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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • 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

Definitions

• the present invention relates to proteonomics More specifically, the invention relates to protan-protein interactions and methods for identifying interacting proteins and the ammo acid sequence at the site of interaction
• Protein-protein interactions govern biological processes that handle cellular information flow and control cellular decisions (e g , signal transduction, cell cycle regulation and assembly of cellular structures)
• the entire network of interactions between cellular proteins is a biological chart of functional events that regulate the internal working of living organisms and their responses to external signals
• a necessary step for the completion of this biological interaction chart is the knowledge of all the gene sequences in a given living organism
• the entire DNA sequence of the homo sapiens genome will be completed at the latest by the year 2003 (29)
• the sequence of a gene does not reveal its biological function nor its position in the biological chart Given the expected number of proteins in the human genome (80,000 to 120,000), the mapping of the biological chart of protein-protein interactions will be an enormous but a rewarding task
• the present invention seeks to overcome the drawbacks of the prior art More specifically, the invention concerns an approach to identify protein-protein interaction domains which differ from the p ⁇ orart Moreover, one approach of the present invention is based on an understanding of the principle that govern protein-protein interactions Such understanding therefore, allows the use of several methods Such a method is exemplified in detail below to identify i) at least one of the exact ammo acid sequences between interacting proteins, n) a number of, possibly all interacting proteins in different cells or tissues, and in) the specific domains (or sequences) between two interacting proteins as targets for isolation of lead drugs Preferably, the method and assay of the present invention enables a determination of i), n) and in) Moreover, unlike the approaches of the prior art, the method described herein, allows for the identification of interacting proteins and the precise ammo acid sequences of interactions in several days as opposed to several months
• the present invention further relates to a novel approach in drug discovery
• Amajor obstacle in drug development for the treatment of diseases has been the identification of target proteins and their functional sites
• R&D research and development
• drugs identified by current approaches often target the active sites in proteins
• Such drugs thus often lead to major side-effects
• P-glycoprotein has been shown to cause multidrug resistance in tumor cell lines selected with lipophihc anticancer drugs
• Analysis of P-gp ammo acid sequence has lead to a proposed model of a duplicated molecule with two hydrophobic and hydrophihc domains linked by a highly charged region of circa 90 ammo acids, the linker domain
• similarly charged domains are found in other members of the P-gp superfamily, the funct ⁇ on(s) of this domain are not known
• this domain binds toother cellular proteins Using overlapping hexapeptides that span the entire ammo acid sequences of the linker domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a direct and specific binding between P-gp1 and 3 linker domains and intracellular proteins is shown herein
• Three different stretches 617 EKGIYFKLVTM 627 , 658 SRSSLIRKR
• a method of identifying a high-affinity interacting domain in a chosen protein, domain thereof or part thereof and the ammo acid sequence thereof comprising a) providing a set of overlapping peptides spanning a complete sequence of the chosen protein, domain thereof or part thereof, covalently bound to a support, b) providing a mixture of proteins and/or a mixtuie of peptides, incubating the set of overlappng peptides of a), with the mixture of b), under conditions enabling the binding between a high-affinity interacting domain in a peptide of the set and one or more proten or peptide of b) to occur, d) washing of any protein-protein interaction which is not a high-affinity interaction of c), and e) identifying which peptide of a) interacts with high-affinity to a protein or peptide of b), thereby identifying the peptide of e) and the sequence thereof as
• overlapping peptides spanning a peptide sequence refers to peptides of a chosen size, based on the sequence of the protein (or part thereof)
• these peptides are synthetic peptides
• the size of the overlapping peptides has a significant impact on the workings of the present invention
• peptides of four contiguous ammo acids appear to significantly increase the low affinity binding of proteins thereto
• larger peptides such as 20 ammo acids or higher, would be expected to increase the proportion of repulsive ammo acids to high affinity ammo acids, thereby maskng or totally inhibiting the binding of specific proteins to the peptides
• the preferred size for the overlapping peptides of the present invention is between 5 and
• support in the context of a support to which the overlapping peptides of the present invention are covalently bound, can be chosen from a multitude of supports found in the art Such supports include CHIPS, plates (e g 96-well plates), glass beads and the like)
• CHIP technology is well-known in the art Reference relating theretomclude Debouck et al , Nat Genet 1999 Jan, 21 (1 Suppl) 48-50, Review, Brownet al , Nat Genet 1999 Jan, 21 (1 Suppl) 33-7, Review, Cheung et al , Nat Genet 1999 Jan, 21 (1 Suppl) 15-9, Review, Duggan et al , Nat Genet 1999 Jan, 21 (1 Suppl) 10-4, Review, Schena et al , Trends Biotechnol 1998 Jul,16(7) 301-6, Review, and Ramsay et al , Nat Biotechnol 1998 Jan, 16(1) 40-4, Review
• nucleic acid molecule refers to a polymer of nucleotides
• Non-limiting examples thereof include DNA (e g genomic DNA, cDNA) and RNA molecules (e g mRNA)
• the nucleic acid molecule can be obtained by cloning techniques or synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense])
• recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments This is often eferred to as genetic engineering
• DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of am o acids which can be referred to as a polypeptide, protein, protein fragment and the like
• amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, whch are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions
• nucleic acid e g DNA or RNA
• physiologically relevant is meant to describe interactions which can take effect to modulate an activity or level of one or more proteins in their natural setting
