EP0990051A1 - Identification de cibles moleculaires - Google Patents

Identification de cibles moleculaires

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Publication number
EP0990051A1
EP0990051A1 EP98945752A EP98945752A EP0990051A1 EP 0990051 A1 EP0990051 A1 EP 0990051A1 EP 98945752 A EP98945752 A EP 98945752A EP 98945752 A EP98945752 A EP 98945752A EP 0990051 A1 EP0990051 A1 EP 0990051A1
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Prior art keywords
library
protein
peptide
ligand
members
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German (de)
English (en)
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Lee Makowski
Diane R. Makowski
Hitesh J. Sanganee
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Florida State University
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Florida State University
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    • 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
    • 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
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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

Definitions

  • the present invention is directed, in general, to a method for the identification of the molecular targets for drugs or toxins in an organism or other biological system.
  • ligands Most drugs or toxins express their activity by binding to proteins. These proteins are referred to as receptors, drug targets or molecular targets (Gies, 1996). Drugs (pharmaceuticals), toxins and other biologically active molecules will be referred to herein as ligands. Identification of the ligand target is the crucial first step in understanding how a ligand affects a biological system. Currently, this identification is usually a long and arduous process. The identification of a ligand' s target is desirable, however, because it provides essential information for the improvement of the drug or assessment of toxicity or side effects.
  • a drug Prior to human testing of a new drug, a drug is tested on animals to evaluate its toxicity. The success of these toxicological screens depends on the efficacy with which the animal model mimics the human systems to be effected. If the molecular targets of the animal are essentially identical to those of humans, the toxicological evaluation in an animal will be an accurate guide to the toxicity of the drug in humans. This is, however, not universally true. Many drugs and toxins are highly species dependent in their action (for instance, aspirin is toxic to mice, Ohdo, et al., 1995). If a list of potential human molecular targets were available prior to testing in animals, one could choose a more appropriate test animal.
  • one potential target of a drug is the enzyme hexokinase (the first enzyme in the glycolytic pathway)
  • the sequences of human and mouse hexokinase could be compared; if these sequences are similar at the postulated drug binding site then a mouse is an acceptable model for the evaluation of the effect of a drug on glycolysis; if not, then use of another animal model would be indicated. Consequently, the ability to predict potential drug binding sites in advance of animal testing would aid in the design and evaluation of toxicological screens. Furthermore, during clinical trials, a list of potential targets would simplify the evaluation of adverse effects of the drug.
  • the gene for the protein may be recovered, cloned into an expression vector, and later sequenced. Although this process will yield the identity of the protein suspected of binding to the ligand, the steps of cell culturing, purification, peptide sequencing, and probing for hybridization of the gene of interest, are all costly and time consuming.
  • the present invention is directed to a process for the identification of a protein which binds to a ligand, the ligand having a molecular weight which is less than 5,000 Daltons and being other than a peptide or protein.
  • the ligand is screened against a library of peptides or proteins each of which is displayed on the surface of a genetic package that contains the corresponding nucleic acid sequence to identify the members of the library which have an affinity for the ligand which is greater than the affinity possessed by other members of the library.
  • Each member of this peptide or protein library is physically linked to a nucleic acid polymer which encodes that member by a genetic packages, which also allows the peptide or protein to interact with the ligand.
  • Those members of the library which have an affinity for the ligand which is greater than the affinity possessed by other members of the library are separated from the library and the nucleic acid sequences which encode these members are determined and translated into peptide sequences or consensus peptide sequences. Proteins which contain the peptide sequences or which correspond to the consensus peptide sequences are then identified by searching protein sequence databases.
  • Ligand as used herein means a small molecule (under 5 kD) which is capable of binding to a protein, preferably other than a nucleic acid, peptide or protein.
  • Protein as used herein means an unbranched amino acid polymer, ranging in size from 3 amino acids (the lowest number for which consensus sequence information would be useful) to any length. Some very long peptides, as defined here, would sometimes be referred to as “proteins” by persons skilled in the art.
  • Genetic package as used herein means any mode of connecting a protein fused with a peptide from a peptide library with the genetic information encoding the peptide fused, while presenting that peptide in such a manner that it may interact with a ligand of interest.
  • a non-exhaustive list of genetic packages includes: phage peptide presentation systems, bacterial pilus presentation systems, yeast surface protein presentation systems, plasmid DNA binding fusion protein systems, and other like modes.
  • Platinum agent as used herein means any molecule which can be used to fix a conjugated molecule to a solid support, including molecules which comprise a solid support.
