Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • 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
  • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the invention relates to gene discovery, in particular, identification of gene function.
• the invention concerns the use of phage display techniques to monitor ligand/receptor interactions where the ligand is a typical small molecule pharmaceutical.
• Phage display is a widely used method for identifying proteins and the genes encoding them which bind to other proteins.
• T7 is commercially available from Novagen and a detailed description of the use of T7 phage is provided in the company's T7 Select ® System Manual and is described in U.S. patents 5,223,409; 5,403,484; 5,571,698; and 5,766,905, all incorporated herein by reference.
• the present invention extends the use of phage display to the interaction of cDNA libraries using traditional pharmaceutical-type small molecules as bait. This extension permits the direct identification of gene functionality by identifying phage which comprise nucleotide sequences encoding proteins that bind non-peptide small molecule ligands which are of known physiological function.
• molecules of unknown function can be used to ascertain their protein targets.
• Such molecules may be natural products, for example, or may be members of a combinatorial library.
• the invention method provides a direct approach to identify proteins and genes encoding them, which proteins interact with a specific small molecule ligand.
• the phage containing nucleotide sequences that encode proteins capable of interacting with a specific small molecule bait can be "pulled out" of, for example, a cDNA library and identified.
• the successful phage can be amplified and sequenced, thus identifying the gene encoding the interacting protein and providing the ability to deduce the amino acid sequence of the protein.
• the invention is directed to a method to identify a nucleotide sequence encoding a protein that binds a non-peptide target which method comprises amplifying and sequencing the nucleotide sequence contained in a bacteriophage, wherein the phage displays a protein which binds to the non-peptide bait.
• the phage is identified by a method which comprises contacting a phage display library comprising a multiplicity of DNA fragments encoding candidate proteins for said binding with said non-peptide bait, the non-peptide bait is coupled to a solid support under conditions wherein said non-peptide bait interacts with a displayed protein to which it binds specifically; and, if necessary, washing said solid support to remove non-specifically bound phage.
• the specifically bound phage is then removed by elution and amplified and the DNA insert encoding the displayed protein sequenced.
• the invention is directed to a method to identify a compound which binds a protein identified by the above-recited method of the invention which method comprises contacting candidate compounds with the protein thus identified and assessing the ability of the compounds to interact with said protein.
• the invention is directed to compounds so identified and to methods of treating conditions characterized by the need to modulate the activity of the protein by administering the identified compound.
• Figure 1 shows a diagram of the method of the invention with one round of amplification.
• Figure 2 shows, schematically, the synthesis of biotinylated FK506.
• Figure 3 is a schematic representation of the T7 display vector and a diagram of the modified T7 gene.
• Figures 4A and 4B show a model combinatorial compound and a schematic for the synthesis of a benzyl cycloadduct as a scaffold supported bait.
• Figure 5 shows the coupling of the benzyl cycloadduct to an affinity support.
• the invention generally employs a phage display system to provide an exposed protein for assay of the interaction with a small molecule.
• the small molecule is coupled to a solid support, preferably, but not necessarily, through a coupling system that is subject to elution using reagents which are independent of the nature of the small molecule.
• the successfully interacting phage are thus selectively bound to the solid support and after washing the support if necessary to remove un-bound phage, can be eluted and recovered for amplification and sequencing.
• phage display systems involve incorporating genes encoding non-viral proteins into the viral genome so as to result in the production of fusion proteins between the desired foreign proteins and a coat protein of the virus.
• the foreign protein when the virus replicates, the foreign protein is displayed at its surface. Viral replication occurs in host cells. In a typical phage display library, a multiplicity of DNA-encoding foreign proteins is inserted and thus viral particles displaying a multiplicity of proteins is produced. This library of displayed proteins is then contacted with "bait" typically bound to a solid support. The successfully interacting displayed proteins will then preferentially be retained on the solid support and the non-binding proteins washed away. The bound protein-displaying phage are then eluted and amplified by culturing them in cells to produce a library enriched in proteins which bind the bait.
• FKBP12 FK506 binding protein
• a particularly useful combinatorial library is disclosed herein.
• the library is based on a scaffold shown in formula 1 of Figure 4A where the defined portions of the molecule comprise the scaffold and R1-R 5 represent variable substituents which can be manipulated at will.
