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WO1999003877A1 - Procedes pour le depliement des proteines au moyen de complexes metalliques - Google Patents

Procedes pour le depliement des proteines au moyen de complexes metalliques Download PDF

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Publication number
WO1999003877A1
WO1999003877A1 PCT/US1998/014630 US9814630W WO9903877A1 WO 1999003877 A1 WO1999003877 A1 WO 1999003877A1 US 9814630 W US9814630 W US 9814630W WO 9903877 A1 WO9903877 A1 WO 9903877A1
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WIPO (PCT)
Prior art keywords
protein
unfolding
preferred
proteins
metmb
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PCT/US1998/014630
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English (en)
Inventor
Ofer Blum
Abed Haiek
Dory Cwikel
Zvi Dori
Thomas J. Meade
Harry B. Gray
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California Institute Of Technology
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Application filed by California Institute Of Technology filed Critical California Institute Of Technology
Priority to EP98934562A priority Critical patent/EP1027369A4/fr
Priority to AU84052/98A priority patent/AU8405298A/en
Priority to CA002297362A priority patent/CA2297362A1/fr
Publication of WO1999003877A1 publication Critical patent/WO1999003877A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents

Definitions

  • the invention relates to methods of unfolding proteins using metal complexes, particularly cobalt-containing Schiff s base compounds.
  • Partially folded proteins are polypeptides with substantial secondary structure in a largely disordered tertiary structure (Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Roder, H. & Colon, W. (1997) Curr. Opin. Struct. Biol. 7, 15-28; P. L. Privalov (1996) J. Mol. Biol. 258, 707-725; Ptitsyn, O. B. (1996) Nat. Struct. Biol. 3, 488-490.). They are often referred to as molten globules ( Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Ptitsyn, O.
  • the Co(III)-myoglobin complex is the first example of an isolable partially folded species obtained from a naturally folded precursor.
  • Suitable denaturing media are basically limited (urea, guanidinium-HCl, acids, salts, organic solvents and detergents), and a large excess of denaturant is needed, resulting in nonphysiological and therefore biologically questionable conditions.
  • the denaturation is generally reversible, as the conditions are chosen to prevent irreversible aggregation of the unfolding proteins.
  • the present invention provides methods of unfolding a protein comprising contacting the protein with a reactive metal complex, wherein said protein exhibits a 5% or greater change in at least one minima or maxima of a circular dichroism (CD) spectra of the protein as a result of binding at least one metal complex to the protein.
  • Preferred embodiments utilize cobalt-containing tetradentate Schiff s base compounds.
  • Figure 1 UV/vis absorption spectral changes during the incubation of metMb and 1 in 0.01 M sodium phosphate, at pH 6.5 and 25 °C, with 15 min intervals between measurements.
  • Figures 3A and 3B (A) CD at 222 nm after incubating apoMb (7.5 ⁇ M) with 1 and 2. (B) Far-UV CD after incubating metMb and apoMb with 11 equivalents of 1. Conditions as given in the Fig. 2 legend.
  • the present invention is directed to the discovery that the addition of metal complexes, particularly Schiff s base chelates of cobalt, to proteins, can result in their binding through the metal of functional moieties of certain accessible amino acids in a protein. This can result in the unfolding of the protein due to the complexing of the functional moiety to the metal complexes, resulting in replacement of some of the protein structure stabilizing interactions by bonding to the metal.
  • metal complexes particularly Schiff s base chelates of cobalt
  • the metal complex compounds outlined herein derive their biological activity by the substitution or addition of ligands to the metal complexes.
  • the biological activity of the complexes results from the binding of a new ligand, generally (but not always) in an axial position, most preferably a nitrogen of the side chain of histidine, although as outlined below, other amino acids may be involved.
  • the amino acid serving as the new ligand of the metal complex is required by the target protein for its structural integrity. This can be due to the involvement of the histidine in structurally important hydrogen bonding, for example, or, as a result of the "bringing together" of two histidines as axial ligands in one metal complex, thus perturbing the structure of the protein.
  • the addition of the metal complexes depicted herein are added to a protein or enzyme, for example, and one or more of the original ligands are replaced by one or more ligands from the protein.
  • This will occur either when the affinity of the protein axial ligand is higher for the metal complex as compared to the original ligand, or when the new axial ligand is present in elevated concentrations such that the equilibrium of ligand binding favors the binding of the new ligand from the protein.
  • This latter possibility may be encouraged by the use of a targeting moiety, as is more fully described below, which increases the presence of the metal complex at or near the surface of the target protein.
  • the nitrogen atom of an imidazole side chain of the amino acid residue histidine, contained within a target protein, is the new axial ligand. While the examples and disclosure herein particularly describe this histidine embodiment, any "reactive amino acid” may serve as the new ligand.
  • a "reactive amino acid” is one which is capable of binding to the metal compounds of the invention as a new ligand.
  • nitrogen of the imidazole side chain of histidine is particularly preferred
  • alternative embodiments utilize the nitrogen atom of the aromatic indole side chain of tryptophan, the sulfur atoms of the side chains of cysteine and methionine, nitrogens of the side chains of arginine, lysine, asparagine or glutamine, the oxygen atoms of the side chains of aspartic and glutamic acids and of tyrosine, glutamine, asparagine, serine and threonine, and potentially the ⁇ bonds of the aromatic residues of phenylalanine, tyrosine and tryptophan, as well as the protein backbone carbonyl and amino groups, as the moieties which may become axial ligands as outlined above.