• DNA molecule or sequence (as well as sometimes the term “oligonucleotide”) refers to a molecule comprised of the deoxy ⁇ bonucleotides adenme (A), guanme (G), thymme (T) and/or cybsine (C), in a double-stranded form, and comprises or includes a "regulatory element” according to the present invention, as the term is defined herein
• oligonucleotide” or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes orsynthetically derived DNA As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction
• Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure
• hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al , 1989, supra and Ausubel et al , 1989, supra) and are commonly known in the art
• a hybridization toa nitrocellulose filter as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 ⁇ g/ml denatured carrier DNA (e g salmon sperm DNA)
• the non-specifically binding probe can then be washed off the filter by several washes in 0 2 x SSC/0 1 % SDS at
• Probes for nucleic acids can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann Reports Med Chem 23 295 and Moran et al , 1987, Nucleic Acids Res , 14 5019 Probes of the invention can be constructed of either nbonucleic acid (RNA) or deoxynbonucleic acid (DNA), and preferably of DNA It is an advantage of the present invention that he detection of the interaction between proteins and/or peptides be dependent on a label Such labels provide sensitivity and often enable automation In one embodiment of the present invention, automation is performed usmgCHIP technology For example, the overlapping peptides, spanning a chosen sequence of a protein, are bound to a CHIP which can then be used to automate a testing for interaction with proteins or peptides Of course
• radioactive nucleotides can be incorporated into peptides or proteins of the invention by several well-known methods A non-limiting example thereof includes in vitro or in vivo labelling of proteins using 35 SMet
• vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be clored Numerous types of vectors exist and are well known in the art
• expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more
• expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host
• the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
• control element sequences such as promoter sequences
• the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences
• Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript
• two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence
• Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transc ⁇ ptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
• Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of nterest This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e g SDS gel electrophoresis, gel filtration, centnfugation, ion exchange chromatography )
• size and charge e.g SDS gel electrophoresis, gel filtration, cent
• the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule
• Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and mtiating transcription of a downstream (3' direction) coding sequence
• the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
• a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
• Eukaryotic promoters will often, but not always, contain "TATA" boses and "CCAT” boxes Prokaryotic promoters contain
• the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether an nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence
• This functional derivative or equivalent may be a natural denvative or may be prepared synthetically
• Such derivatives include ammo acid sequences having substitutions, deletions, or additions of one or more ammo acids, provided that the biological activity of the protein is conserved
• derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained
• the substituting ammo acid as chemico-physical properties which are similar to that of the substituted ammo acid
• the similar chemico-physical properties include, similarities in charge, bulkmess, hydrophobicity, hydrophyhcity and the like
• the term “functional derivatives" is intended to include “fragments", "se
• a conservative mutation or substitution of an am o acid refers to mutation or substitution which maintains 1) the structure of the backbone of the polypeptide (e g a beta sheet or alpha- helical structure), 2) the charge or hydrophobicity of the ammo acid, or 3) the bulkmess of the side chain More specifically, the well-known terminologies "hydrophihc residues” relate to serme or threonine "Hydrophobic residues" refer to leucme, isoleucme, phenylalanine, valme or alanme "Positively charged residues” relate to lysine, arginme or hystidme Negatively charged residues" refer to aspartic acid or glutamic acid Residues having "bulky side chains” refer to phenylalanine, tryptophan or tyrosine
• Peptides, protein fragments, and the like in accordance with the present invention can be modified in accordance with well-known methods dependently or independently of the sequence thereof
• peptides can be derived from the wild-type sequence exemplified herein in the figures using conservative ammo acid substitutions at 1 , 2, 3 or more positions
• conservative ammo acid substitutions is well-known in the art which relates to substitution of a particular ammo acid by one having a similar characteristic (e g aspartic acid for glutamic acid, or isoleucme for leuc e)
• non-conservative ammo acid substitutions can also be carried out, as well as other types of modifications such as deletions or insertions, provided that these modifications modify the peptide, in a suitable way (e g without affecting the biological activity of the peptide if this is what is intended by the modification)
• a list of exemplary conservative ammo acid substitutions is given herembelow
• Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, lie, D-lle, Orn, D-Orn
• variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention
• a variant according to the present invention can be identified with a method of the present invention It can also be designed to formally test for the conservation of particular ammo acids (e g by synthesizing a variant or mutant peptide) These variants can also be tested as part of the full length sequence of the protein in order to validate the interaction
• ammo acids e g by synthesizing a variant or mutant peptide
• these variants can also be tested as part of the full length sequence of the protein in order to validate the interaction
• the skilled artisan will understand that having identified a region of a chosen protein asa region which is involved in high-affinity protein ⁇ nteract ⁇ on(s) enables an in vitro mutagenesis (or a testing of related peptide sequences) ofthis region to identify and dissect the structure/function relation of this region
• Such methods are well- known in the art
