifically.
  • This binding can be demonstrated by a competition assay between the peptide and the parent phage or by using a biotinylated peptide which is then detected using a streptavidin-alkaline phosphatase conjugate (SA-AP) .
  • SA-AP streptavidin-alkaline phosphatase conjugate
  • This conjugate could be used to screen through large numbers of low molecular weight compounds to find those that bind at the DNA binding site of UL44. This could be done several ways.
  • the first approach is using the assay shown here. First the target protein, in this case UL44 or a UL44 fusion protein would be immobilized on a solid surface (i.e. a microtiter plate) . The peptide would be complexed with SA-AP and this complex added to a well containing the immobilized target at the same time that a small molecular weight compound is added. After allowing time for binding, the wells would be washed with buffer and the substrate for the conjugate (PNPP) could be added and allow for any conjugate in the well to convert the clear substrate to the colored product .
  • PNPP substrate for the conjugate
  • the amount of color is proportional to the amount of SA-SP in the well. If a small molecular weight compound bound to the target at the same place as the surrogate ligand: SA-AP complex, then a reduced amount of color would be detected because the compound and the peptide : SA-SP complex can not occupy the same space. This would be the identification of a potential drug lead.
  • This method could be used in an automated format to screen large numbers (from 1,000 to 1,000,000) of compounds for those which have the property of binding to a target in the same place as the surrogate ligand and preventing the labeled surrogate ligand from binding to the target.
  • the peptides here could be used in a precomplexed format (as shown above) or they may be used as monomeric ligands and then detected in a second step.
  • This example illustrates the use of phage display to isolate surrogate ligands targeted to a biologically relevant site on a viral protein and the utility of using this ligand in a high throughput screen of small molecules for the discovery of potential therapeutic drug leads. Similar experiments can be done with any viral protein which is a target for antiviral therapy.
  • the protein was produced by cloning and expressing the target in bacteria.
  • the cloned protein could be expressed in any system including but not limited to bacteria, yeast, baculovirus, vaccinia virus, CHO cells (Chinese hamster ovary cells) , HeLa, fibroblasts, adenovirus or any other expression system in which the target protein is produced in such a way that it presents an active conformation to the potential surrogate ligands.
  • the protein could also be made in vi tro by transcription and translation using any of a variety of RNA polymerases in conjunction with lysates from reticulocytes, wheat germ or any other source of enzymatic machinery for the translation of RNA to protein. If the target protein were small enough or if a synthetic scheme could be devised to produce it de novo, the target could also be a completely synthetic molecule.
  • peptides generally do not cross the plasma membrane of cells.
  • technology is available to deliver peptides into cells with high efficiency using liposomes, electroporation, micro-injection, etc.
  • This in vi tro assay was originally developed in the HSV-1 system (Hernandez and Lehman, 1990) , and we will adapt and validate the assay for the HCMV replication system.
  • the in vi tro HSV-1 system utilizes three virus-coded proteins: the DNA polymerase (HSV UL30) , the processivity factor (HSV UL42) , and the single-stranded DNA-binding protein (HSV UL29) .
  • the template used for the in vi tro replication system is single- stranded M13mpl8 DNA to which an oligonucleotide primer (5'- GTTTTCCCAGTCACGAC-3' ) is annealed.
  • the oligonucleotide is generally used for DNA sequencing and is commercially available (New England BioLabs) .
  • the replication assay is an origin-independent primer extension reaction.
  • the t HSV-1 polymerase and single-stranded DNA-binding protein extend the primer annealed to M13 DNA, producing chains of variable length and only a small quantity of completely double-stranded DNA where the primer has been extended the full length of the M13 circular DNA. In contrast, with addition of the processivity factor, most of the product is full length, double-stranded M13 DNA.
  • the production of variable length versus full length DNA products is monitored by the electrophoretic separation of reaction products in agarose gels (Hernandez and Lehman, 1990) .
  • the coding sequence for the HCMV polymerase (UL54) and single-stranded DNA-binding protein (UL57) will be amplified from HCMV genomic DNA as described above for the processivity factor (UL44) .
  • the UL54 and UL57 coding regions will be validated by automated DNA sequence analysis, and cloned into pBlueBacHis2 (Invitrogen) .
  • the plasmids will then be used to construct baculovirus recombinants for expression of the replication proteins in Sf9 insect cells.
  • Expressed proteins carry two tags at their 5' ends, one the Xpress leader peptide (Asp Leu Tyr Asp Asp Asp Asp Lys) is easily detected with a monoclonal antibody in ELISA assays and the other includes a six histidine binding site that has a high affinity for divalent cations.
  • Nickel-chelating resins will allow us to purify the recombinant proteins in one step.
  • the HCMV equivalent of the HSV-1 replication assay will then be optimized using purified proteins.
  • we When we have successfully implemented the in vi tro origin- independent primer extension assay, we will titrate peptides that we have shown to interact with the processivity factor into the reaction to test for their ability to perturb the interaction.
  • inhibitory effects will be assayed by testing the ability of active peptides to inhibit the activity of mammalian DNA polymerase alpha (partially purified from HeLa cells by sequential chromatography on Q- Sepharose and double-stranded DNA cellulose, Owsianka et al . , 1993) or the E. coli Klenow polymerase (commercially available) on the M13 -primer complex.
  • mammalian DNA polymerase alpha partially purified from HeLa cells by sequential chromatography on Q- Sepharose and double-stranded DNA cellulose, Owsianka et al . , 1993
  • E. coli Klenow polymerase commercially available
  • HCMV helicase/primase subunits UL105, UL 70, UL 101-102
  • baculovirus vectors infect with the mixture of baculoviruses expressing the HCMV proteins equivalent to the HSV-1 proteins listed above, produce extracts and test their activity. Peptides could then be tested for their ability to inhibit origin- independent replication in the extracts.
  • cells could be infected with a set of plasmids expressing the complete set of eleven factors needed to reconstitute HCMV oriLyt-dependent replication (Pari and Anders, 1993; Pari et al . , 1993).