  • moieties may depend on the pH of the solution containing the protein or enzyme, since in the protonated state, many of these moieties are not good electron donors suitable as ligands. Thus, for example, non-physiological pHs may be used in protein production schemes. Under normal physiological conditions, this binding is effectively irreversible, although, as outlined below, the use of relatively high concentrations of certain reagents can remove the metal complexes from the proteins.
  • the present invention provides methods of unfolding a target protein comprising contacting the protein with a reactive metal complex.
  • unfolding herein is meant that a protein loses at least a portion of its tertiary structure and/or secondary structure.
  • Unfolding in this context can also refer to a conformational change of the protein leading to an alteration in the biological activity. Unfolding can be complete (i.e. an effective denaturation to a generally linear chain of amino acids), or partial.
  • Partial unfolding includes, but is not limited to, a complete loss of tertiary structure with the retention of at least some secondary structure, a partial loss of tertiary structure with no loss of secondary structure, and a partial loss of tertiary structure and a partial loss of secondary structure.
  • increases in secondary structure, or non-native structures may be seen.
  • the unfolding can be measured in a number of ways, as will be appreciated by those in the art. Preferred methods include, but are not limited to, changes in absorption spectra, changes in circular dichroism (CD) spectra, changes in fluorescence, changes in nuclear magnetic resonance (NMR) spectra, and changes in small angle X-ray scattering (SAXS) spectra.
  • unfolding is measured as a change in circular dichroism (CD) spectra, and serves to give a measure of the relative quantities of the secondary structure components of a protein.
  • CD circular dichroism
  • absorption spectra as is known in the art, native proteins have a characteristic CD spectra, with small changes unique to each particular protein. The shape of the spectra curve, as well as the maxima and minima, provide information about the protein.
  • peaks present in the 200 to 250 nm wavelength (“far UV”) range are generally a "w” shaped spectra with troughs around 222 and 208 being indicative of the presence of ⁇ -helical structures, and a "v” shaped spectra with a trough around 217-220 nm being indicative of ⁇ -sheet structures.
  • Scans in the "near UV” range i.e. 250 - 300 nm, give information about tertiary structure.
  • Other parts of the spectrum, as around 410 nm for heme proteins, may yield structural information as well. See description in Freifelder "Physical Biochemistry", 2nd Ed., Freeman, NY 1982, Ch. 16 pp 573-602 for UV CD; and Strickland CRC Critical Reviews in Biochemistry 1974 2:113- 175. For use in protein folding studies see Kelly et al., Biophys. Biochem. Acta 1997 133, 161-185.
  • the sample is prepared as is known in the art.
  • a far UV CD the protein solution is placed in a cuvette and the spectrum is taken.
  • a typical concentration can be 10 ⁇ M, in a 1 mm cuvette or 1 ⁇ M in a 10 mm cuvette. The concentration needed depends on the light path length through the sample, the protein size and on the solvent properties.
  • a far UV CD is taken similarly, with about a 10 fold higher concentration or pathlength.
  • a spectrum of the buffer alone should be subtracted to obtain the final spectrum.
  • a CD spectra is generally obtained prior to the addition of the metal complexes used in the invention, the complexes are added, and a second spectra is obtained.
  • unfolding is present when changes of at least about 5% of at least one maxima or minima of the spectra are observed, with changes of at least about 10% being preferred, and at least about 25-50 % being particularly preferred, and larger changes also possible.
  • changes in this context includes both increases and decreases.
  • changes in more than one maxima or minima are preferred.
  • unfolding may be measured as a result in changes in the shape of a CD curve.
  • shape of a CD spectra can indicate the percentages of each type of secondary structure: -helix, antiparallel ⁇ -sheet, and ⁇ -turn. Changes in the shape of the curve, therefore, can show unfolding to a protein containing altered structures and random coils.
  • unfolding may be measured as a result of changes in fluorescence.
  • fluorescence-based assays There are generally two different types of fluorescence-based assays to measure unfolding.
  • proteins are specifically altered, generally covalently, with two different fluorochromes (i.e. a fluorescence emitting donor and absorber) to allow fluorescence resonance energy transfer (FRET) as is known in the art (see Freifelder, supra, Ch. 15, pp537-572). That is, the emission spectra of the first fluorochrome overlaps the excitation spectra of the second fluorochrome.
  • fluorochromes i.e. a fluorescence emitting donor and absorber
  • FRET fluorescence resonance energy transfer
  • exciting the first fluorochrome results in a much attenuated fluorescence signal.
  • This may be used to detect unfolding as generally the unfolding of the protein results in a distance increase between any two particular residues; thus, generally, unfolding results in less fluorescence quenching due to the greater distance of the two fluorochromes.
  • unfolding may be monitored by following the fluorescent signal.
  • monitoring of unfolding is done by first measuring the fluorescence of the protein in the absence of the metal complex, and then the metal complex is added and the experiment is repeated.
  • a change of at least about 5% of the fluorescence signal is preferred, with at least about 10% being particularly preferred, and at least about 25-50% being especially preferred.
  • the deficiency of the fluorescence quenching is strongly dependent on the overlap of the emission and absorption spectra of the fluorochromes, and on the distances; thus, a much improved "picture" of unfolding is obtained if the donor and acceptor are moved around the protein and different effects are observed.
  • the proteins are not modified with fluorescence labels as above, but rather external fluorescent dyes are used whose fluorescence depends on their environment. That is, the dye is substantially non-fluorescent in a certain environment (i.e. a polar environment), but upon a change in medium (i.e. association of the dye with hydrophobic areas of the protein), the dye exhibits a change in fluorescence intensity. Accordingly, upon the unfolding of a protein, hydrophobic areas of the protein are exposed, allowing an increase in fluorescence to be detected.
