Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B30/00—Methods of screening libraries
  • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • G01N2333/245—Escherichia (G)

Definitions

• the present invention relates to a method of selecting a candidate drug compound having affinity for biological receptors.
• Cell surfaces e.g. the surfaces of erythrocytes, endothelial cells, tumour cells, etc have characteristic binding sites for biological ligands. Frequently such binding sites are the result of the secondary structure, i.e. the folding conformation, of cell surface protons, and binding to these triggers a response by the cell.
• One example is the ST receptors which are found only on the apical brush border membranes of the cells lining the intestinal tract of placental mammals.
• a variety of bacteria such as Eschericia coli, Vibrio cholerae, Citrobacter freundii and Yersinia enterocolitica, which may infect the mammal gut produce homologous peptide toxins which bind to ST receptors and trigger a cascade of biochemical processes eventually leading to fluid secretion into the intestinal lumen and hence diarrhoea.
• These ST enterotoxins are a major cause of infectious diarrhoeal disease in developing countries, the fourth leading cause of mortality and morbidity in the pediatric population worldwide.
• These enterotoxins typically contain 18 or 19 amino acid residues, are stable to proteases and maintain their bioactivity even after incubation at 100°C for 15 minutes. Examples of such heat stable ST enterotoxins are listed in Table 1 below:
• E.Coli STa has disulphide bridges between the Cys residues at positions 5 and 10, 6 and 14 and 9 and 17.
• Such cell receptor binding oligopeptides are of interest both for therapeutic and diagnostic purposes.
• an oligopeptide capable of binding to a cell surface receptor may be coupled to a therapeutically or diagnostically effective moiety and serve as a biological vector to deliver that moiety to sites possessing such cell surface receptors.
• a radionucleotide capable of detection in a nuclear imaging technique e.g. scintigraphy, SPECT or PET
• SPECT single photonucleotide
• PET PET
• the receptor occurs solely or predominantly on undesired cells, e.g. tumour cells
• a cytotoxic dose of radiation can likewise be delivered to the site of concern using radiation emitting vector-bound radionuclides .
• the ST receptors occur naturally only in the intestinal lumen and are found elsewhere in the body only as a result of metastases of colon cancers.
• Parenteral administration of a radionuclide-tagged ST oligopeptide can be used to detect and treat such metastases (see US-A-5518888 and W095/11694) .
• native oligopeptides as vectors for therapeutic or diagnostic moieties is problematic, for example because the native oligopeptide will bring about its natural consequences on binding at the cell surface receptors.
• an ST antagonist might be administered orally so as to suppress diarrhoea.
• Analogs of the native ligands may also be sought which have greater stability in vivo, to labelling conditions, or to storage, etc. or which have a longer blood pool residence time in vivo.
• the generation of a candidate receptor - (ant) agonist may typically proceed from modification of a native ligand, by generation of a non-peptidic analog of the native ligand, by selection from a random library of biological oligomers, or by generation of a chemical structure which fits both geometrically and functionally into the receptor pocket where this has been elucidated.
• drug candidate development will generally involve computer aided molecular design (CAM- D) .
• Modification of known native ligand structures generally proceeds via single amino acid alterations, e.g. deletions, insertions or exchanges.
• single amino acid alterations e.g. deletions, insertions or exchanges.
• the number of possible single amino acid modifications is immense and the technique is accordingly highly time-consuming.
• the present invention is directed to an improved method of selecting a candidate drug compound with receptor binding ability in which a novel combination of rational and combinatorial drug design techniques is used.
• the invention provides a method for selecting a candidate drug compound, said method comprising: i) identifying a starting oligopeptide having a binding affinity for a cell surface or other receptor site; ii) generating a plurality of homologous oligopeptides having single site amino acid modifications relative to said starting oligopeptide and identifying modification sites which produce oligopeptides having an increase (or decrease) in the ratio of binding affinity between oligopeptide and said receptor site to biological response of binding between oligopeptide and said receptor site; iii) identifying components of said starting oligopeptide responsible for the secondary structure thereof ; iv) for modification sites identified in step (ii) selecting a set of amino acids, e.g.