  • Amplification as used herein means the replication of the genetic package displaying a member of a peptide library and containing DNA encoding that member of the library.
  • Texane as used herein denotes compounds containing the A, B and C rings (with numbering of the ring positions shown herein):
  • FIGURE 1 shows the number of amino acids in contact with a bound ligand for selected proteins, as determined an analysis of their three dimensional structure, with a "contact" criteria of a maximum separation of 4 angstroms and 5 angstroms.
  • FIGURE 2 shows the ELISA data demonstrating binding of Bcl-2/GST and Bcl-X L to taxol and various controls.
  • Bcl-X L lacks homology to SEQ. ID NO. 3 in the putative binding site.
  • FIGURE 3 shows the circular dichroism spectrum of Bcl-2/GST with and without taxol. This spectrum shows that the Bcl-2/GST fusion protein undergoes a substantial conformational change in the presence of taxol, unlike GST alone.
  • FIGURE 4 shows the results of the dioxin binding ELISA experiments of example 3.
  • SEQ ID NO. 1 lists the sequence of a tetramer consensus peptide sequence identified from the second round of affinity selection enrichment in example 1.
  • SEQ ID NO. 2 lists the sequence of a tetramer consensus peptide sequence identified from the second round of affinity selection enrichment in example 1.
  • SEQ ID NO. 3 lists the sequence of a pentamer consensus peptide sequence identified from the second round of affinity selection enrichment in example 1.
  • SEQ ID NO. 4 lists the sequence of a pentamer consensus peptide sequence identified from the second round of affinity selection enrichment in example 1.
  • a ligand on a molecular target is dependent on its energy of interaction with the target, or binding energy. This is usually characterized as a binding constant or a dissociation constant.
  • a dissociation constant is used.
  • K D the dissociation constant
  • K D [P] [L] / [PL] (2) wherein [P] and [L] are the concentrations of protein and ligand, respectively, and [PL] is the concentration of the protein-ligand complex.
  • [P] and [L] are the concentrations of protein and ligand, respectively, and [PL] is the concentration of the protein-ligand complex.
  • the most intuitive characterization of K D is that it corresponds to the concentration of ligand at which half of the proteins retain bound ligand.
  • Physiologically relevant binding constants are generally smaller than 10-50 micromolar, and are often in the nanomolar range. Small molecules bind to the accessible surface of a protein, usually in a pocket or groove where the contact area between the ligand and the protein will be larger; the larger the contact area, the greater the number of bonds that can be made between the ligand and the protein, and the smaller the dissociation constant.
  • a protein is a polypeptide chain consisting of an unbranched polymer of amino acids. To form a native conformation, the protein chain folds into a complex structure in which amino acids from different portions of the amino acid chain interact with one another. Usually, but not always, the binding site is made up of amino acids from several different regions of the protein chain. Sometimes the presence of the ligand can stabilize disordered portions of the protein chain as it wraps around the ligand.
  • the first step in the identification of potential ligand targets is the screening of peptide or protein libraries for sequences that exhibit relatively high affinity for a particular ligand compared to other sequences in the library.
  • Most protein or peptide libraries are expressed on the surface of a genetic package which provides a base protein on which the library peptide is presented.
  • a protein or foreign peptide may be displayed on the surface of a phage, bacterium, yeast cell or other genetic package through the insertion of nucleic acid corresponding to the sequence of the peptide or protein into the genome (DNA or RNA) of the genetic package or vehicle of choice.
  • a physical or logical connection between each peptide and the nucleic acid that encodes the peptide is desirable. Usually, this is accomplished by fusing the DNA sequence corresponding to a peptide with the gene encoding a surface protein of the genetic package.
  • a genetic package with a foreign protein or peptide on its surface may be propagated. After rounds of screening for affinity to the ligand of interest and reculturing bound candidates, such a connection allows identification of the genetic material encoding interesting peptides.
  • proteins or peptides with the desired binding properties are obtained, their sequences can be obtained by sequencing the corresponding nucleic acid within the genetic package. The sequences thus identified may correspond to sequences within proteins that bind to the ligand. To optimize the probability of obtaining positive results, several libraries representing numerous scaffolds should be used.
  • the first are random peptide libraries, like that used in example 1.
  • all, or a portion of, the nucleic acid inserted into the phage genome is randomized (e.g. by chemical synthesis of partially or completely random oligonucleotides then inserted into the genome). This technique is typically used to generate 5-12 random amino acids on the surface of a phage particle.