  • Suitable dyes include, but are not limited to, 8-anilino-l-naphthalenesulphonate (ANS). See for example Englehard et al., Protein Sci.
  • fluorescence monitoring may not be possible with all metal complexes, as some serve as fluorescent quenchers.
  • unfolding is measured as a change in UV absorption spectra.
  • Some native proteins for example, heme containing proteins
  • a characteristic absorption spectra that contains at least one maxima ( ⁇ max ), generally characteristic of the protein.
  • ⁇ max a characteristic absorption spectra that contains at least one maxima
  • reactive metal complex herein is meant a metal complex that is capable of binding to a reactive amino acid, as defined above, and causing unfolding of the protein containing the reactive amino acid.
  • the metal complexes of the invention comprise a metal ion and chelator, as are more fully described below. Without being bound by theory, it appears that the metal complexes useful in the invention have several important characteristics.
  • the metal complexes used in the invention may only bind one side chain, if hydrogen bonds of the side chain are structurally important.
  • the metal complexes of the invention are preferably small, and in some cases may be relatively hydrophobic, enough to at least partially penetrate into the protein. Without being bound by theory, it appears that the metal complexes penetrate into the hydrophobic interior of a protein to disrupt at least one structurally important residue; once the first unfolding event has occurred, others can then follow, as additional residues become exposed to the solvent containing the metal complex.
  • preferred chelators that utilize R substitution groups generally utilize small and/or hydrophobic groups.
  • the chelators may exhibit regiospecific hydrophilicity/hydrophobicity; that is, R groups on one "side" of the chelate may be hydrophobic, and the other "side” may comprise one or more hydrophilic residues (for example, in Structure 1, R 7 , R 8 , RQ and R ⁇ are hydrophobic, and at least one of R perpetrat R 2 , R 3 and R !0 is hydrophilic, although other combinations resulting in amphiphathic characteristics are also possible, as will be appreciated by those in the art).
  • R groups on one "side” of the chelate may be hydrophobic
  • the other "side” may comprise one or more hydrophilic residues (for example, in Structure 1, R 7 , R 8 , RQ and R ⁇ are hydrophobic, and at least one of R perpetrat R 2 , R 3 and R !0 is hydrophilic, although other combinations resulting in amphiphathic characteristics are also possible, as will be appreciated by those in the art).
  • this regiospecific hydrophilicity/ hydrophobicity can allow the metal complex to more efficiently interact with the protein or enzyme, which generally displays both hydrophobic and hydrophilic regions.
  • the complex may be added to a test protein, for example myoglobin, and the ability of the metal complex to cause protein unfolding may be measured.
  • the chelator has a number of coordination sites containing coordination atoms which bind the metal ion, to ensure that not all ligands will dissociate, and, in some cases, to labilize the axial ligands.
  • the number of coordination sites, and thus the structure of the chelator depends on the metal ion.
  • the choice of the metal ion in turn depends on the identity of the reactive amino acid, with preferred metals being selected from the group consisting of cobalt (either Co(I), Co(II) or Co(III)), copper (including Cu(I) and Cu+2 or Cu(II)), nickel (including Ni+2 or Ni(II)), palladium (including Pd+2 or Pd(II)) and platinium (including Pt+2 or Pt(II)), with silver (Ag) and gold (Au), as well as Rh, Ir, Ru, Fe, Os, Cr, Mn, Zn, Mo, Ru, and Cd, being possible in some embodiments as described herein.
  • cobalt either Co(I), Co(II) or Co(III)
  • copper including Cu(I) and Cu+2 or Cu(II)
  • nickel including Ni+2 or Ni(II)
  • palladium including Pd+2 or Pd(II)
  • platinium including Pt+2 or Pt
  • suitable metal ions include gold, nickel, palladium, platinum and copper, as these metals have a strong propensity to bind sulfur preferentially to other elements such as oxygen, nitrogen and carbon. Consequently these complexes will preferentially bind to the sulfur atom of a cysteine or methionine residue.
  • chelators that can be used in the methods of the invention.
  • preferred metal complexes can bind at least two different side chains of the target protein, and thus, in general, when n is the number of coordination sites of the metal ion, the chelator provides n-2, n-3 or n-4 coordination atoms, and the remaining sites are filled by ligands that provide preferably a single coordination atom each, to allow for the substitutional lability of the ligands.
  • the chelator is a Schiff s base compound.
  • Schiff s base herein is meant a substituted imine.
  • Schiff s bases are generally the condensation products of amines and aliphatic aldehydes forming azomethines substituted on the nitrogen atom.
  • Schiff s base compounds can be di-, tri- and tetravalent, with a tetravalent Schiff s base being generally depicted in Structure 1, below.
  • Particularly preferred in this embodiment are cobalt containing complexes, and particularly preferred compounds are outlined in U.S. Patent Nos.
  • Structure 1 depicts the metal as cobalt, but other metals, particularly d 6 metals, can also be used in Structure 1. It should be noted that if Co(II) is used, the axial ligands may not be present, but upon oxidation of the Co(II) to Co(III), two axial ligands will be picked up. In addition, the two oxygen atoms may be replaced with either sulfur or selenium atoms as well, and the nitrogen by phosphorus.
  • L, and L 2 are axial ligands, also called neutral coordinating ligands herein, and each of the R groups is a substitution group.
  • axial ligand herein is meant a ligand L, or L 2 located at either the fifth or sixth coordination sites, generally depicted in Structure 1 above, in the equatorial plane defined by the chelate ad the metal.