• step (iii) selecting at least one non-natural amino acid which mimics the secondary structure of the component; vi) generating a first combinatorial library of oligopeptides having at said identified modification sites amino acid residues corresponding to said selected sets of amino acids and preferably having at the sites of said components amino acid residues corresponding to said non-natural amino acids; vii) identifying members of said first library having a binding affinity value above a predetermined minimum and optionally having a relatively high (or low) ratio of binding affinity to biological response; optionally viii) changing, by expanding and/or contracting, the sets of amino acids for said modification sites or non-natural amino acids for said components and generating a further combinatorial library of oligopeptides having members not present in said first library, and identifying members of said further library having
• amino acids and oligoamino acid motifs which are likely to result in poor in vivo stability or short blood pool lifetime and to seek to exclude these from the libraries to be tested.
• amino acids or amino acid sequences will generally be ones to avoid or replace:
• Tyr-Gly peptides with free ⁇ -amines
• Pro-x (where x is a natural amino acid) ; x-Phe-x; peptides with free ⁇ - carboxyls; x-Lys; Lys-Lys; Gln-Gln; x-Arg; Arg-Arg; Gly- Gly; Ala-Ala; and x-Tyr.
• R is an optional substituent, e.g. optionally hydroxylated C ⁇ . 10 alkyl, aralkyl, aryl or aralkyl
• an N-terminal Asn is identifiable as a helical end cap (capable of hydrogen-bonding to a nearby amide carbonyl , here of
• Tyr 4 which can stabilize a helix, here the 3 10 helix.
• an ST receptor agonist would require the functional groups at the N terminus to adopt a similar three-dimensional configuration .
• Thr or Ala is usually associated with a Type I beta turn. Such a sequence occurs at positions 11-13 of E.Coli STa. Other mutations for Asa or Ala generally give rise to other forms of ⁇ -turn or to ⁇ -turns. Mutations disrupting the Type I ⁇ turn in E.Coli STa give rise to agonists (e.g. Alal3 Gly, Asnll Val or Asnll Gly) or antagonists (e.g. Prol2 Gly) . (By XX-n-YY is meant that XX" is changed to YY") .
• the C-terminus of the E.Coli STa is also associated with secondary structure, namely a Type II ⁇ turn.
• the amino acid combinations responsible for secondary structure can in general either be predicted from known structures or can be determined by X-ray crystallography. (See “Protein folds: a distance-based approach”, Eds. Bohr and Brunak, CRC Press, NY (1995) and “Protein Folding” Eds. Gierasch and King, AAAS, Washington DC, (1990)).
• the generation of the first library in step vi) it is possible to use restricted sets of amino acids by panning one or more restricted libraries produced by modification at a restricted set of modification sites or secondary structure component sites, e.g. by panning a "sub- library" in which one, more or all of the secondary structure component sites are left unchanged or one in which one or more of the least promising (or indeed the most promising) of the modification sites are left unchanged.
• the sets of amino acids for the modification/component sites will typically be changed to avoid amino acids not found to be effective in the first library and to substitute amino acids which are functionally similar to the successful amino acids, e.g. amino acids omitted from the sets used in generation of the first library either to keep down the overall library size or because they are relatively expensive to purchase or complex to fabricate .
• amino acids omitted from the sets used in generation of the first library either to keep down the overall library size or because they are relatively expensive to purchase or complex to fabricate .
• the first library one might select a single member of a homologous group of amino acids, e.g. using a chloro compound as a representative of a group of halogenated compounds, and if this is a successful amino acid then other members of the homologous group would be added in for the generation of the further library.
• the sets of amino acids, for the modification sites may include natural (L) amino acids, as well as non-natural amino acids, e.g. D-acids, ⁇ -chain length homologues, ⁇ -chain substitution homologues, etc.
• the sets of amino acids used in the generation of the first library may, and in general will, also include the amino acid present in the particular modification/component site in the starting oligopeptide.
• the starting oligopeptide used in the method of the invention may be a native (i.e. naturally produced) oligopeptide (e.g. E.Coli STa) known to have a binding affinity for a cell surface or other receptor site and known to produce a particular biological response.
• the starting oligopeptide may be a non-native oligopeptide, e.g. one known from the literature or one found using library screening techniques, e.g. one identified by the use of a phage display library.
• the method of the invention will be used to identify a candidate receptor antagonist; however in certain circumstances, e.g. where a drug which provokes biological response is desirable, the method of the invention will be used to identify a candidate drug compound which is a receptor agonist.
• Generation, panning and if required deconvolution of the combinatorial libraries used in the method of the invention may be performed using known techniques.
• oligopeptide synthesis will be carried out on a solid phase, and binding affinity will preferably be assessed m the solution phase, e.g. following release of library members from the solid phase. Where this is done, the solid phase bound library can either be released to produce a solution containing the library members or solutions containing individual library members. In the latter case deconvolution is not required, in the former it is.