• the interaction domains of 2 proteins having been identified, it is thus possible for the skiled artisan to identify and/or design variants having a modified affinity
• chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention
• moieties could affect the physico-chemical characteristic of the derivative (e g solubility, absorption, half life and the like, decrease of toxicity)
• Such moieties are exemplified in Remington's Pharmaceutical Sciences (e g 1980) Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art
• allele defines an alternative form of a gene which occupies a given locus on a chromosome
• a “mutation” is a detectable change in the genetic material which can be transmitted to pursehter cell
• a mutation can be, for example, a detectable change in one or more deoxy ⁇ bonucleotide
• nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutations and experimentally induced mutations exist
• the res ⁇ t of a mutations of nucleic acid molecule is a mutant nucleic acid molecule
• a mutant polypeptide can be encoded from this mutant nucleic acid molecule
• purified refers to a molecule having been separated from a cellular component
• a purified protein has been purified to a level not found in nature
• a “substantially pure” molecule is a molecule that is lacking in most other cellular components
• molecule As used herein, the terms “molecule”, “compound” or “ligand” are used interchangeably and broadly to refer to natural, synthetic or semi- synthetic molecules or compounds
• molecule therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like
• Non limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents
• the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling, combinatorial library screening and the like
• the terms “rationally selected” or “rationally designed” are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention
• macromolecules having non-naturaly occurring modifications are also within the scope of the term "molecule”
• peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above Similarly, in a preferred embodiment, the polypeptidomime
• Libraries of compounds are well-known in the art Libraries of peptides are also available Such libraries can be used to build an overlapping set of peptide sequences spanning a chosen domain, protein or part thereof
• the recitation “indicator cells” refers to cells that express, in one particular embodiment, two interacting peptide domains of the present invention, and wherein an interaction between these proteins or interacting domains thereof is coupled to an identifiable or seectable phenotype or characteristic such that it provides an assessment or validation of the interaction between same
• the mdicat ⁇ cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of these interacting domains
• the cells can be yeast cells or higher eukaryotic cells such as mammalian cells (WO 96/41169)
• the indicator cell is a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (Ausubel et al , 1994, supra) and can be used to test a compound or a library thereof
• a reporter gene encoding a selectable marker or an assayable protein can be operably linked to
• Non-limiting examples of such fusion proteins include a hemaglutmin fusions, Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions
• GST Gluthione-S-transferase
• MBP Maltose binding protein
• it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused Such protease cleavage sites between two heterologously fused polypeptides are well known in the art
• Signal peptides frcm diverse organisms are well known in the art Bacterial OmpA and yeast Suc2 ae two non limiting examples of proteins containing signal sequences
• the interaction domains of the present invention can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better desgn and identification of modulating compounds
• some derivative or analogs having lost their biological function of interacting with their respective interaction partner may still find utility for example for raising antibodies
• Such analogs or derivatives could be used for example to raise antibodies to the interaction domains of the present invention
• These antibodies cculd be used for detection or purification purposes
• these antibodies could also act as competitive or non-competitive inhibitor and be found to be modulators of an interaction identified in accordance with the present invention
• a host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e g a DNA construct) when such DNA has been introduced inside the cell
• the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained
• the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents
• the DNA segmenfe or proteins according to the present invention can be introduced into individuals in a number of ways For example, erythropoietc cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the
• the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (e g DNA construct, protein, molecule), the response and condition of the patient as well as the severity of the disease
• composition within the scope of the present invention contain the active agent (e g protein, nucleic acid, or molecule) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects
• the nucleic acids in accordance with the present invention car be administered to mammals (e g humans) in doses ranging from 0 005 to 1 rrg per kg of body weight per day of the mammal which is treated
• Pharmaceuticaly acceptable preparations and salts of the active agent are within thescope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed Mack Ed )
• the amount administered should be chosen so as to avoid adverse side effects
• the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient Typically, 0 001 to 50 mg/kg/day will be administered to the mammal
• Figure 1 shows the principle of protem-protem interaction
• the plus signs (+) indicate the regions of high-affinity binding
• the minus signs (-) indicate the regions of high-repulsive forces
• Figure 2 is a schematic representation of a method of identification of high-affinity binding sequences according to one embodiment of the present invention A, the different shapes represent different proteins ma total cell lysate
• the signs are like for Figure 1 B, small overlapping peptides that cover the entire sequence (or a segment) of protein A will be synthesized directly on denvatized wells of 96-well polypropylene plates Following peptide synthesis, metabohcally radiolabeled total cell lysate is added to each well containing the various peptides and incubated in an incubator buffer C, The dark filled circles represent the radiolabeled proteins from total cell
• Figure 3 is a schematic representation of a method of identification of high-affinity binding sequences according to another embodiment of the present invention.