  • Co(II) compounds have up to four coordination atoms, although it is possible that other molecules (usually the solvent) may be weakly associated in one or both axial ligand positions.
  • Co(III) compounds have up to six coordination atoms, of which two are defined herein as axial ligand positions.
  • the complex is synthesized or formulated with two particular axial ligands, and then when the complex is added to a protein, for example, the original axial ligand or ligands are replaced by one or more ligands from a protein. This will occur either when the affinity of the protein axial ligand is higher for the metal complex as compared to the original axial ligand, or when the new axial ligand is present in elevated concentrations such that the equilibrium of axial ligand binding favors the binding of the new axial ligand from the protein.
  • Co(III) complexes are made with axial ligands that can be substituted with other ligands.
  • Co(II) compounds of the invention are preferably synthesized with no axial ligands.
  • certain moieties such as the nitrogen atom of the imidazole of the side chain of histidine, within the protein can become an axial ligand, resulting in a tightly-bound protein-cobalt compound complex. This occurs when the Co(II) compound, with its four coordinating atoms from the Schiff s base, binds an imidazole moiety, for example, and is oxidized to a Co(III) compound.
  • this may be considered a redox reaction, since the Co(II) compound is oxidized to a Co(III) compound upon binding to the protein.
  • the imidazole axial ligand serves as a fifth coordinating atom, and is tightly bound.
  • the axial ligands or neutral coordination ligands are preferably water soluble groups having weak to intermediate ligand field strength (that is, they are substitutionally labile in that the axial ligands can be replaced by the reactive amino acid side chains, such as the nitrogen atom of the imidazole side chain of histidine).
  • ligands may be arranged in a spectrochemical series according to the magnitude of the field strength.
  • suitable axial ligands include, but are not limited to, halides, amine groups, water, dimethyl sulfoxide, any bulky ligand, alcohols, alkoxides, thioethers, carbonyl bound compounds, hydrophylic olefines, etc.
  • two axial ligands when two axial ligands are present, they may be the same or different.
  • each of the R groups is a substitution group.
  • Suitable R substitution groups include a wide variety of groups, as will be understood by those in the art.
  • Each R group may be independently selected from the group include hydrogen, halides, alkyl groups (including substituted alkyl groups and heteroalkyl groups), aryl groups (including substituted aryl and heteroaryl groups), organic acids, glycols, alcohols, amines, amides, esters, ethers, nitro groups, aldehydes, sulfur containing moieties, phosphorus containing moieties, cyano moieties, and targeting moieties.
  • some positions i.e.
  • R 4 and R 5 in Structure 1) designated above may have two R groups attached (R' and R' '), although in a preferred embodiment only a single non-hydrogen R group is attached at these positions.
  • two adjacent R groups may be bonded together to form ring structures together with the carbon atoms of the chelator.
  • alkyl group or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position.
  • the alkyl group may range from about 1 to about 30 carbon atoms (Cl -C30), with a preferred embodiment utilizing from about 1 to about 20 carbon atoms (Cl -C20), with about Cl through about C12 to about C15 being preferred, and Cl to C5 being particularly preferred, although in some embodiments the alkyl group may be much larger.
  • alkyl group also included within the definition of an alkyl group are cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.
  • Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and silicone being preferred.
  • Alkyl includes substituted alkyl groups.
  • substituted alkyl group herein is meant an alkyl group further comprising one or more substitution moieties "R", as defined above.
  • aryl or “aromatic” groups or grammatical equivalents herein is meant an aromatic monocyclic or polycyclic hydrocarbon moiety generally containing 5 to 14 carbon atoms (although larger polycyclic rings structures may be made) and any carbocylic ketone or thioketone derivative thereof, wherein the carbon atom with the free valence is a member of an aromatic ring.
  • Aromatic groups include arylene groups and aromatic groups with more than two atoms removed. For the purposes of this application aromatic includes heterocycle.
  • Heterocycle or “heteroaryl” means an aromatic group wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen, sulfur, phosphorus, boron and silicon wherein the atom with the free valence is a member of an aromatic ring, and any heterocyclic ketone and thioketone derivative thereof.
  • heterocycle includes thienyl, furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl, isoquinolyl, thiazolyl, imidozyl, etc.
  • amino groups or grammatical equivalents herein is meant -NH 2 , -NHR and -NR 2 groups, with R being as defined herein.
  • nitro group herein is meant an -NO 2 group.
  • sulfur containing moieties herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and sulfides (-RSR-).
  • phosphorus containing moieties herein is meant compounds containing phosphorus, including, but not limited to, phosphines and phosphates.
  • silicon containing moieties herein is meant compounds containing silicon.
  • ether herein is meant an -O-R group.
  • Preferred ethers include alkoxy groups, with - O-(CH 2 ) 2 CH 3 and -O-(CH 2 ) 4 CH 3 being preferred.
  • ester herein is meant a -COOR group.
  • halogen or “halide” herein is meant bromine, iodine, chlorine, or fluorine.
  • aldehyde herein is meant -RCHO groups.
  • alkyl alcohol herein is meant -OH groups, and alkyl alcohols -ROH.
  • the alkyl alcohol may be primary, secondary or tertiary, depending on the alkyl group.
  • the alkyl alcohol is a straight chain primary alkyl alcohol, generally containing at least 3 carbon atoms.
  • Preferred alkyl alcohols include, but are not limited to, n-propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-heptyl alcohol, or n-octyl alcohol.