• Deconvolution techniques have been widely described in the literature m recent years but where deconvolution is required according to the present invention this will preferably involve orthogonal scanning, i.e. the use of sub-libraries to work back and identify the relevant oligopeptide structures.
• the libraries will preferably be created on polymer beads using the split-and-mix technique.
• the libraries may be generated by the multi-pin method, or they may be generated on an extensive (e.g. sheet-like) substrate using the spot technique or using masks and photo-deprotection. In these latter two techniques, the identity of the library member is determined by its location on the substrate.
• a combined solution phase library can be created by simultaneous release of all substrate bound oligopeptides - otherwise individual library members may be released into separate containers, e.g. by punching out pieces of substrate from the oligopeptide growth sites for the different oligopeptides or by pressing the substrate against an array of tubes and releasing the different oligopeptides into their adjacent tubes.
• a selection for binding affinity may be carried out before biological response is determined, thus reducing the number of compounds for which biological response is determined. This will be particularly preferable where testing for biological response requires a tissue sample or a live animal. (Binding affinity will generally be determinable using only cell cultures or tissue samples) . Thus before step (ix) , if a selection for a biological response has not already been carried out in steps (vii) or (viii) , such a selection will generally be effected.
• standard assays may be used, e.g. turbidification, competitive binding against labelled receptor binding agents, etc.
• For biological response one may use an assay appropriate to the particular response as well as to the animal model if the response is being tested in vivo. Design and performance of such assays are well within the normal skill of those skilled in the technical field.
• the drug candidate may be manufactured, optionally conjugated to a therapeutically or diagnostically effective moiety as discussed above.
• a pharmaceutically acceptable carrier or recipient e.g. water for injections, physiological saline, Ringers solution, tabeletting aids, sweeteners, pH adjusters, viscosity modifiers, propellants, bulking agents, etc
• methods of treatment or diagnosis using them are all deemed to form further aspects of the invention.
• an oligopeptide candidate drug compound Following identification of an oligopeptide candidate drug compound using the method of the invention, it may be possible to produce an analog in which the peptide backbone structure is wholly or partially replaced by a non-peptide backbone, e.g. replacing NH-CH-CO-NH-CH-CO-
• R ⁇ n are peptide side chains
• backbone components by equivalently R ⁇ n side chain substituted backbone components in which some or all of the peptide bonds are replaced, e.g. by backbone structures such as
• amide oxo groups are optionally replaced by CH 2 , CS or CHOH groups and amide NH groups are optionally replaced by -0-, -S-, CH 2 or CHOH groups or wherein an amide group is optionally replaced by a 5- or 6- membered homo or most preferably ring nitrogen- containing hetero-cyclic group) or wherein the peptide structure is replaced in part or full by a peptoid structure , i . e . wherein amide oxo groups are optionally replaced by CH 2 , CS or CHOH groups and amide NH groups are optionally replaced by -0-, -S-, CH 2 or CHOH groups or wherein an amide group is optionally replaced by a 5- or 6- membered homo or most preferably ring nitrogen- containing hetero-cyclic group) or wherein the peptide structure is replaced in part or full by a peptoid structure , i . e . where
• the native ST enterotoxin scaffold has undesirable physicochemical characteristics which reduce its attractiveness for use as a vector for therapeutic or diagnostic moieties, eg the multiple disulphides, the diarrhoeagenic activity and its short blood pool residence time.
• therapeutic or diagnostic moieties eg the multiple disulphides, the diarrhoeagenic activity and its short blood pool residence time.
• a number of mutations have been made with retention, to a greater or lesser degree, of the binding affinity and biological response.
• This disulphide pair is able to tolerate the loss of the amino function at Cys 5 or the reversal of its stereochemistry (ie D-Cys replacement) with no detectable loss in biological response.
• the stereochemistry of Cys 10 however is important since its replacement with D-Cys results in a 5000 fold loss of biological response as compared with E.Coli STa.
• Cys 5 Cys 10 disulphide is replaced with a carbo-sulphide analog (resulting in a simpler bicyclic oligopeptide) only a 100 fold loss in biological response is observed.
• This disulphide cannot tolerate substitution of either Cys by a D-Cys without a major loss (>770 fold) in biological response.
• Mutations which vary the hydrogen bonding capacity (eg Asp or Gin substitutions) or the length of the side chain in position 7 appear to have relatively little effect on biological response.