• A shows a schematic representation of the interaction between Protein A and Protein B.
• B small overlapping peptides that cover the entire sequence (or a segment) of Protein A will be synthesized directly on derivatized wells of 96-well polypropylene plates. Following peptide synthesis, a radiolabelled Protein B (synthesized from in vitro transcription-translation reaction mix) are added to each well containing the various peptides and incubated in an incubation buffer.
• C the dark filled circles represent the radiolabeled Protein B that has been added to all the wells of the 96-well plates to identify high-affinity binding sequences on Protein A.
• the high affinity binding sequences are in those wells in which Proteh B (radiolabeled protein in dark) is still bound to the peptides from Protein A.
• E four high affinity binding sequences between Protein A and Protein B are identified in rows 1 , 3, 6 and 8. The wells that contain the high-affinity binding sequences are identified by radiolabeled counting and SDS-PAGE;
• Figure 4 is a schematic representation of a method of selection of drugs that specifically inhibit the binding of protein A to B according to one embodiment of the present invention.
• A shows a schematic representation of the interaction between Protein A and Protein B.
• B peptides that encode high-affinity binding sequences are used as LEAD sequences for the selection of specific drugs that inhibit the association between Protein A and Protein B and ultimately the function of the complex.
• Figure 6 shows the protein binding to overlapping hexapeptides encoding P-gp1 linker domain Overlapping hexapeptides that encode the linker domain of HP-gp1 were synthesized on polypropylene rods and used to identify proteins that bind to these peptides A total of 90 plus two control hexapeptides for P-gp1 were incubated with total cell lysate from P 5 S] methionine metabohcally labeled cells (see methods) All bound proteins were eluted from the peptide-fixed rods and resolved on 10% SDS PAGE Lanes 1 to 92 show the p 5 S] methionine bound proteins from P-gp1 The migration of the molecular weight markers is shown to the left of gels
• Figure 7 shows the effects of different detergents or high salt on the binding of proteins to P-gp1 hexapeptides Metabohcally radiolabeled proteins bound to hexapeptides (hexapeptides 50 to 53) from P-gp1 linker domain were eluted in the presence of increasing concentrations of anionic detergent (0 12% - 0 5% SDS), Zwittenonic detergent (20 mM - 80mM CHAPS) or Salt (0 3 M - 1 2 M KCI)
• the y-axis represents the amount of radioactivity eluted from a pool of three hexapeptides (50 to 53)
• Figure 8 shows the effects of CHAPS on the binding of proteins to the overlapping hexapeptides encoding P-gp1 linker domain
• Overlapping hexapeptides of the linker domain of HP-gp1 were incubated with total cell lysate from S] methionine metabohcally labeled cells extracted with 10 mM CHAPS
• Bound proteins were eluted from the peptide-fixed rods and resolved on 10% SDS PAGE
• Lanes 1 to 92 show the p 5 S] methionine bound proteins to P-gp1 linker domain The migration of the molecularweight markers is shown to the left of gels
• Figure 9 shows the protein binding to overlapping hexapeptides encoding P-gp3 linker domain Overlapping hexapeptides that encode the linker domain of HP-gp3 were synthesized on polypropylene rods and used to identify proteins that bind to these peptides A total of 88 plus two control hexapeptides for P-gp3 were incubated with total cell lysate from p 5 S] methionine metabohcally labeled cells All bound proteins were eluded from the peptide-fixed rods and resolved on 10% SDS PAGE Lanes 1 to 90 show the f 5 S] methionine bound proteins from P-gp3 The migration of the molecular weight markers is shown to the left of gels Figure 10 shows the sequence alignment of three binding regions of P-gp1 and P-gp3 linker domains Alignment of P-gp1 and P-gp3 linker domains is shown using a single-letter code for ammo acids The regions of high
• Figure 11 shows the two high affinity binding hexapeptides
• Two high affinity binding sequences 658 RSSLIR 663 and 66E SVRGSQ 67 from P-gp1 linker domain were resynthesized and incubated with total cell lysate from f 5 S] methionine metabohcally labeled cells following 24 hour or 48 hour incubation times
• Bound proteins were eluted from peptide-fixed rods and resolved on 10% SDS PAGE The migration of the molecular weight markers is shown to the left of the figure
• Figure 12 shows the effects of different carrier proteins as blocking agent of unspecific binding
• Total cell lysates from f 5 S] methionine metabohcally labeled CEM cells were used as is or made 1 % gelatin, 0 3% BSA or 3% BSA
• the cell lysates were incubated with a high affinity binding hexapeptide 658 RSSLIR 663 from P-gp1 linker domain
• the bound proteins were eluted with SDS sample buffer and resolved on 10% SDS PAGE The migration of the molecular weight markers is shown to the left of the figure