  • Peptides would be introduced into cells by electroporation, which has been used to introduce functional small polypeptides into cells with high efficiency (Kashanchi et al . , 1992) and tested for their ability to inhibit the reaction.
  • Combinatorial chemical libraries will be screened to identify compounds that block macromolecular interactions of UL44. We will screen the benzodiazepine library described above and other chemical diversity libraries.
  • the compound libraries will be screened by placing individual compounds in microtiter plates wells that have been coated with GST-UL44 fusions and adding the molecular probe. While one can use biotinylated peptides as probes within screening assays, we have found that this can be made much more "user friendly" for compound screening by using an expression vector pMY to which peptides or protein modules can be fused to bacterial alkaline phosphatase (BAP) (described above) .
  • BAP bacterial alkaline phosphatase
  • Ligand/BAP fusion protein also encodes the
  • FLAG epitope we include as a control well for each compound examined a test for any apparent non-specific activities by examining the effects of the compound upon binding the Ligand/BAP fusion to immobilized anti-FLAG mAb Ml.
  • Adey, N. B., and Kay, B. K. (1996) Gene 169, 133-134.
  • Blond-Elguindi S., Cwirla, S., Dower, W. , Lipshutz, R., Sprang, S., Sambrook, J., and Gething, M.-J. (1993) Cell 75, 717-728.
  • Protein Kinases are important regulators of intracellular processes and play a central role in signal transduction. One of the most important controlling points is the regulation of protein kinase C. Its various isozymes are involved in many processes which regulate cell growth and hence it is an attractive target for an antitumor agent . To explore this possibility we have isolated surrogate ligands specific for human protein kinase C beta II.
  • Baculovirus produced Protein Kinase C ⁇ l ⁇ was obtained from L. Ballus (Sphinx Pharmaceutical Corp.) and was immobilized as described in Example 1 and affinity selection of phage was carried out as in example 1 with several slight modifications. Elution was found to be more efficient if the initial pH 2.0 glycine treatment was carried out with buffer prewarmed to 50° C and was immediately followed by an elution at pH 12 with prewarmed 100 mM ethanolamine for 10 minutes. The eluted phage was pooled, neutralized, and amplified in 1 ml of 2X YT containing E. coli DHFaF' in a 2 ml Megatiter plate in which each 2 ml well is arranged in a 96 well array. Results :
  • the peptides fall into 3 classes.
  • the first and largest class are peptides from the X 10 C library and are characterized by a loop of 4 amino acids constrained by two cysteine residues . There are conserved residues both inside the loop and flanking the N terminal cysteine.
  • the second group of peptides also came from the X 10 C library and is characterized by 2 residue loop. the conserved sequence is a hexapeptide in which the only variance between residues is a valine vs. isoleucine substitution on one of the peptides.
  • the third group consists of three peptides with little primary sequence conservation.
  • phage ELISA To place the different clones in a relative order based on the affinity they have for the target, we carried out a set of phage ELISA with decreasing concentrations of phage. The results are shown in Figure 12.
  • the phage exhibit a fairly wide range of binding affinities, needing anywhere from 0.5 ⁇ l to 10 ⁇ l of phage supernatant to exhibit half maximal binding.
  • This example uses a eukaryotic cellular protein kinase as a target for which we have isolated artificial ligands.
  • the peptide sequences shown above could easily be used to set up a screen for small molecules which bind at the same site.
  • the artificial ligand could be used in any of the ways discussed in example 1.
  • selections may also be done in the presence of one or more cofactors or regulators of the enzymes function.
  • PKC we could have carried out the selection in the presence of diacyglycerol or phorbol esters to activate the enzyme. This would result in the enzyme taking on a different conformation and may alter the ligands that are obtained. This strategy may be altered to target a specific site by eluting the phage with the known ligand. To do this, I would carry out. all of the binding and amplification steps as above, however, the elution step would be replaced by an extended incubation in the presence of large amounts of the natural ligand (i.e. Phorbol) .
  • the pool of phage from the final round of selection could be "sorted" by adding the natural ligand first followed by the phage.
  • the binding of the natural ligand would prevent the phage binding to a specific site but not at others. I would then take the supernatant which contains the unbound phage and test individuals for binding. In this way you can enrich for phage to a specific known site. Sequence analysis of these phage would then yield a cluster of peptides which would describe the ligand binding site.
  • phage could be eluted with the compound at each round of selection or the target could be blocked by the compound before phage from the final round of selection were allowed to bind. Both of these approaches would give rise to artificial ligands which bound to the site blocked by the compound. These ligands could then be used in a high throughput screen to find additional compounds which bind to the same or overlapping sites .
  • Example 3 Isolation of Peptides which bind to human MDM2. Subcloning of human MDM2 cDNA into GST expression vector.
  • the MDM2 (mouse double minute protein) is involved in the control of cell growth by interacting with the protein product of the tumor suppressor gene p53.
  • p53 acts as a sensor of DNA damage and uncontrolled cellular proliferaction and prevents cell growth by activating a number of gene products which cause either cell cycle arrest or programmed cell death (apoptosis) .
  • MDM2 interacts with the N terminus of p53 and prevents it from activating these genes. Thus overexpression of MDM2 leads to uncontrolled cell growth.
  • One potential place for pharmacological intervention in this system is the disruption of the MDM2 - p53 interaction.
  • a fusion protein for affinity selection we subcloned the MDM2 cDNA from pQEll-hMDS by digestion with BamHI and EcoRV and ligating the cDNA into pGEX5X-l (Pharmacia) cut with BamHI and Smal to produce pGSThMDM2.
  • the deletion construct expressing GST fused to the first 139 amino acids of MDM2 was made by digesting pGSThMDM2 with BsrGI and Xhol, blunting the ends with Klenow and religating. All clones were verified by restriction enzyme mapping and DNA sequence analysis.
  • the fusion proteins were produced according to standard protocols from the manufacturer and were used as is or cleaved with thrombin.