• the scaffold containing the desired substituents can conveniently be coupled to a solid support through a linker such as that shown in Figure 5.
• the coupling of the bait to the solid support is covalent and the elution of the specifically bound phage comprises the use of a solution containing a compound which mimics the bait and thus displaces the coupled specifically bound phage or a change in solvent conditions.
• a successful experiment resulting in retrieval of a compound which binds a benzyl cycloadduct of formula 1 is set forth below in Example 2.
• cDNA libraries can be assembled from a variety of sources, including single tissues such as brain, liver, stomach, prostate tissue, breast tissue, and the like from a variety of mammals or can be derived from any source, including yeast, invertebrates, plants, or prokaryotes. Libraries of the greatest interest are derived from mammals, particularly humans.
• the tissue may be derived from an organism which is considered "normal,” or the organism or tissue may exhibit an abnormal condition, such as inflammation, tumor growth, hypertrophy, and the like. Techniques for preparing cDNA libraries are well known and such libraries can be obtained from very small samples, including single cells. Cell cultures may also be used.
• Various cDNA libraries are also available commercially.
• the phage system can be selected from a variety of well known display systems, including the classical Ml 3 system as well as ⁇ phage, T4 and T7. Particularly preferred are the class of lytic phage, and in particular those in which the inserted nucleotide sequence tails the nucleotide sequence encoding a coat protein.
• the advantage of such tailed inserts is that the presence of a stop codon in the inserted nucleotide sequence does not prevent display of the encoded amino acid sequence as a contiguous fusion.
• the advantage of lytic phage resides in the ease with which the displayed protein can contact a selection means.
• One disadvantage of the Ml 3 systems is that the phage containing the display protein must be secreted from E. coli in order to contact the selection system, thus conferring an advantage on normally secreted proteins, for example.
• lytic phage because the host cell is lysed, can be immediately contacted with, for example, an affinity support.
• the target protein In order for the method to be successful, the target protein must be displayed on the phage at a sufficient level that it can successfully be retrieved by the small molecule bait.
• the phage display library will contain no more than 10 7 different displayed proteins, preferably no more than 10 6 different displayed proteins, more preferably no more than 10 5 different displayed proteins, and most preferably no more than 10 4 different displayed proteins.
• the selecting "bait" is a small molecule in the classical sense of that term.
• the molecule includes, for example, pharmaceuticals which are natural products or which are prepared synthetically, but which are not peptides or proteins.
• the non-peptide bait includes polyketides such as FK506 and erythromycin, synthetic drugs such as cefaclor, steroids such as finasteride, and the multiplicity of non-peptide molecules found in the L ⁇ .S Pharmacopoeia, the contents of which are incorporated herein by reference, to supply a non-limiting list of small molecules included within the scope of the invention.
• the small molecule bait need not be a pharmaceutical or natural product of known function, but can be a new compound or an arbitrarily chosen compound whose function in biological systems, if any, is not known.
• a particularly useful source of such molecules is a combinatorial library wherein a multiplicity of compounds on a similar scaffold is constructed.
• the small molecule bait is used to select a phage bearing a nucleotide sequence which encodes a protein that interacts with the bait by contacting a suspension of phage particles with a solid support to which the bait is coupled.
• the solid support can be any of a multiplicity of such supports, including agarose, polystyrene or other polyvinyl compounds, magnetic beads, and the like.
• the bait may be coupled covalently to the support or may be noncovalently bound by a system which permits the release of the entire complex between the bound phage and bait.
• a column derivatized with N-hydroxysuccinimide can be used to couple covalently a carboxylic acid function on the bait molecule.
• the complex can be eluted in a manner not specific to the bait - for example, by supplying an excess of the linker.
• Illustrated below is a system wherein the bait is covalently bound to biotin which serves as a linker to noncovalently adsorb to an avidin derivatized column. After washing with buffer to remove non-bound phage, the phage/bait complex can be removed by treating the column with excess biotin.
• the bait may be covalently coupled to other linkers such as polyhistidine which noncovalently associates with nickel chelates permitting removal or elution using excess polyhistidine, or can be coupled to glutathione which associates with a glutathione-S-transferase system coupled to the support or vice versa.