• Figure 13 shows the purification of a 57 kDa protein Total cell lysate was incubated with fifty P-gp1 hexapeptides 658 RSSLIR 663 and 669 SVRGSQ 674 Samples containing the 57 kDa protan (P57) from one hundred hexapeptide incubation mix were pooled and resolved on 10% SDS PAGE The resolved proteins were transferred to PVDF membrane and stained with Ponseau S The migration of the molecular weight markers is shown to the right of the figure Figure 14 shows the western blot analysis with anti-tubuhn monoclonal antibodies Total cell lysate from CEM cells and proteins eluded from the high affinity binding hexapeptides of P-gp1 linker domain (P57) were resolved on SDS PAGE and transferred to nitrocellulose membrane One half of the membrane was probed with anti- ⁇ and anti- ⁇ tubuhn monoclonal antibodies The migration of the molecular weight markers is shownto the left of the figure
• Figure 15 shows the helical wheel presentations of the high affinity binding region of P-gp1 and P-gp3 linker domains
• the single-letter am o acid code for the high affinity binding region of P-gp1 and P-gp3 linker domains are shown
• the positively charged ammo acids on one side of the helix have been circled
• dimenzation There is an abundance of examples in biology whereby protein-protein interactions are essential for activation or inhibition of function (10)
• Non-limiting examples of homo- or heterodimers include, growth factor receptors (9), membrane transport proteins (2, 7, 18), tumor suppressor proteins (16), and proteins that mediate apoptosis (23)
• dynamic dimenzation is a common theme in the regulation of signa " transduction
• Some of the functional consequences of dimenzation include, increased proximity for activation of single transmembrane cell surface recepto ⁇ (e g , EGF receptor (9)) and differential regulation by heterodime ⁇ zation [e g , BCL2 family of proteins (23)]
• the protein concentration in living cells is very high and is in the range of 10-30 mg/ml At this high protein concentration, most if not all proteins should interact precisely and specifically with other cellular proteins Someof the interacting proteins act as inhibitors of function, while others may be activators [e g The BCL2-BAX family of proteins, (23)] Moreover, the cycling of a given protein between activator and inhibitor association will require the association- dissociation process to occur rapidly For example, when protein X is associated with an inhibitor protein I, the domains (small sequences) that are required for the association of protein X with an activator protein A may not be easily accessible in the X-l complex Therefore, current methods to identify associated protein (i e , the two- hybrid system and similar approaches) may not be able to identify all associated proteins In other words, current methods, when successful, may only identify some but not all functional domains and their associated proteins By contrast, using the peptide scanning approach, the method of the present invention is capable of identifying all functional domains or high-affinity interacting domains of protein X
• the present approach and methodology used to identify discontinuous strings of sequences between two or more interactive proteins is a scanning overlapping peptide approach Using this approach, a large number of short overlapping peptides which cover the entire ammo acid sequence of the a given protein "the bait" are synthesized in parallel on an inert solid support [See Figure 2]
• the rationale for synthesizing a large number of overlapping peptides as opposed to a discontinuous peptide library is based on the fact thd one does not know a priori what exact sequence of a given protein will contain the high affinity binding sites and the repulsive sequences Therefore, a discontinuous peptide approach will often lead to the presence of both high affinity binding sequences and repulsive sequences in the same peptide Such peptides will net bind to potential interacting proteins with high affinity Moreover, the use of overlapping peptides also provides internal controls for unspecific binding For example, using overlapping peptides, the high affinity bindmgsequences will give
• P- glycoprotem is a membrane protein (8) that confers resistance to anticancer drugs and therefore is responsible for the failure of chemotherapy
• P- glycoprotem has been shown to function by preventing the accumulation of chemotherapeutic drugs in tumor cells, the exact mechanism of how this protein functions and what are the associated proteins that modulate its functionare not known
• it is of interest to identify proteins that interact with P-glycoproten such as to enable an inhibition of binding between P-glycoprotein and its associated proteins, thereby potentially modulating its function in resistanttumor cells
• it was of interest to identify those proteins which bind to the linker domain of P-glycoprotein Thus, in this particular example, a domain of a chosen protein was used The linker domain, encodes a region of about 90 ammo acids
• those specific overlapping peptides represent high affinity binding sequences in the P-glycoprotein linker domain (or other chosen domains or non- chosen domains), and