  • Enrichment for binders monitored by pool ELISA. After 3 rounds of selection, the only libraries which showed an enrichment for binding phage were the H and W libraries. 95 clones were tested, 48 from the H library and 47 from the W library. Strong binding was observed on 5 from the H library and 28 from the W library. Testing the specificity of these 33 phage against a variety of proteins showed that 17 of them bound to give strong signals and were very specific.
  • the DNA sequences for the displayed phage were determined and are shown below:
  • GAPWNWEKKEL ADPRLPVEREL MDGSGGERNSMW PMRTEWAVGSES These peptides sequences can be placed in two groups. The first group align between themselves to form the consensus sequence FxDyWqdL where the upper case residues are completely conserved. This sequence aligns perfectly with a sequence with the human or mouse p53 protein that has been shown to interact with the N terminal portion of hMDM2 by biochemical studies and crystallography (Leng et al 1995 and Kussie et al 1996) . The other peptides have limited homology to each other and do not align with peptide sequences from p53 or any other protein in genbank .
  • N-Terminal 130 amino acids of MDM2 are sufficient to inhibit p53 -mediated transcriptional activation. Oncqgene 10 :1275-1282.
  • Transfer RNA synthetases catalyze the ATP dependent charging of a tRNA molecule with a specific amino acid. These charged tRNAs are then utilized in translation for the production of new proteins. These enzymes are required for growth in all organisms and are quite different from bacteria to humans. They thus represent an attractive target for antimicrobial compounds. We set out to isolate surrogate ligands to the E. coli proline synthetase and test to see if these surrogate ligands were targeted to the active site of the enzyme .
  • Each of the isolated phage display peptides constrained by two cysteines with an intervening loop of 8 residues with several conserved residues both inside and outside the loop.
  • the one clone isolated from the P library also contained a peptide with identical constraints.
  • the codon structure of the DNA encoding the displayed peptide shows that this phage is not a contaminant from the X 10 C library.
  • the fixed C residue in the X 10 C library uses the codon TGC, however, in the phage isolated from the P library this C residue is encoded by a TGT codon, clearly demonstrating that this clone did indeed arise from the P library.
  • FIG. 13 shows that the peptide is specific for E. coli ProRS and that the TP does not bind other peptides. This interaction can be disrupted in a dose dependent fashion by a non- biotinylated peptide of the same sequence ( Figure 14) . In addition, the binding of this peptide to ProRS is dependent on time and peptide concentration ( Figure 15) .
  • peptides designed from these phage are directed to random regions on the target protein or if they target active sites and regions of other biological interactions.
  • the peptides used in the above ELSAs were added to a charging assay to assess its affects on enzymatic activity of the target. Although the time of preincubation with the peptide needed was long (about.1.5 hours for 50% inhibition using 530 ⁇ M peptide), the peptide effectively inhibited the activity of the enzyme with an Ki of greater than 500 ⁇ M. This inhibition is competitive in respect to proline, thus these peptides are directed to the active site of the enzyme. We did not isolate any phage expressing peptides which bound to other regions of the target.
  • the environment is such that disulfide bonds are reduced.
  • the displayed peptides are likely to be linear inside of the cell and then circularize after being exposed on the outside of the cell. This means that the peptide would not take on the same conformation inside the cell as it is displayed on mature phage that are used in panning. If a peptide expressed inside of E. coli bound to an E. coli protein and inhibited its function and this protein were essential, then that peptide would be selected against during the process of making and propagating the phage display library (because these phage are grown on an E. coli host) .
  • the X 10 C library must display a cyclized peptide which exhibits a different conformation inside of the cell and therefore would not bind to the protein in question, in this case the E. coli proline tRNA synthetase.
  • the tyrosyl tRNA synthetase is an attractive target for new antibiotics. We set out to isolate surrogate ligands for this synthetase and to determine where these peptides were targeted on the enzyme .
  • phage ELISAs showed enrichment for binding phage in the D, F, W, N, P, CWL, PHD7 (random 7-mer, New England Biolabs) and PHD12 (random 12-mer New England Biolabs) libraries. Individual clones from each of these libraries was tested for specific binding and the peptide displayed were deduced from the DNA sequence. The results are shown below:
  • Peptides corresponding to TyrRSl through TryRS6 were synthesized and tested in several assays.
  • Peptide TyrRSl was biotinylated and was used for a standard ELSA as described above. This peptide binds to TyrRS specifically (Fig. 16), other peptides do not bind to the target TyrRS (Fig 17), and the binding is dependent on time and concentration (Figs 18- 19) .
  • these peptides are directed to a potential area of target drug interaction. If this peptide can be displaced by a small molecular weight compound, the compound is likely to be a drug lead candidate.
  • Effective inhibitors of this target are available for our use and they mimic an intermediate in the reaction of charging Tyr tRNA. These inhibitors are tyrosyladenylate compounds .
  • One such inhibitor was tested for its ability to disrupt the phage: target interaction with all of the phage isolated. We determined that the inhibitor is an effective competitor for phage binding on a majority of group 1 phage .
  • the ability of the compound to inhibit the binding of the TyrRSIB peptide to TyrRS was determined.
  • a standard ELSA was carried out except that various concentrations of the compound were added to surrogate ligand before adding the mixture to the immobilized target in a well of a microtiter dish. The results are shown in Figure 20. It is clear that the compound inhibits the binding of the surrogate ligand to the target in a dose dependent manner.
  • a related compound, a prolyladenylate that inhibits another synthetase (ProRS) had no effect on the binding of the surrogate ligand.
  • oocytes which span the membrane often need to be in a membrane to take on the correct conformation for a biologically active protein. This presents a problem for conventional techniques designed to find artificial ligands targeted to the native form of the protein.
  • One possible solution to this problem is the use of live cells to express the receptor of choice and then use the whole cell as the way to present the target to the library of artificial ligands.
  • One system in which to do this is the oocyte from Xenopus laevis. I would first clone the receptor of interest into a vector from which RNA could be produced in vi tro using bacterial or phage RNA polymerases. This RNA would then be injected into oocytes and the oocytes then incubated to allow the production of protein.