  • ethylene glycol or "(poly)ethylene glycol” herein is meant a -(O-CH 2 -CH 2 ) n - group, although each carbon atom of the ethylene group may also be singly or doubly substituted, i.e. -(O-CR 2 -CR 2 ) n -, with R as described above.
  • Ethylene glycol derivatives with other heteroatoms in place of oxygen i.e. -(N-CH 2 -CH 2 ) n - or -(S-CH 2 -CH 2 ) n -, or with substitution groups are also preferred.
  • organic acid or grammatical equivalents herein is meant an alkyl group containing one or more carboxyl groups, -COOH, i.e. a carboxylic acid.
  • the alkyl group may be substituted or unsubstituted.
  • Cl - C20 alkyl groups may be used with at least one carboxyl group attached to any one of the alkyl carbons, with Cl - C5 being preferred.
  • the carboxyl group is attached to the terminal carbon of the alkyl group.
  • Other preferred organic acids include phosphonates and sulfonates.
  • a preferred organic acid is propionic acid.
  • alkyl alcohol herein is meant an alkyl group containing one or more alcohol groups, similar to the alkyl acids. As defined above, the alkyl group may be substituted or unsubstituted.
  • the alkyl alcohol may be primary, secondary or tertiary, depending on the alkyl group.
  • the alkyl alcohol is a straight chain primary alkyl alcohol, generally containing at least 2 carbon atoms.
  • Preferred alkyl alcohols include, but are not limited to, ethanol, n-propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-heptyl alcohol, or n-octyl alcohol.
  • preferred alkyl alcohols have an alcohol group attached to the terminal carbon of the alkyl group.
  • alkyl thiol herein is meant an alkyl group containing a thiol (-SH) group at any position, with terminal positions preferred as for acids and alcohols.
  • carbonyl oxygen herein is meant an oxygen double bonded to a carbon atom.
  • phosphonyl oxygen herein is meant an oxygen double bonded to a phosphorus atom.
  • targeting moiety herein is meant a functional group that will specifically interact with the target protein, and thus is used to target the metal complex to a particular target protein. That is, the metal complex is covalently linked to a targeting moiety that will bind or associate, preferably specifically, with a target protein.
  • the metal complexes used in the invention may include a polypeptide inhibitor that is known to inhibit a protease, thus effectively increasing the local concentration of the metal complex around the target protein.
  • Suitable targeting moieties include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, hormones including proteinaceous and steroid hormones, growth factors, receptor ligands, antigens and antibodies, and the like.
  • polypeptide herein is meant a compound ranging from about 2 to about 15 amino acid residues covalently linked by peptide bonds.
  • Preferred embodiments utilize polypeptides from about 2 to about 8 amino acids, with about 4 to about 6 being the most preferred.
  • the amino acids are naturally occurring amino acids in the L- configuration, although amino acid analogs are also useful, as outlined below.
  • the polypeptide may be only a single amino acid residue.
  • the polypeptide may be larger, and may even be a protein, although this is not preferred.
  • the polypeptide is glycosylated.
  • polypeptide Also included within the definition of polypeptide are peptidomimetic structures or amino acid analogs.
  • non-naturally occurring side chains or linkages may be used, for example to prevent or retard in vivo degradations.
  • the amino acid side chains may be in the (R) or D-configuration.
  • the amino acids, normally linked via a peptide bond or linkage i.e. a peptidic carbamoyl group, i.e. -CONH-, may be linked via peptidomimetic bonds.
  • These peptidomimetic bonds include CH 2 -NH-, CO- CH 2 , azapeptide and retroinversion bonds.
  • nucleic acid or "oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs, and mixtures of different nucleic acid analogs may be made.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
  • nucleoside includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
  • carbohydrate herein is meant a compound with the general formula C x (H 2 O) y .
  • Monosaccharides, disaccharides, and oligo- or polysaccharides are all included within the definition and comprise polymers of various sugar molecules linked via glycosidic linkages.
  • Particularly preferred carbohydrates are those that comprise all or part of the carbohydrate component of glycosylated proteins, including monomers and oligomers of galactose, mannose, fucose, galactosamine, (particularly N-acetylglucosamine), glucosamine, glucose and sialic acid, and in particular the glycosylation component that allows binding to certain receptors such as cell surface receptors.
  • lipid as used herein includes fats, fatty oils, waxes, phospholipids, glycolipids, terpenes, fatty acids, and glycerides, particularly the triglycerides. Also included within the definition of lipids are the eicosanoids, steroids and sterols, some of which are also hormones, such as prostaglandins, opiates, and cholesterol.
  • Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid-stimulating hormone, calcitonin, chorionic gonadotropin, cortictropin, follicle-stimulating hormone, glucagon, leuteinizing hormone, lipotropin, melanocyte-stimulating hormone, norepinephrine, parathryroid hormone, vasopressin, enkephalins, seratonin, estradiol, progesterone, testosterone, cortisone, and glucocorticoids.
  • Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others.
  • preferred chelators are relatively small and hydrophobic. Accordingly, preferred R substitution groups include hydrogen, small alkyl groups, halides, OH, OH, NHR, CN, COOH, and COO-Na+. When hydrophilic or larger R groups are used, preferred embodiments utilize chelators that have only 1 to 3 of these groups, with 1 being preferred.
  • M is a transition metal ion
  • A is either nitrogen, phosphorus, sulfur or oxygen
  • E is oxygen, sulfur, nitrogen, phosphorus or selenium
  • D is carbon, boron (B) or phosphorus (P).
  • X is either a counter-ion or a neutral coordinating ligand.