• linkers such as polyhistidine which noncovalently associates with nickel chelates permitting removal or elution using excess polyhistidine, or can be coupled to glutathione which associates with a glutathione-S-transferase system coupled to the support or vice versa.
• a first round selection occurs when the phage display library containing the cDNA nucleotide sequences is contacted with the affinity support coupled to the non- peptide, small molecule bait, washed if necessary with, for example, buffer to remove non-specifically bound phage, and then eluted according to a procedure based on the mechanism used for coupling the bait to the affinity support.
• the eluted and thus selected phage members of the library can then conveniently be assessed for homogeneity by, for example, size separation of the cDNA inserts.
• the selected phage from the first round can be amplified and sequenced using standard techniques. Additional rounds of selection involve the same steps of contact with affinity support labeled with bait, wash, elution, and analysis, including, if desired, amplification and sequencing.
• the number of rounds of selection desirable will depend on the purpose of the experiment. As shown below, only two rounds of selection were required to obtain an apparently homogeneous population of a binding protein with a high affinity for the
• FK506 bait More weakly bound proteins may be obtained by analyzing the results of the first round of selection. On the other hand, if only weakly binding proteins exist in the library, several rounds of selection may be necessary to retrieve them with sufficient probability.
• Retrieval of a phage which binds specifically to a small molecule ligand is useful in many contexts. First, it provides a direct method of revealing the cellular targets of small molecules, including natural products. This permits classification of proteins into groups as well as the identification of interactions that may have previously been unknown with respect to a pharmaceutical, thus revealing the nature of any side effects exhibited by administering the drug. Identification of the molecular targets involved in these side effects permits the design of formulations and co-administered drugs to alleviate such effects.
• Identification of additional targets for a pharmaceutical or other compound may reveal desirable interactions of these molecules with target proteins as well.
• a pharmaceutical or other compound such as a member of a combinatorial library
• the identified protein may be used in screening assays for identification of compounds which also interact with it.
• Combinatorial libraries can readily be screened against such proteins, as well as can individual compounds. This permits identification of successful potential therapeutics which can also be assessed using the same methods for their side effects or lack thereof. For example, identification of the receptor which interacts with a pharmaceutical known to reduce inflammation would permit screening for other anti-inflammatory drugs.
• the invention is directed to compounds identified by these screening assays and to methods to treat conditions mediated by the target proteins identified by the method of the invention.
• Bovine brain has traditionally been a good tissue source for the FKBP protein [Harding, M.W., et al, Nature (1989) 341 :758-760]. As display cloning only requires a small amount of mRNA for library construction, human brain was chosen as the mRNA source. Purified human brain poly A + mRNA (obtained from normal whole cerebral brain tissue of a 50 year old Caucasian male, Clontech, Inc., Palo Alto, CA) was randomly primed in the first-strand synthesis followed by second-strand synthesis to yield double-stranded complimentary DNA. The cDNA was doubly digested with EcoRI and Hindlll endonucleases and size fractionated.
• the purified cDNA was then directionally ligated into T7 Selectl-lb vector arms and subsequently in vitro packaged into phagemids (Novagen, Madison, WI).
• a diagram of the manner in which the DNA segments are inserted is shown in Figure 3.
• the insert is downstream of the code protein (CP10) so the presence of a stop codon in the cDN A transcript does not destroy the ability of the phage to display the protein.
• Ligation efficiencies were evaluated with a small aliquot of the packaged phage and indicated a total of 3.3 x 10 6 plaque forming units.
• the culture was then inoculated with 10 ml (9 x 10 phage) of the human brain cDNA library stock described above.
• the culture was incubated at 37°C until completely lysed.
• a bio-affinity column was prepared based on monomeric avidin bound to agarose, which has a relatively low affinity for biotin (K d approximately 10 "7 mol). This permits selective release of any biotin-bound species. The elution is thus independent of the FK506-protein interaction because the elution depends on competition of biotin for the entire complex containing biotinylated FK506 and phage.
• each member of the cDNA library is represented by approximately 10 5 phage.
• the cDNA phage supernatant (100 ml) was poured over each column, followed by washing with PWB (100 ml) and distilled water (10 ml).