  • the oocytes (probably 1-10 per binding reaction), now with the receptor of interest on the cell surface would be mixed with the library of artificial ligands and binding allowed to occur.
  • the oocytes would be washed to remove the non-specific binding ligands and then the ligands would be eluted using a change in pH, salt concentration or another treatment which would break the interaction.
  • the ligands would then be amplified and subjected to further rounds of selection.
  • the positive selection described above may give rise to ligands specific to the receptor of interest, however, it may be necessary to use a negative selection to remove the ligands binding to proteins on the surface of the oocyte. This would be accomplished by binding the pool of artificial ligands to an oocyte which has not been injected with any RNA and thus is presenting only native oocyte proteins on its surface. Any ligands which bind to the oocyte would be removed and the ligands remaining in the supernatant would be used for subsequent rounds of positive selection. This negative selection may be carried out before or after each round of positive selection or it may only be required once during the selection process. In any case the concept is to remove artificial ligands which are binding to oocyte proteins and leave the pool of ligands enriched for those which bind to the receptor of interest .
  • the enrichment for ligands binding to the receptor can be monitored utilizing a modified ELISA procedure.
  • oocytes injected and expressing the receptor would be place in the well of a microtiter dish and individual artificial ligands added.
  • phage expressing peptides a phage clone grown from an isolated plaque would be used.
  • the oocyte would be washed and the ligand would be detected by convential methods, usually and antibody conjugated to an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • non- injected oocytes would be subjected to the same clone in a parallel well and the signals compared.
  • Clones giving a higher signal in the well containing the inject oocyte would be considered positive (they bind to the receptor in a specific manner) and those with equal signals in both wells bind to a protein normally on the oocyte surface.
  • the clones which bind would be sequenced and compared for common elements.
  • RNA polymerases in conjunction with lysates from reticulocytes , wheat germ or any other source of enzymatic machinery for the translation of RNA to protein. It may be advantageous to produce and isolate the protein in an environment that promotes proper protein folding.
  • Example 7 Isolation and Use of nucleic acids as surrogate ligands (prophetic)
  • peptide based artificial ligands are a powerful approach in the paradigm of drug screening discussed here. However, there may be some targets for which it will be difficult to isolate peptide ligands. In this case it may be preferable to use a DNA or RNA based aptamer as the ligand, especially as one can work with very large (complexities of > 10 14 ) DNA or RNA libraries.
  • Ligands could be isolated by several different screening methodologies (US patents 5,270,163; 5,475,096; 5,567,588; 5,595,877; and 5 , 637 , 459 ) . For example, the starting libraries for a DNA library would have defined sequences on each end of 10 to 30 bases flanking a random core of 10 to 100 bases.
  • Primers complementary to the defined sequences on each end would be used to amplify the library and one would have a tag (such as biotin) .
  • the double stranded DNA would be bound to a matrix (streptavidin agarose) and denatured to release ssDNA.
  • the target protein would be incubated with a starting library of single stranded DNA (ssDNA) and the aptamers allowed to bind.
  • Protein : aptamer complexes would then be collected by filtration through nitrocellulose or nylon membranes which will bind protein with a very high capacity but have a low affinity for ssDNA.
  • the unbound aptamers would be washed away through the filter with an excess of buffer leaving only the aptamers which bound to the original target protein.
  • These aptamers would be eluted by one of several methods (pH shock, phenol extraction, SDS treatment or heat) , precipitated with ethanol and then amplified by PCR to synthesize a new pool for use in the next round of selection.
  • This process would be repeated from once to 20 times. The number of times this would be carried out is determined by monitoring the enrichment for binders after each round or after every other round of selection. This could be accomplished in several ways. The most often used approach is to radioactively label a small percentage of the library and monitor the fraction of the library retained on the filter after each round. An alternative method is to use a primer in the amplification reaction which would allow the aptamer to be detected. Two examples of this are rhodamine and digoxigenin. Rhodamine is detected directly by fluorescence and DIG is detected by an antibody which is either directly or indirectly coupled to an enzymatic or fluorescence readout.
  • a labeled primer would allow the detection of aptamer binding to target in a standard ELISA format in which the target protein is immobilized in the well of a plate, the aptamer is added and allowed to bind and is then detected using one of the methods mentioned above.
  • the final pool would be amplified and cloned into a plasmid which allows for the rapid sequencing of the inserts. This could be done by using restriction sites in the primers which are compatible with those in the vector, however it would be preferable to take advantage of the additional "A" residue added by many thermostable polymerases to clone the products into a "T" tagged vector. This is desirable because of the possibility of the aptamer containing the restriction site used for cloning, which would result in the loss of all or a portion of the aptamer upon cloning. Individual surrogate ligand aptamers would be prepared by amplification from plasmid DNA using tagged primers.
  • the resulting ligands would be tested for binding to the target protein as well as against several other unrelated proteins as controls for specificity.
  • DNA from clones that bind specifically to the target protein and give strong signals will be prepared for automated DNA sequencing.
  • the sequences will then be aligned and searched for regions of homology.
  • the regions of linear sequence homology are likely to be representations of secondary and tertiary structures which are required for the specific interaction of the aptamer with the target .
  • the ligand could be labeled with rhodamine or DIG as described above. Alternatively they could be labeled as described by Pitner (US Patent 5,650,275 and US Patent 5,641,629) . Screens for drug leads can be carried out with the nucleic acid surrogate ligands in the manner illustrated in the above examples.
  • Example 8 Peptides which bind to Agrobacterium faecaelis ⁇ - glucosidase, carboxypeptidase, alcohol dehydogenase, and E. coli Pro RS.