  • R is a substitution group as outlined herein, or may be absent when A is oxygen.
  • R 2 is a substitution group as outlined herein, carbonyl oxygen, phosphonyl oxygen, or -OR when A is boron.
  • R 3 is a substitution group as outlined herein, or -OR when A is boron or phosphorus, or is absent when R 2 is carbonyl oxygen.
  • the other R groups are substitution groups as outlined herein.
  • A, E, X and M will depend on a variety of factors. Since, in a preferred embodiment, the metal complexes of the invention are neutral, i.e. uncharged, the collective charge of the A, E, X and M moieties preferably equal zero. Thus, as is depicted herein, the choice of A and E will determine whether X is a counter-ion or a ligand. Thus, when A and E are such that they both carry a negative charge (for example when A is oxygen and R, is absent, and E is sulfur, oxygen, or selenium, with R 8 being absent) then X is a neutral ligand.
  • a and E when one or the other of A and E is negatively charged, and the other is neutral, X is a counter-ion.
  • X is a counter-ion.
  • a or E should carry a negative charge.
  • preferred embodiments utilize both A and E with negative charges; A as nitrogen (with R, present) and E as oxygen, sulfur or selenium, with R 8 being absent; or A as oxygen (R, absent) and E as oxygen or nitrogen with R 8 present.
  • Suitable counter-ions include, but are not limited to, halides; -OR; -SR; SO 4 2-, PF 6 -, BF 4 -, Bar4, RCCO, citrate, and -NHR, where R is a substituent group as herein defined, preferably alkyl and aryl. It should be noted that the choice of the counter-ion can influence conformational changes, so chaotropic and kosmotropic anions are included.
  • neutral coordinating ligand herein is meant a neutral molecule capable of donating electrons to a metal to form a metal-ligand complex without a formal change in oxidation state.
  • Suitable neutral coordinating ligands include, but are not limited to, water (H 2 O), dioxane, THF, ether (ROR), thioether (RSR), amine (NR 3 ) and phosphine (PR 3 ), with R being any number of groups but preferably an alkyl group.
  • E is oxygen, sulfur or selenium
  • R 3 is hydrogen
  • X is a counter-ion.
  • E is oxygen, sulfur, or selenium and X is a neutral coordinating ligand.
  • E is oxygen, sulfur, or selenium
  • X is a neutral coordinating ligand
  • E is oxygen, sulfur, or selenium and X is a neutral coordinating ligand.
  • E is nitrogen, oxygen or sulfur, and X is a counter-ion.
  • the metal complexes of the invention have the formula depicted below in Structure 8: Structure 8
  • M is a transition metal ion selected from the group consisting of Co, Cu, Ag, Au, Ni, Pd and Pt
  • E is oxygen, sulfur, or selenium, with oxygen being preferred.
  • Rq, R 10 , R n , R 12 , R )3 , R )4 , R 15 and R ]6 are each independently a substitution group as defined herein, although R,, and R ]2 together may form a cycloalkyl or aryl group.
  • R 15 and R 16 together may form a cycloalkyl or aryl group.
  • the metal complexes of the invention have the formula depicted below in Structure 9:
  • M is a transition metal ion with an oxidation state of +1, preferably Cu(+1), Au(+1), or Ag(+1).
  • X is a counter-ion.
  • R 25 , R 26 , R 27 , R 2g , R 29 , R 30 , R 3] , R 32 and R 33 are independently substitution groups, that may, with an adjacent R group forms a cycloalkyl or aryl group.
  • the metal complexes of the invention have the formula depicted below in Structure 10: Structure 10
  • M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Au+2 being preferred.
  • X is a counter-ion.
  • R 35 , R 36 and R 37 are independently hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, a targeting moiety, or, together with an adjacent R group forms a cycloalkyl (preferably heterocycloalkyl, with the heteroatom being nitrogen, oxygen, or sulfur) substituted cycloalkyl, aryl, or substituted aryl groups.
  • At least one R 35 , R 36 , R 37 or the R substituents of the cycloalkyl or aryl group is a targeting moiety, with polypeptides and nucleic acids being preferred.
  • preferred embodiments include the structures depicted below:
  • the R group on the nitrogen atom may be an R group as defined herein or it may be hydrogen.
  • the metal complexes of the invention have the formula depicted below in Structure 12: Structure 12
  • M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Cu, Ni, Pd and Pt being preferred.
  • X is a counter-ion.
  • R 38 , R 39 , R 40 , R 41 , R 42 and R 43 are independently hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, or a targeting moiety. In a preferred embodiment, at least one of R 38 to R 43 is a targeting moiety.
  • the metal complexes of the invention have the formula depicted below in Structure 13:
  • M is a transition metal ion selected from the group consisting of Cu, Ag, Au, Ni, Pd and Pt, with Cu, Ni, Pd and Pt being preferred.
  • E is oxygen, sulfur or selenium, with oxygen being preferred.
  • Each X is independently a counter-ion.
  • R 44 is hydrogen, halogen, alkyl, alkyl alcohol, alcohol, alkyl thiol, alkyl acid, alkyl amine, amine, aryl, or a targeting moiety. In a preferred embodiment, at least one of R 3 to R 43 is a targeting moiety.
  • A, B, C and D are independently single or double bonds, with the latter being preferred.
  • the R groups are independently substitution groups, and as above, two adjacent R groups may together form a cycloalkyl or aryl ring.
  • target protein herein is meant a protein that is to be unfolded using the methods of the invention. Without being bound by theory, it appears that target proteins that may be unfolded using the methods of the invention.