• the associated proteins which bound to the high affinity binding sequences can be isolated in large quantities for the purpose of determining their identity by N-termmal ammo acid sequencing by Edman degradation (28) or the like Briefly, the sequences of the overlapping peptides that bounda given protein are resynthesized on a solid support and kept fixed thereto Total cell lysate from p 5 S]meth ⁇ on ⁇ ne metabohcally radiolabeled cells is added to the solid support containing the fixed high affinity sequence peptides and incubated as described above Following washing steps to remove unbound material, the associated protein is isolated in large amounts following an elution step with SDS-containmg buffers (see below) The purified associated protein is now ready for am o acid sequencing Of course, should further purification steps be required, they are well known to the skilled artisan The purified protein is ran on SDS-PAGE and the resolved protein is transferred to PVDF membrane as previously described (1 ) Other methodsfor ammo acid sequence determination can also be easily applied (26)
• metabohcally radiolabeled proteins as “the prey” to interact with the overlapping peptides encoding "the bait”, increases the sensitivity of this technique and allows the identification of interacting proteins with binding affinities of 10 "10 - 10 12 M for a standard 50 kDa protein which encodes one to ten radiolabeled methionine residues (20) Method to Use High Affinity Binding Sequences in High Throughput Assays to Screen for Lead Compounds
• MDR multidrug resistant tumor cells
• P-gp MDR1 P-glycoprotein
• MRP1 multidrug resistance-associated protein
• the P-gp gene family is made up of three structurally similar isoforms in rodents (classes I, II, and III) and two isoforms in humans (classes I and III) (14) Gene transferstudies suggest functional differences among these structurally similar isoforms For example, only the P-gp isoforms of classes I and II confer the MDR phenotype (18, 73), while theclass III isoforms do not (7, 65)
• the class III isoforms mediatethe transfer of phosphatidylcholme from the inner to the outer leaflet of the plasma membrane (i e , "flipase") (61 , 67)
• P-gp distribution is restricted mainly to tissues with secretory functions (55, 76) Its polarized localization to apical surfaces facing a lumen in the adrenal gland, liver, kidney intestine suggests a normal transport or detoxification mechanism Moreover, hematopoietic stem cells and specific lymphocyte subclasses also express high levels
• MDR-reversmg agents include verapamil, quinidme, Ivermectm, cyclosponns, and dipy ⁇ madol analogues to name but few (23, 26)
• mdr-reversing agents e g , verapamil or quinidme
• P-gp was shown to be a substrate for protein kmases C and A (2 9) Moreover, it has been demonstrated that agents, which modulate protein kmase C activity, modulate P-gp phosphorylation and its MDR-mediate phenotype (4, 8)
• PMA phorbol ester (a protein kinase C activator) was shown to increase the MDR phenotype and drug efflux in MCF7 breast cancer cells
• sodium butyrate treatment of SW620 human colonic carcinoma cells was shown to result in a large increase in P-gp expression without a concomitant increase in drug-resistance or -efflux
• P-gp in SW620 cells was also shown to be poorly phosphorylated following sodium butyrate treatment (3)
• Predenvatized plastic rods, active ester and polypropylene trays were purchased from Cambridge Research Biochemicals (Valley Stream, NY) Peptides were synthesized on solid polypropylene rods as previously described (27, 28) Briefly, the F-moc protecting group on the predenvatized polypropylene rods as solid support (arranged in a 96-well formate) was removed by incubation with 20% (v/v) piperidine in dimethylformamide (DMF) for 30 minutes with shacking Following the deprotection of the ⁇ -alanme spacer on the polypropylene rods, Fmoc protected ammo acids were dissolved in HOBt/DMF and added to the appropriate wells containing deprotected rods Coupling of ammo acids was allowed to take place for 18 hours at room temperature after which the rods were washed in DMF (1X2 minutes), methanol (4 X 2 minutes), and DMF (1 X 2 minutes) The coupling of the second am o acid required
• CEM Drug sensitive
• CEM/VLB 1 ° Drug sensitive cells
• ⁇ -MEM media supplemented with 10% fetal calf serum (Hyclon Inc ) as previously described (6) All cells were examined for Mycoplasma contamination every three months using the Mycoplasma PCR kit from Stratagene Inc San Diego, CA
• CEM or CEM/VLB 1 ° cells at 70-80% confluency were metabohcally labeled with 3 ! S] methionine (100 ⁇ Ci/ml) for 6 hours at 37C in methionine-free ⁇ -MEM media
• hypotonic buffer 10 mM KCI, 1 5 mM MgCj,, 10 mM T ⁇ s-HCI, pH 7 4) containing protease inhibitors (2 mM PMSF, 3 ⁇ g/ml Leupeptm, 4 ⁇ g/ml pepstatin A and 1 ⁇ g/ml aprotinm
• PMSF phosphate buffered saline
• protease inhibitors 2 mM PMSF, 3 ⁇ g/ml Leupeptm, 4 ⁇ g/ml pepstatin A and 1 ⁇ g/ml aprotinm
• f ⁇ S] methionine labeled proteins from total cell lysate were mixed with equal volume of 3-6%BSA in phosphate buffered saline (PBS) and incubated with overlapping hexapeptides covalently fixed to polypropylene rods The peptides and total cell lysate were incubated overnight at 4°C. The rods were then removed and washed four times in PBS.