  • Affinity selections were carried out as in example 3 except that the protein was presented in several ways. At first the protein was immobilized on Immulon 4 plates (Dynex) as in previous examples, however, repeated attempts failed to isolate phage which bound specifically to the target. It was found that the target exhibited greatly reduced activity when bound to Immulon 4 plates, making it likely that it was denatured when bound to plastic. To circumvent this problem, two approaches were used, both of which utilized biotinylated protein. Protein
  • Biotinylated proteins were separated from the excess biotinylation reagent on a Sephadex G-50 micro-spin column
  • phage libraries were mixed with 1 ⁇ g biotinylated target protein in solution and allowed to incubate at room temperature for 4 hours.
  • Phage protein complexes were then captured on streptavidin paramagnetic beads (Promega, cat# Z5482) by adding beads and incubating the tubes while rotating at room temperature for 30 minutes. The complexes were then drawn to the sides of the tubes using a magnet and the beads were washed with TBST containing 5 mM biotin. Beads were washed once in the first round of panning and 3 times for each additional round. Phage were eluted and amplified using the sequential procedure described in example 3.
  • phage libraries were mixed with 1 ⁇ g biotinylated target protein that was first captured on streptavidin paramagnetic beads. After the protein was bound, the beads were washed once with TBST containing 5 mM biotin to block the remaining binding sites on streptavidin. The libraries and beads were then mixed at room temperature on a rotator for 4 hours and washed, eluted and amplified as above. Phage ELISAs were carried out by first coating a microtiter plate (Immulon4, Dynex) with 1 ⁇ g streptavidin, followed by blocking with BSA. Biotinylated protein was then added'to the wells and allowed to bind to the streptavidin for 1 hour. The plates were washed with TBST and used for phage ELISAs.
  • peptides fall into two clusters based on homology. Different methods of immobilizing the protein produced similar results: peptides from both classes were isolated from solution or bead displayed target and identical peptides were isolated from the two methods. Peptides in the second cluster were isolated predominantly from the W and P fixed residue libraries, a finding which is in concordance with the PWP motif in the conserved sequence. Phage presenting peptides in the first group came from the N, K, and R libraries, again consistant with the consensus sequence in this cluster. The only conserved residues in this motif for which a fixed residue library was screened were D and W. In both cases, the consensus sequence extends for greater than 5 residues to the carboxy terminal side making it impossible for these libraries to encode peptides which would bind to the target .
  • Similar affinity selection procedures could be carried out using glycogen phosphorylase a and yeast hexokinase .
  • enzyme activity could be monitored in the absence and presence of phage displaying peptides or using synthetic peptides to determine if they are effective inhibitors.
  • competitions between the phage/synthetic peptides and inhibitors or substrates could be carried out to determine if the surrogate ligands identified are binding at the active site of the target.
  • These surrogate ligands could be used to format a competitive binding assay used to search for small molecular weight inhibitors of each of these targets as described in the above examples. From the data presented here it is clear that some proteins are very sensitive to inactivation upon binding to plastic.
  • streptavidin coated microtiter plate or beads in conjuction with biotinylated target protein provides one alternative method for the presentation of the target protein.
  • Other methods of target presentation such as using an antibody to an epitope tag or a ligand: fusion protein combination, as well as others may be advantageous in the selection of phage and in the screen for small molecule inhibitors.
  • Example 9 (Hypothetical) Identification of Inhibitors of the B7:CD28 Complex.
  • B7 is a potent adjuvant for the priming of T cell in vi tro, and more importantly, in vivo .
  • CD28 is a relatively high affinity receptor for B7. Engagement of CD28 on the surface of a T cell results in increased activity in a cREL dependent pathway. Blocking B7/CD28 interaction with antibody to B7 inhibits signal transduction, and results in a failure to efficiently prime T cells. Clearly compounds which block this interaction would have potent immunologic properties.
  • the proteins will be purified by simple affinity chromatography with glutathione-agarose (Pharmacia) . If we have difficulty producing the external domain as a GST fusion, we will clone it into an Ig fusion construct as has been successfully done with CTLA4-Ig and CD28-Ig [Peach, 1995 #9] . We will produce this fusion construct in mammalian cells and purify it from supernatants using protein A disks.
  • Phage-displayed random peptide libraries within a manner similar to that described in Example 1, will be screened for binding to immobilized GST-CD 28 fusion proteins using affinity selection techniques.
  • the binding of individual isolates to the GST-CD28 fusion proteins will be evaluated by a simple enzyme linked immunosorbent assay (ELISA) with a goat anti-phage antibody conjugated to horseradish peroxidase (Pharmacia) .
  • ELISA enzyme linked immunosorbent assay
  • Pharmacia horseradish peroxidase
  • Consensus sequences of CD28 binding peptides will be determined in a manner similar to that described for UL44 in Example 1. By comparisons of sequences of the phage from the apparent "preferred" library with those from the other libraries we should be able to obtain a definition for an optimal CD28 binding peptide ligand. These consensus sequences are anticipated to be useful in computer searches such as those using the PROSITE system and the SWISS Protein database to identify novel CD28 ligands. We anticipate that some of the
  • CD28 binding peptides may have similarity to the CD28 binding domains within B7.1 and B7.2. Based on published observations
  • dendritic cells or EBV transformed B cells
  • IL-2R IL-2R
  • 2R up regulation is an early event in T cell activation. It may provide a more rapid and sensitive readout than proliferation .
  • PBMCs peripheral blood monocytes
  • stimulator cells in the presence of a varying numbers of stimulator cells.
  • dendritic cell stimulators from an MHC mismatched individual by growing dendritic cells from PBMC in IL-4 and GMCSF [Romani, 1994 #119; Xu, 1995 #122] .
  • Such cultures are markedly enriched in DC and are potent MLC stimulators.
  • Cultures will be tested for the number of Class II + , B7 + cells by flow cytometry. Peptide will be added at increasing concentrations to the cultures, and the response measured. Negative controls will consist of irrelevant peptides of similar size and amino acid composition.