  • the target protein should have reactive amino acids on the surface.
  • suitable target proteins include, but are not limited to, enzymes (including hydrolases such as proteases (including, but not limited to, serine (including, but not limited to, plasminogen activators and other therapeutically relevant mammalian serine proteases as well as bacterial serine proteases such as subtilisins) aspartyl, metal, acid and cysteine proteases (including, but not limited to cathepsins (including cathepsins B, H, J, L, N, S, K, O, T and C, (cathepsin C is also known as dipeptidyl peptidase I), interleukin converting enzyme (ICE), calcium-activated neutral proteases, calpain I and II); carbohydrases, lipases; isomerases
  • hydrolases such as proteases (including, but not limited to, serine (including, but not limited to, plasminogen activators and other therapeutically relevant mammalian serine proteases as well as bacterial serine proteases
  • influenza virus influenza virus
  • paramyxoviruses e.g respiratory syncytial virus, mumps virus, measles virus
  • adenoviruses e.g. respiratory syncytial virus
  • rhinoviruses e.g. coronaviruses
  • reoviruses e.g. togaviruses (e.g. rubella virus)
  • parvoviruses poxviruses (e.g. variola virus, vaccinia virus)
  • enteroviruses e.g. poliovirus, coxsackievirus
  • hepatitis viruses including A, B and C
  • herpesviruses e.g.
  • Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C.perfringens; Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G. lambliaY. pesiis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis;
  • Bordetella e.g. B. pertussis; Treponema, e.g. T. palladium; and the like
  • proteinaceous hormones and cytokines many of which serve as ligands for cellular receptors
  • EPO erythropoietin
  • TPO thrombopoietin
  • the interleukins including IL-1 through IL- 17
  • insulin insulin-like growth factors
  • IGF-1 and -2 epidermal growth factor
  • EGF epidermal growth factor
  • transforming growth factors including TGF- ⁇ and TGF- ⁇
  • human growth hormone transferrin, epidermal growth factor (EGF), low density lipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, human chorionic gonadotropin, cotrisol, estradiol, follicle stimulating hormone (F
  • the metal complexes are contacted with the target protein under conditions that allow the binding of the metal complex to the protein, and the mixture is allowed to incubate for some period of time.
  • contacted or “added” herein is meant that the solutions containing the two are mixed, with homogeneous solutions being preferred.
  • the salt concentration, buffer composition and concentration, heat, pressure and pH can all be varied. Low pH (i.e. 5.5) generally facilitates the reaction.
  • the number of metal complexes bound to any particular protein depends on both the number of reactive amino acids and the concentration of the added metal complex; thus, for example, using less than stochiometric ratios of the metal complex can allow a partial unfolding in some cases.
  • the metal complexes are added to the protein in the absence of any significant amounts of traditional denaturants, such as high salt concentrations, guanidinium-HCl, detergents, etc. That is, the proteins do not significantly unfold unless the metal complex is present.
  • binding herein is meant the formation of a coordination bond. That is, the metal complex gives up at least one, and preferably two, of its substitutionally labile ligands in favor of binding one or more amino acid side chains from the target protein.
  • the metal complexes bind to the proteins, generally to solvent accessible reactive amino acids. As outlined above, amino acid side chains that are buried within the interior of a folded protein may become exposed as the protein unfolds.
  • metmyoglobin yields a partially folded protein isolated in a biologically relevant medium; while some proteins in their natural state may be partially folded, this Co(III)- myoglobin complex is the first example of an isolatable partially folded species obtained from a naturally folded precursor.
  • Mechanistic studies indicate that the irreversibility and selectivity of unfolding by 1 originate in the strong bond formed preferentially between cobalt and an imidazole nitrogen of a histidine. Our findings open the way for applications based on the unique properties of molten globules as toxin-like prodrugs.
  • UV/vis absorption spectra were acquired on a Hewlett Packard HP 8452 diode array spectrophotometer.
  • CD spectra were measured on an Aviv 62DS spectropolarimeter at 25 °C (1 mm cell for far-UV CD; 10 mm cell for near-UV CD). Measurements were made on the incubation mixtures, without additional treatment, to prevent dissociation of weakly bound Co(III) complexes. Note, however, that removal of free 1 by dialysis did not change the spectrum of the incubation product of metMb with 11 equivalents of 1.
  • the far UV circular dichroism shows a 50% decrease in the ⁇ -helical secondary structure of metMb (Fig. 2b) (14) that parallels the decrease in the Soret band (Fig. 2a).
  • the loss of the near-UV CD indicates diminished packing around the aromatic amino acids.
  • At an initial 1 :metMb ratio of 11 : 1 virtually no near-UV CD signal is observed.
  • the reaction is irreversible in the sense that extensive dialysis leaves an average of 6 cobalt complexes associated with the protein, and that the product retains 1 for prolonged periods in solution even in the absence of free 1.
  • External reagents reverse the reaction.
  • Excess dithionite reduces the hemin to ferroheme, and causes cobalt dissociation from the protein (probably by reducing Co(III) to labile Co(II)).
  • a large excess of imidazole leads to slow, incomplete recovery of the Soret band.
  • Excess hemin does not affect the product UV/vis spectrum.
  • ApoMb reacts with 1 much faster than metMb, and a smaller 1 to apoMb ratio completes the transformation (about 6:1 L.apoMb (Fig. 3a)).
  • the product is identical with the one obtained from metMb and excess 1 (overlapping far-UV CD (Fig. 3b), with 6 Co(III) complexes binding to the protein).
  • the observation that these products are the same indicates that 1 causes hemin dissociation from the active site of metMb.