• PBS phosphate buffered saline
• the bound proteins were eluted by incubating the peptide-fixed rods in 1X SDS sample buffer for 60 minutes at room temperature with shacking
• the peptides-fixed rods were regenerated by incubation in PBS, containing 2% SDS and 1 mM ⁇ - mercaptoethanol at 65°C in a sonicator for 30 minutes Following the latter incubation, the rods were washed for five minutes in 65°C ionized water and two minutes in 65°C methanol
• the peptides-fixed rods are now ready for the next round of screening
• p 5 S] methionine labeled proteins from total cell lysate were mixed with equal volume of 3%BSA in phosphate buffered saline containing KCI (300 mM to 1200 mM), SDS (0 12% to 2%), or CHAPS (20 mM to 160 mM) and incubated with covalently fixed peptides as described
• Protein fractions (100-150 ⁇ l) were resolved on SDS-PAGE using the Laemmh gel system (47) Br/iefly, proteins were dissolved in 1X solubihzation sample buffer I (62 5 mM T ⁇ s-HCI, pH 6 8, containing 2% (w/v) SDS, 10% (w/v) glycerol and 5% ⁇ -mercaptoethanol) and samples were electrophoresed at constant current Gel slabs containing the resolved proteins were fixed in 50% methanol and 10% acetic acid Polyacrylamide gels containing [ 35 S] methionine proteins were exposed to Kodak x-ray film following a thirty- mmute incubation in an AmplifyTM solution (Amersham Inc )
• proteins were transferred to nitrocellulose membrane in Tns-glycine buffer in the presence of 20% methanol for Western blot analysis according to the procedure of Towbm et al , (71)
• Nitrocellulose membrane was incubated in 5% skim milk/PBS prior to the addition of anti- ⁇ or anti- ⁇ tubuhn monoclonal antibodies (0 5 ⁇ g/ml in 3% BSA, Amersham, Inc ) Following several washes with PBS, the nitrocellulose membrane was incubated with goat anti-mouse peroxidase conjugated antibody and immunoreactive proteins were visualized by chemiluminescence using ECL method (Amersham Inc )
• the 57 kDa associated protein was purified using a block of polypropylene rods with two high affinity binding peptides Briefly, the peptide- fixed rods were incubated with total cell lysate as indicated above, however, in this case the carrier substance was gelatin (1 %) The bound proteins were eluded in 100 mM phosphate buffer, pH 7 4 containing 2% SDS and 0 1 % ⁇ - mercaptoethanol The eluted proteins were precipitated by mixing with 9 volumes of ice cold ethanol and incubated at -20 °C Following a high speed centnfugation of the latter sample (15 minute cent ⁇ fugationat 15,000 Xg, at 4°C), the precipitated proteins were resuspended in 1%SDS in PBS and mixed with equal volume of 2X SDS Laemmli sample buffer (47) Protein samples were resolved on 10% SDS PAGE and transferred to PVDF membrane The migration of the 57 kDa band was visualized by staining the PVDF membrane with pons
• P-gp is a tandemly duplicated molecule made up of two halves with each encoding forsix transmembrane domains and an ATP binding domain The two halves of P-gp are linked by a linker domain
• the P-gp gene family in man is encoded by two isoforms, P- gp 1 and P-gp 3 (or mdr 1 and mdr 3, (14)) However, as indicated earlier, only P-gp 1 confers an MDR phenotype Moreover, although P-gp 1 and 3 share about 80% am o acid sequence homology (73), the linker domain is the most variable domain among the two isoforms with 66% ammo acid sequence homology. To determine if the P-gp 3 linker domain binds to the same or different proteins, overlapping hexapeptides encoding P-gp 3 linker domain were synthesized on polypropylene rods and their binding to soluble proteins was examined as indicated above Figure 9 shows the profile of binding proteins to the hexapeptides of P-gp 3 Interestingly, a similar molecularweight protein (57 kDa) also bound to the hexapeptides from P-gp 3 However, the binding to some hexa
• Figure 12 shows the effects of no blocking carrier, 1 % gelatin and 0.3% or 3% BSA on the binding of the hexapeptides to the 57 kDa protein. These results of this experiment were surprising in that no carrier protein was required to reduce the unspecific binding (figure 12). The latter established binding conditions were used to isolate large amounts of 57 kDa protein that bound to several copies of hexapeptides 658 RSSLIR 663 and 669 SVRGSQ 674 .