  • the combination of PMA plus anti-CD28 treatment will serve as a positive control and indicate the maximal T cell response. All data points will be collected in triplicate, and data will be analyzed using standard statistical methods. We expect that antagonist peptides will result in a shift in the dose response curve towards higher stimulator cell concentrations for an equivalent T cell response. Peptides with agonist activity would be expected to shift the dose response to lower numbers of stimulator cells. We anticipate that these peptides may function either as agonist, activating CD28, or antagonists, blocking CD28 dependent responses. Any peptides that fail to show blocking activity will be tested for agonist activity using suboptimal doses of anti-CD3 to stimulate T cells. Those peptides which can act as agonists will showed enhanced responses. Anti CD28 antibody will be a control for these responses .
  • Programmed cell death also known as apoptosis, is both a normal and abnormal cellular process.
  • normal cell death that occur during embryo development, such as formation of digits in limbs, destruction of larval muscles during metamorphosis of the moth, sculpting of the mouth, removal of misconnected neurons, and removal of thymocytes that recognize self antigens.
  • Defects in the control of cell death have also been described as the basis of certain forms of autoimmunity, escape of cancer cells from growth control, and immortalization of virally infected cells, to name a few.
  • the molecular basis of apoptosis is considered by many scientists to be critical in understanding normal and abnormal growth control.
  • the molecules involved in cell death can be defined, they will serve as valuable targets with which to develop therapeutic drugs that may be used to block or induce apoptosis. These drugs may have uses in treating certain forms of autoimmune disease, fighting bacterial and viral infection, promoting survival of damaged neurons, and battling cancer.
  • TNF tumor necrosis factor
  • NGF nerve growth factor
  • Fas receptor also known as APO-1 and CD95
  • Rb a variety of immune cell receptors
  • Rb a variety of immune cell receptors
  • Rb a variety of nematode neuronal guidance protein
  • THe extracellular domains of the family all share from three to six cysteine-rich domains.
  • DD death domain
  • NGF-R LNGSAGDTWRHLAGELGYQPEHIDSFTHE ACPVRALLA
  • TNF and Fas receptors their DD's have been shown to be critical in activating cell death.
  • TNF and Fas ligand their DD's have been shown to be critical in activating cell death.
  • cDNA clones encoding either receptor are introduced into various cell lines, these cells will undergo apoptosis in the presence of TNF or the Fas ligand (Nagata and Golstein, 1995) .
  • the transfected receptors have mutations in their DD's, apoptosis fails to occur in the presence of the extracellular signal (Tartaglia et al . , 1993) . Truncations of as little as 10 amino acids from the N-terminus or three from the C-terminus of the domain are inactivating.
  • DED death effector domain
  • GST glutathione S-transferase
  • GST fusions to mutant DD's will be useful as negative controls in experiments examining the specificity of peptide-DD interactions (i.e., the peptide ligands should bind poorly, or not at all, to the inactive DD's) .
  • the mutations will be engineered by replacing a pair of oligonucleotides during the assembly of the DD's for cloning (above) .
  • the libraries will be screened according to standard techniques (Kay et al . , 1993; Adey and Kay, 1996.
  • several micrograms of GST-DD fusion protein will be immobilized in ELISA style microtiter plates. After non-specific protein binding are blocked with excess protein (i.e., BSA, Pierce Chemical SuperBlock) , approximately 10 11 phage are added to each well. After several hours incubation at 4°C, the liquid is discarded from the wells with 200 mM glycine (pH2) which denatures the protein-phage complex. Bacteria are infected with the released phage after the pH is neutralized and cultured overnight.
  • BSA Pierce Chemical SuperBlock
  • the infected cells release phage, ⁇ 1000 per minute per bacterium, so that the titer of the final culture is 10 12 plaque forming units per ml. This constitutes one round of screening. The process is repeated three times in series, and the resulting phage are grown as isolates.
  • DDs are ⁇ 31 % identical in amino acid sequence. Definition of an optimal DD peptide ligand will be useful in computer searches (http : //expasy .hcuge . ch/sprot/scnpsit2.html) of possible cellular ligands.
  • dissociation constants will be quantitated on a BIAcore system, see Karlsson, Anal. Biochem., 228:274-280 (1995) and Raghavan, Structure, 3:331-3 (1995).
  • peptides isolated from phage-displayed libraries are anticipated to have lO ⁇ M to lOnM dissociation constants. If desired to determine the importance of individual residues of the peptide ligands, and thereby faulitate the design of more efficient libraries for the target in question, we may prepare a set of alanine-scanned variants and measure their affinities.
  • the synthetic peptides will be used in competition experiments as well. Soluble peptides will be added to radioactive ( 3S S-methione labelled) cell lysates incubated with various GST-DD fusion proteins. A similar experiment was performed with SrcSH3 peptide ligands to demonstrate that they bound in the same way as natural ligands (Sparks et al . , 1994) . If the peptides bind to the DD, little or no cellular protein should bind to the GST-DD fusion protein when it is recovered by chromatography over glutathione-agarose, resolved by SDS- PAGE, and autoradiographed . Non-binding peptides will serve as negative controls.
  • the WW domain is newly described protein motif of 38 amino acids, typified by two conserved tryptophan residues (Sudol et al . , 1995), which binds proline-rich (Chen and Sudol, 1995) .
  • COLT is the preferred way of identifying other DD domain containing proteins in the human or other genomes .
  • Peptides corresponding to DD ligands will be synthesized with biotin, complexed to streptavidin-linked alkaline phosphatase, and used to screen ⁇ human cDNA libraries. Such as T cell (Jurkit) and HeLa cDNA libraries.
  • Lambda plaques expressing DD-containing proteins should form blue plaques on nitrocellulose filters when exposed to the peptide complex and NBT and BCIP.
  • the cDNA inserts of such plaques will be rescued (Short et al . , 1988) and their nucleotide sequences determined by dideoxy sequencing.
  • a high throughput screen will be used to screen combinatorial libraries of benzodiazepines, peptoids, and other small chemicals.
  • Such a screen will be based on fusing the peptide ligands identified as described above to the enzyme alkaline phosphatase (AP) and then looking for compounds that inhibit binding of the peptide ligand-AP fusion to GST-DD protein immobilized in microtiter plate wells.