  • the dissociated hemin is not separated from the cobalt-myoglobin product by dialysis, as confirmed by iron atomic abso ⁇ tion measurements. It probably binds nonspecifically to the unfolded protein, as observed by Hargrove, M. S. & Olson, J. S.
  • near-UV CD Gast, K., Damaschun, H., Misselwitz, R., M ⁇ ller-Frohne, M.; Zirwer, D. & Damaschun, G. (1994) Eur. Biophys. J. 23, 297-305; Irace, G., Bismuto, E., Savy, F. & Colona, G. (1986) Arch. Biochem. Biophys. 244, 459- 469; Fink, A. L., Oberg, K. A. & Seshadri, S.
  • the partially unfolded Co(III)-myoglobin can be described as a molten globule ( Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229; Ptitsyn, O. B. (1996) N t. Struct. Biol. 3, 488- 490; Ewbank, J. J., Creighton, T. E.; Hayer-Hartl M. K. & Hartl F. U. (1995) Nat. Struct. Biol. 2, 10-11.), since it retains much secondary structure but very little tertiary structure (Fig. 2d). As all free denaturant can be removed, this is in every sense an isolated, kinetically stable, partially folded protein.
  • the small changes in wavelength (6 nm shift) and extinction coefficient of the ⁇ - ⁇ * abso ⁇ tion band of 1 indicate that it binds to a nitrogen donor of the protein. While these values are similar for ammonia, alkylamines, pyridine, and substituted imidazoles as axial ligands ( ⁇ ottcher, A., Takeuchi, T., Hardcastle, K. I., Meade, T. J., Gray, H. B., Cwikel, D., Kapon, M. & Dori, Z. (1997) Inorg. Chem. 36, 2498-2504; Costa, G., Mestroni, G., Tauzher, G. & Stefani, L. (1966) J.
  • Molten globules are superior to folded proteins in their ability to translocate across or insert into membranes (van der Goot, F. G., Lakey, J. H. & Pattus, F. (1992) Trends Cell Biol. 2, 343-348;, as they have increased affinity for hydrophobic surfaces (Ptitsyn, O. B. (1995) Adv. Prot. Chem. 47, 83-229;.
  • An isolable partially folded protein can, therefore, be a powerful new type of pro-drug, functioning in a manner similar to that suggested for some bacterial toxins (van der Goot, F. G., Lakey, J. H. & Pattus, F. (1992) Trends Cell Biol. 2, 343-348;.
  • Metal-ion-induced unfolding may also be utilized for selective protein precipitation, thereby aiding the formation of solid inclusion bodies, which could lead to improvements in large-scale protein biosynthesis (Betts, S., Haase-Pettingell, C. & King, J. (1997) Adv. Prot. Chem. 50, 243-264;.

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Abstract

L'invention concerne des procédés pour le dépliement des protéines au moyen de complexes métalliques, en particulier des composés du type base de Schiff renfermant du cobalt. Les protéines peuvent être des protéines virales ou de la myoglobine, et le dépliement peut être iréversible dans des conditions physiologiques. Les protéines dépliées présentent des modifications spectrales dans l'absorption des rayons ultraviolets/de la région visible du spectre (voir la figure) et une modification égale ou supérieure à 5 % dans au moins un minimum ou un maximum de spectre du dichroïsme circulaire des protéines.
PCT/US1998/014630 1997-07-16 1998-07-16 Procedes pour le depliement des proteines au moyen de complexes metalliques WO1999003877A1 (fr)

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EP98934562A EP1027369A4 (fr) 1997-07-16 1998-07-16 Procedes pour le depliement des proteines au moyen de complexes metalliques
AU84052/98A AU8405298A (en) 1997-07-16 1998-07-16 Methods of unfolding proteins using metal complexes
CA002297362A CA2297362A1 (fr) 1997-07-16 1998-07-16 Procedes pour le depliement des proteines au moyen de complexes metalliques

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106483086A (zh) * 2016-09-21 2017-03-08 大连大学 一种利用激光诱导肌红蛋白还原的方法

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Publication number Priority date Publication date Assignee Title
US5106841A (en) * 1986-05-13 1992-04-21 Chai-Tech Corporation Antiviral compositions and method for their use
WO1996018402A1 (fr) * 1994-12-15 1996-06-20 California Institute Of Technology Composes de base de schiff au cobalt

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5106841A (en) * 1986-05-13 1992-04-21 Chai-Tech Corporation Antiviral compositions and method for their use
WO1996018402A1 (fr) * 1994-12-15 1996-06-20 California Institute Of Technology Composes de base de schiff au cobalt

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Title
BLUM O., ET AL.: "ISOLATION OF A MYOGLOBIN MOLTEN GLOBULE BY SELECTIVE COBALT(III)-INDUCED UNFOLDING.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 95., 1 June 1998 (1998-06-01), US, pages 6659 - 6662., XP002913686, ISSN: 0027-8424, DOI: 10.1073/pnas.95.12.6659 *
LOUIE A. Y., MEADE T. J.: "A COBALT COMPLEX THAT SELECTIVELY DISRUPTS THE STRUCTURE AND FUNCTION OF ZINC FINGERS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 95., 1 June 1998 (1998-06-01), US, pages 6663 - 6668., XP002913687, ISSN: 0027-8424, DOI: 10.1073/pnas.95.12.6663 *
See also references of EP1027369A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106483086A (zh) * 2016-09-21 2017-03-08 大连大学 一种利用激光诱导肌红蛋白还原的方法

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