• mutations which change the stereochemistry of the Glu 7 residue eg D-Glu substitution
• result in significant loss of biological response There is minimal difference observed with mutations replacing Glu' by helix disrupting Pro or helix promoting Ala indicating that the 3 10 helix observed in the X-ray structure of native ST is not a required secondary structure.
• Glu 7 may be replaced by cyclic analogs of aspartic acid, eg Pro (a reduced pyroglutamic acid) .
• acceptable Glu 7 replacements include Asp, Gin, Ala and Pro.
• the major trigger for enterotoxigenicity appears to be the Type I ⁇ turn of the component Asn n -Pro 12 -Ala 13 . Mutation here can destroy both binding affinity and biological response. However, it is feasible to find mutations that, while dropping the binding affinity cause an increase m the binding affinity to biological response ratio. Such mutations include for example D-
• G A, N, P, Y, E, R, L, dA, dN, dP, dY, dE, dR, dL or equal mixtures of all 15 amino acids.
• Asn 11 - Pro 12 -Ala 13 may be replaced by the ⁇ -turn mimetic
• the combinatorial libraries may be synthesized using solid state peptide synthesis, eg on beads or sheets of polymers such as polystyrenes, polystyrene copolymers (eg PEG-polystyrene polymers), PEGs, celluloses (eg paper, cotton, etc) , carbohydrates (eg dextrins) , or polyacrylamides, or controlled pore glass.
• the substrate will be a material which is swellable in aqueous and organic solvents, eg a PEG-polystyrene copolymer.
• the invention provides a process for the solid state synthesis of an oligopeptide on a resin substrate, optionally followed by release of the oligopeptide from the substrate, characterised in that as said substrate is used an acetamidomethyl-protected polymer.
• the oligopeptide can be cleaved from such a resin using a reagent such as iodine.
• C-terminal cysteine residues may conveniently be attached to the resin surface.
• an ST enterotoxin analog may be prepared as follows:
• succinamic acid (19.9g) and 0.018 mole KOH (l.Og) are dissolved in a solution of formaldehyde:water (7:3 v/v; 12 ml, 0.012 moles).
• the mixture is stirred at 70°C for 5 minutes, then overnight at room temperature and the acidified with 6N HC1 to pH 7.0.
• the solution is rotovapped to a syrup and redissolved in ethylacetate, then dried over MgS0 4 . Crude product is obtained by filtration and concentration of the filtrate.
• each vessel has a sintered frit and stopcock for filtration
• 150 mmoles of FMOC-Cys (Sum) -resin (compound IV) is added 150 mmoles of FMOC-Cys (Sum) -resin (compound IV).
• the FMOC group is removed and the resin washed according to normal protocols (see G.B. Fields et al . "Principles and practice of solid phase peptide synthesis" in "Synthetic Peptides: a user's guide” W.H. Freeman & Co., New York, Editor Gregory A. Grant) for each reaction.
• Each reaction vessel is then individually reacted with an excess of one of the representative protected amino acids (Gly, D and L versions of Ala, Asn, Pro, Tyr, Glu, Arg, Leu) .
• Each of the individual # 1 dipeptide resins is split equally into 15 individual reaction vessels, deprotected, fully coupled with each of the representative amino acids and then recombined. This cycle is repeated for each amino acid position to be explored, as shown in Figure 2 of the accompanying drawings. Evaluation of the bioactivity of each of these peptide sets will enable determination of which amino acid is most preferable in the first incorporated position.
• the underlined amino acids have been chosen to represent their respective subsets.

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Citations (8)

• 1997
  • 1997-08-20 GB GBGB9717652.3A patent/GB9717652D0/en active Pending
• 1998
  • 1998-08-20 JP JP2000510029A patent/JP2001516029A/ja not_active Withdrawn
  • 1998-08-20 AU AU88173/98A patent/AU8817398A/en not_active Abandoned
  • 1998-08-20 CA CA002300903A patent/CA2300903A1/en not_active Abandoned
  • 1998-08-20 AU AU88168/98A patent/AU8816898A/en not_active Abandoned
  • 1998-08-20 EP EP98939765A patent/EP1005655A2/en not_active Withdrawn
  • 1998-08-20 WO PCT/GB1998/002504 patent/WO1999009416A2/en not_active Application Discontinuation
  • 1998-08-20 WO PCT/GB1998/002510 patent/WO1999009417A2/en active Application Filing
• 2000
  • 2000-02-18 NO NO20000811A patent/NO20000811L/no unknown

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