• the elution step for each column was identical and involved treatment with 10 ml of 5 mM biotin and collecting the eluate in 0.5 ml aliquots. A small amount (10 ml) from each fraction was used for titer determination.
• the initial fractions from the avidin column contained a phage titer of approximately 40 x 10 4 pfu as compared to only 5 x 10 4 pfu in the initial fractions of the biotin pretreated column.
• the titers rapidly declined in subsequent fractions of the avidin column.
• the FK506 biotin treated column showed an elevated titer relative to the biotin control column in later fractions.
• fractions from the FK506 selection were combined and used to inoculate 100 ml of log phase BLT5403 cells and incubated at 37°C until completely lysed.
• An FK506-biotin affinity column was prepared as described above and the remaining 25 ml of the amplified phage were poured over the column. The column was washed with PWB (100 ml) and distilled water (10 ml), then eluted with
• DNA was isolated from the phage supernatants using standard lambda DNA purification resin (Wizard Lambda Preps DNA Purification System, Promega Corp., Madison, WI). Two primers, T7-up (5'-GGAGCTGTCGTATTCCAGTC-3') and T7- down (5'-AACCCCTCAAGACCCGTTTA-3') anneal to the vector sequence immediately flanking the cloning site and were used with standard PCR conditions to evaluate the relative size of the insert region of the isolated clones.
• T7-up 5'-GGAGCTGTCGTATTCCAGTC-3'
• T7- down 5'-AACCCCTCAAGACCCGTTTA-3'
• the synthesis of the small molecule bait which is a benzyl cycloadduct is outlined in Figure 4B.
• the diazoimide (3) was prepared by the procedure of Savinov, S.N., et al, Chem. Commun. (Cambridge) (1999) 1813-1814.
• the benzyl cycloadduct was then coupled to an agarose support which had been activated with an N-hydroxy succinimide and linked with aminocaproic acid as outlined in Figure 5.
• Treatment of (4) with hydrazine yielded hydrazide (5).
• the N- hydroxy succinimide (NHS) activated aminocaproic acid linked agarose support (3.3 mg, 0.2 mM) was treated with hydrazide (5) (20 ml, 100 mM, 50% DMSO/water) to provide benzyl scaffold agarose (6).
• the human brain cDNA library described in Example 1 was used to inoculate log phase BLT5615 cells (Novagen, Inc.), containing 1 mM IPTG. The culture was incubated at 37°C until lysis was observed. The lysate was immediately treated with a protease cocktail (1% v/v 8.6 mg Sigma P8465 in 200 ml 20% DMSO/water) and adjusted to 10 mM DTT, 1% BSA, and 0.5 M NaCl. The solution was centrifuged at
• a 1 ml aliquot of the freshly prepared phage lysate was allowed to batch incubate with the probe support (6) (10 ml) for 30 minutes.
• the support was column washed with 1 ml of phage wash buffer (PWB) (0.05% Tween-20 in PBS) followed by batch incubation with 500 ml PWB for 5 minutes.
• PWB phage wash buffer
• the bound phage were batch eluted by treating the support with 100 ml of a 10 mM solution of amide (2) in Luna Broth containing 1% BSA for 5 minutes.
• the overall first round phage titer for the specific elution with scaffold 2 was 6xl0 5 pfu, (plaque forming units) while the titer of the last wash was 7xl0 5 pfu.
• the lack of significant titer increase was due to the wash step using a small amount of detergent (0.01% Tween-20), which contributes to a higher titer in the wash solution.
• a second round of affinity selection was performed to allow the amplification of any binding proteins of lower affinity that might require more than one round of selection in order to be detected.
• the second round specific elution titer increased to 33xl0 5 pfu, while the last wash titer remained low (6xl0 5 pfu), indicating a potential phage enrichment.
• a confirmatory experiment was performed to validate the ability of the ⁇ domain of Fi ATP synthase to bind the benzyl cycloadduct.
• two phage systems were mixed in a 1 : 1 ratio - a phage containing no insert and expressing no surface protein and a phage expressing the Fi ⁇ ATP synthase at its surface.
• the mixture was subjected to one round of selection as described above and assayed, resulting in an approximate two-fold increase in the Fj ⁇ ATP phage as compared to control.

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