  • Another screen will be to fuse the DD directly to the AP and then staining beads that have the combinatorial compounds attached.
  • Adey, N. B., and Kay, B. K. (1996). Identification of calmodulin-binding peptide consensus sequences from a phage- displayed random peptide library. Gene 169, 133-134.
  • Nuclear receptors are proteins that bind hormone ligands causing them to bind to DNA and activate the expression of a subset of genes. Examples of nuclear receptors are estrogen receptor, retinoic acid receptor and glucocorticoid receptor. These proteins are of important pharmacological interest due to their impact on the control of cellular proliferation. For example, there are tumors whose continued growth depends upon estrogen being present.
  • nuclear receptors Due to there function, nuclear receptors have at least two biologically relevant sites: the hormone binding pocket and the DNA binding site. In addition, many of the receptors form homo- and heterodimers providing a third protein :protein interaction site. In each case, the binding sites are very specific: the hormone binding pocket will bind only the appropriate hormone, the DNA binding site will interact with a specific sequence element and only certain proteins will interact to form dimers .
  • surrogate ligands can be isolated to each of these domains and the resulting surrogate ligands can be used in a screen to discover small molecule agonists/antagonists to the receptors activity.
  • Estrogen receptor was purchased from Panvera Corporation and was immobilized directly on Immulon4 plates (Tynatech) . Affinity selection for phage binding to the receptor was carried out as described in example using TyrRS. A number of libraries showed enrichment using a pool phage ELISA. Interestingly, the X5LX5 library was sensitive to competition with estradiol . Individual clones were tested from each of the enriched libraries for specific binding to estrogen receptor as well as sensitivity to competition with estradiol. A number of specific binding phage were isolated and the binding of a subset of these was sensitive to estradiol.
  • a peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding sited on integrins . J. Cell Biology 130:1189-1196.
  • Bottger V., A. Bottger, S.F. Howard, S.M. Picksley, P. Chene, C. Garcia-Echeverria, H-K. Hochkeppel and D.P. Lane.. 1996. Identification of novel mdm2 binding peptides by phage display. Oncogene 13:2141-2147.

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Abstract

L'invention concerne l'identification de composés dans un bibliothèque de composés, qui peuvent être à l'origine de l'activité biologique d'une protéine réceptrice cible même lorsque les ligands à l'origine de l'activité par liaison à ce récepteur ne sont pas encore connus. Le procédé consiste: (1) à cribler au moins une bibliothèque combinatoire de substituts potentiels pour des éléments (de préférence des peptides ou des acides nucléiques) se liant à la protéine cible (PT) et donc pouvant être utilisés pour le ligand inconnu des étapes (2) et (3); (2) à cribler au moins une bibliothèque complémentaire, de préférence une bibliothèque combinatoire, (qui peut comporter, entre autres, des peptides ou des acides nucléiques et donc appelée parfois 'bibliothèque de composés') pour des composés qui inhibent la liaison d'un ou plusieurs substituts de l'étape (1) de PT, et éventuellement (3) à déterminer si le composé inhibiteur est à l'origine ou non de l'activité biologique de ladite PT.
PCT/US1997/019638 1996-10-31 1997-10-31 Identification de medicaments au moyen de bibliotheques combinatoires complementaires WO1998019162A1 (fr)

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EP97948137A EP0937253A1 (fr) 1996-10-31 1997-10-31 Identification de medicaments au moyen de bibliotheques combinatoires complementaires
CA002270394A CA2270394A1 (fr) 1996-10-31 1997-10-31 Identification de medicaments au moyen de bibliotheques combinatoires complementaires
JP52072798A JP2001510453A (ja) 1996-10-31 1997-10-31 相補的組み合わせライブラリーを使用する薬剤の同定
AU54268/98A AU744444B2 (en) 1996-10-31 1997-10-31 Identification of drugs using complementary combinatorial libraries
US09/069,827 US6617114B1 (en) 1996-10-31 1998-04-30 Identification of drug complementary combinatorial libraries
US10/656,250 US20050069951A1 (en) 1996-10-31 2003-09-08 Identification of drugs using complementary combinatorial libraries

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WO1999058561A1 (fr) * 1998-05-14 1999-11-18 Pasteur Merieux Serums & Vaccins Mimotopes du virus de l'hepatite c
EP0959136A1 (fr) * 1998-05-20 1999-11-24 Introgene B.V. Délivrance ciblée via un transporteur d' acides aminés cationiques
EP0960942A2 (fr) * 1998-05-20 1999-12-01 Introgene B.V. Délivrance ciblée via un transporteur d'acide aminé
WO1999063944A3 (fr) * 1998-06-08 2000-02-10 Advanced Medicine Inc Nouveaux agents therapeutiques modulant les recepteurs des oestrogenes
WO2000039585A1 (fr) * 1998-12-28 2000-07-06 Sunesis Pharmaceuticals, Inc. Identification de ligands de type petites molecules organiques, destines a former des liaisons
US6344443B1 (en) * 1998-07-08 2002-02-05 University Of South Florida Peptide antagonists of tumor necrosis factor alpha
WO2002017948A3 (fr) * 2000-08-30 2002-06-13 Syngenta Mogen Bv Lutte contre les ravageurs des cultures et les parasites animaux au moyen d'un compose a absorption neuronale directe
US6586190B2 (en) 2000-08-18 2003-07-01 Syngenta Participations Ag Parallel high throughput method and kit
US6589738B1 (en) 1999-11-09 2003-07-08 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
WO2003059943A2 (fr) * 2002-01-18 2003-07-24 Karo Bio Ab Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes
WO2002004956A3 (fr) * 2000-07-12 2003-09-12 Karo Bio Usa Inc Procede d'identification de peptides de liaison sensibles a la conformation et utilisations correspondantes
WO2002014858A3 (fr) * 2000-08-17 2003-10-30 Nanogen Recognomics Gmbh Procede de production et d'identification d'effecteurs appropries de molecules cibles a l'aide de bibliotheques de substances
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
WO2004092199A2 (fr) * 2003-04-11 2004-10-28 Cue Biotech, Inc. Procede d'identification de modulateurs de la signalisation de recepteur couple a une proteine g
EP1572863A2 (fr) * 2001-03-14 2005-09-14 Cue Biotech Procede d'identification d'inhibiteurs de signalisation du recepteur complexe a la proteine g
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US8575070B2 (en) 2006-02-20 2013-11-05 Phylogica Limited Methods of constructing and screening libraries of peptide structures
WO2014033136A1 (fr) 2012-08-27 2014-03-06 Cemm - Research Center For Molecular Medicine Of The Austrian Academy Of Sciences Composés aminohétéroaryles en tant qu'inhibiteurs de mth1
WO2019241435A1 (fr) * 2018-06-12 2019-12-19 The J. David Gladstone Institutes Compositions pour interruption de circuit de rétroaction transcriptionnelle de virus de l'herpès et utilisations correspondantes
CN114076826A (zh) * 2020-08-20 2022-02-22 中国科学院大连化学物理研究所 一种微球辅助的基于蛋白加热沉淀的药物靶蛋白筛选方法

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EP1125905A1 (fr) * 2000-02-16 2001-08-22 Pepscan Systems B.V. Synthèse de segments
GB0916749D0 (en) * 2009-09-23 2009-11-04 Mologic Ltd Peptide cleaning agents

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WO1997029372A1 (fr) * 1996-02-07 1997-08-14 Conjuchem, Inc. Bibliotheques de marquage par affinite et leurs applications
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Cited By (29)

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Publication number Priority date Publication date Assignee Title
WO1999058561A1 (fr) * 1998-05-14 1999-11-18 Pasteur Merieux Serums & Vaccins Mimotopes du virus de l'hepatite c
EP0959136A1 (fr) * 1998-05-20 1999-11-24 Introgene B.V. Délivrance ciblée via un transporteur d' acides aminés cationiques
EP0960942A2 (fr) * 1998-05-20 1999-12-01 Introgene B.V. Délivrance ciblée via un transporteur d'acide aminé
WO1999060147A3 (fr) * 1998-05-20 1999-12-29 Introgene Bv Administration ciblee au moyen d'un transporteur d'acide amine cationique
EP0960942A3 (fr) * 1998-05-20 2000-01-12 Introgene B.V. Délivrance ciblée via un transporteur d'acide aminé
WO1999063944A3 (fr) * 1998-06-08 2000-02-10 Advanced Medicine Inc Nouveaux agents therapeutiques modulant les recepteurs des oestrogenes
US6344443B1 (en) * 1998-07-08 2002-02-05 University Of South Florida Peptide antagonists of tumor necrosis factor alpha
WO2000039585A1 (fr) * 1998-12-28 2000-07-06 Sunesis Pharmaceuticals, Inc. Identification de ligands de type petites molecules organiques, destines a former des liaisons
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
US6589738B1 (en) 1999-11-09 2003-07-08 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
WO2002004956A3 (fr) * 2000-07-12 2003-09-12 Karo Bio Usa Inc Procede d'identification de peptides de liaison sensibles a la conformation et utilisations correspondantes
WO2002014858A3 (fr) * 2000-08-17 2003-10-30 Nanogen Recognomics Gmbh Procede de production et d'identification d'effecteurs appropries de molecules cibles a l'aide de bibliotheques de substances
US6586190B2 (en) 2000-08-18 2003-07-01 Syngenta Participations Ag Parallel high throughput method and kit
WO2002017948A3 (fr) * 2000-08-30 2002-06-13 Syngenta Mogen Bv Lutte contre les ravageurs des cultures et les parasites animaux au moyen d'un compose a absorption neuronale directe
EP1572863A4 (fr) * 2001-03-14 2009-06-24 Cue Biotech Inc Procede d'identification d'inhibiteurs de signalisation du recepteur complexe a la proteine g
US7208279B2 (en) 2001-03-14 2007-04-24 Caden Biosciences, Inc. Method for identifying inhibitors of G protein coupled receptor signaling
US7294472B2 (en) 2001-03-14 2007-11-13 Caden Biosciences Method for identifying modulators of G protein coupled receptor signaling
EP1572863A2 (fr) * 2001-03-14 2005-09-14 Cue Biotech Procede d'identification d'inhibiteurs de signalisation du recepteur complexe a la proteine g
WO2003059943A2 (fr) * 2002-01-18 2003-07-24 Karo Bio Ab Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes
WO2003059943A3 (fr) * 2002-01-18 2004-03-11 Karobio Ab Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes
WO2004092199A2 (fr) * 2003-04-11 2004-10-28 Cue Biotech, Inc. Procede d'identification de modulateurs de la signalisation de recepteur couple a une proteine g
WO2004092199A3 (fr) * 2003-04-11 2009-04-30 Cue Biotech Inc Procede d'identification de modulateurs de la signalisation de recepteur couple a une proteine g
US8575070B2 (en) 2006-02-20 2013-11-05 Phylogica Limited Methods of constructing and screening libraries of peptide structures
US9567373B2 (en) 2006-02-20 2017-02-14 Phylogica Limited Methods of constructing and screening libraries of peptide structures
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
WO2014033136A1 (fr) 2012-08-27 2014-03-06 Cemm - Research Center For Molecular Medicine Of The Austrian Academy Of Sciences Composés aminohétéroaryles en tant qu'inhibiteurs de mth1
WO2019241435A1 (fr) * 2018-06-12 2019-12-19 The J. David Gladstone Institutes Compositions pour interruption de circuit de rétroaction transcriptionnelle de virus de l'herpès et utilisations correspondantes
CN114076826A (zh) * 2020-08-20 2022-02-22 中国科学院大连化学物理研究所 一种微球辅助的基于蛋白加热沉淀的药物靶蛋白筛选方法

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AU5426898A (en) 1998-05-22
EP0937253A1 (fr) 1999-08-25
AU744444B2 (en) 2002-02-21
CA2270394A1 (fr) 1998-05-07

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