• Figure 13 shows purified 57 kDa protein on SDS-PAGE stained with Coomassie blue.
• the latter purified protein was transferred to PVDF membrane and stained with Ponceau S to localize the position of the 57 kDa protein.
• the Ponceau S stained band that migrated with the expected molecular mass was cut out and used for direct N-terminal sequencing (22).
• the first seven rounds of Edman degradation showed two sequences of MREVISI and MREIVHI. These two sequences differed only by three amino acids (VIS instead of IVH). Comparison of the two sequence with known protein sequences using FastA protein search engine, showed the latter sequences to encode the first seven N-terminal amino acids of ⁇ - and ⁇ -tubulins.
• tubulins as the 57 kDa protein was consistent with the apparent molecular mass and the potential degradation products that were observed following long incubation periods
• Western blot analysis was preformed on hexapeptide-bound 57 kDa protein and total cell lysate resolved on SDS PAGE and transferred to nitrocellulose membrane The nitrocellulose membrane was then probed with anti ⁇ -tubuhn and anti- ⁇ -tubuhn monoclonal antibodies, respectively
• Figure 14 shows the results of the Western blot analysis Consistent with the sequencing results, both tubuhn subunits ( ⁇ and ⁇ ) were recognized in the lanes containing the hexapeptide bound proteins
• ⁇ and ⁇ -tubuhn both tubuhn subunits ( ⁇ and ⁇ ) were recognized in the lanes containing the hexapeptide bound proteins
• the overlapping peptide-based method of the present invention provides the proof of principle to the hypothesis which states that the region between two interacting proteins consists of high affinity binding sequences and repulsive sequences as well as the fact thatsuch a method can be used efficiently and successfully to identify and characterize domains and sequences of interacting proteins
• the balance of high affinty and repulsive forces determine whether two proteins will form stable complex
• the use of short overlapping peptides allows the identification of such high affinity binding sequences between bait and prey proteins
• the rationale for using short and overlapping peptides to isolate high affinity binding sequences is essential to the success and efficiency of the proof of the principle described herein For instance, larger peptides could contain both high affinity and repulsive binding sequences in one peptide sequence such that the net force of interaction is negative Moreover, the use of overlapping peptides that differ in one ammo acd from the previous or next peptide reduces
• the binding of 57 kDa protein to three different regions in P-gp1 and P-gp3 linker domains is consistent with the herein proposed hypothesis to explain protein interactions (see principle of protein-protein interactions)
• the high affinity binding domains vary in sizes from 10 -26 ammo acids in length
• two of the three high affinity binding domains shared considerable sequence identity
• the third high affinity binding region of the linker domains f 58 SRSSLIRKRSTRRSVRGSQA 67 versus 648 KAATRMAPNGWKSRLFRHSTQKNLKNS 674 ) shared no homology in their primary ammo acid sequence
• helical wheel presentation of these two domains show a cluster of positively charged residues on one face of the helix while a cluster of serine/threonme residues on the other side (see figure 15)
• the region of highest binding affinity to the 57 kDa protein encodes the three putative phosphorylation sites in P-gp
• EXAMPLE 11 The overlapping peptides spanning method is not limited to Pgp- interacting proteins
• the overlapping peptide approach of the present invention has been further validated with Annexin I, a soluble and membrane associated protein, as opposed to P-glycoprotein, a st ⁇ ctlytransmembrane protein Annexin is thus structurally and functionally different from P-glycoprotein
• Annexin I is a member of a large family of intracellular soluble and membrane associated proteins that bind phosphohpids in a reversible and calcium-dependent manner
• Various members of the Annexn family have been implicated in a number of different intracellular processes including vesicular trafficking, membrane fusion exocytosis, signal transduction, and ion channel formation and drug resistance
• the method of the present invention was setout to identify its interacting proteins and the precise ammo acid sequences that mediate Annexin I interactions thereto
• overlapping peptides corresponding to the entire ammo acid sequence of Annexin I were synthesized on a solid support as described above In this case, overlapping heptapeptides, as opposed to hexapeptides were used The peptides were then incubated with total cellular proteins isolated from MCF7 breast tumor cells that were metabohcally labeled with [35S] methionine Following several washes, the bound proteins were eluted and resolved on SDS- PAGE as outlined above The results are consistent with previous results with P-glycoprotein, as the method leads to the identification of several islands of Annexin I am o acid sequences (data not shown) which interacted with five proteins ranging in molecular masses from 10 kDa to 200 kDa (specifically, -10 kDa, -29 kDa, -85 kDa, -106 kDa and -200 kD
• the present invention is shown to enable the simple and efficient identification of high-affinity protein interaction as well as enabling the simultaneous identification of the precise ammo acid sequence of at least one of the interacting partners

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