  • the condensation of the trimers to form the oligocodons is done essentially as described for conventional synthesis employing activated mononucleosides as building blocks. See generally, Atkinson and Smith, 1984, Oligonucleotide synthesis (J.J. Gait, ed.), pp. 35-82. This procedure generates a population of oligonucleotides for cloning that is capable of encoding an equal distribution (or a controlled unequal distribution) of the possible peptide sequences, and minimizes the accidental synthesis of stop codons. Schatz, et al., U.S. Pat. No. 5,498,530.
  • Screening a random library requires sequencing of a large number of clones in order to identify a consensus sequence appearing in several clones.
  • the multiple appearance of a sequence or similar sequences in clones isolated from a random library is required to distinguish a sequence with high affinity relative to the remainder of the library from one with relatively low affinity that has been isolated by chance due to, for instance, non-specific binding during affinity selection.
  • Identification of commonly occurring sequences which are the result of desirable growth properties as opposed to desirable binding properties can be accomplished by the statistical analysis of one hundred randomly chosen members of the library.
  • Computer software which screens these one hundred random sequences with over twenty amino acid property scales (including such properties as hydrophobicity, flexibility, etc.) detects inherent biases in each library and identifies library members which predominate in number due to advantageous growth properties.
  • the consensus binding sequence identified by screening of random peptide libraries does not, usually, constitute the entire random insert.
  • the remainder of the randomized insert provides for variation in the scaffolding around the consensus sequence and/or completion of the binding site. For instance, a 5 amino acid consensus embedded in a 12 amino acid insert will be attached to as many as 8x20 7 (1.024xl0 10 ) possible scaffolds in the library.
  • a pentapeptide can be displayed in an extraordinarily large number of conformations. Some of these could correspond to the conformation that it adopts on the surface of a protein that constitutes a natural target for the ligand.
  • a second method for constructing libraries is to insert DNA from a cDNA library.
  • a cDNA library is constructed from the messenger RNA within a particular tissue, and may even be constructed by PCR from a single cell. The mRNA is isolated from the tissue, and a reverse transcription is used to synthesize the DNA that corresponds to the sequence of the mRNA.
  • Successful screening of a cDNA library provides a list of potential target proteins, all of which correspond to complete, expressed proteins.
  • the sequence of the identified protein is in the sequence data bases, the information about binding will add to the information already known about the protein.
  • the protein sequence is not in the data base, the sequence will represent the identification of a new protein. False positives can arise from misfolded proteins binding to ligand in non-physiological ways and proteins that bind non-specifically to the ligand.
  • False negatives can arise from proteins that are misfolded and do not bind in their normal physiologically relevant fashion, from proteins not represented in the library due to non-expression at the time the mRNA was reverse-transcribed, and from proteins that are censored from the library because they are fatal to the system expressing them.
  • Successful screening of a random peptide library will result in the identification of one or more consensus sequences that exhibit affinity to the ligand. Proteins that are already characterized and contain this sequence will be identifiable by a search of the sequence data banks such as GenBank and SWISS-Prot. Some of the identified proteins will contain the consensus sequence in a conformation that will not bind the ligand; these are false positives. Other proteins may display the sequence in a manner that does bind to the ligand.
  • ligand-binding proteins can exhibit binding constants that may or may not fall within the range of physiological relevance. False negatives will occur for all proteins not yet sequenced and placed in the data bases: however, the number of false negatives will decrease substantially as the work of the Human Genome Project continues. Other false negatives will occur when the libraries that are screened do not adequately mimic the environment of the ligand binding site in a protein.
  • a cDNA library has both advantages and disadvantages over random libraries. Every clone isolated in the screen corresponds to a protein, so the number of irrelevant sequences obtained is small. And usually the cDNA library can give you the entire sequence of the protein target. However, only proteins being actively expressed in a cell have a chance of being detected in the screen; an unknown number of proteins will fold incorrectly on the surface, and not be detected (or provide false positives); membrane proteins or proteins that form large macromolecular assemblies are unlikely to fold properly on the phage surface; and proteins from other species, early development proteins or rarely expressed proteins would each need to be screened for with different libraries.
  • Peptide libraries displayed on a genetic package can be screened for peptides that display a relatively high affinity for a particular ligand by a variety of affinity purification processes including biopanning (as used in example l)(Kay et al., 1996; Yu, Y., et al., 1996, Methods Enzymol. 267:3-27; Petrenko, V.A., et al., 1996, Protein Engineering, 9:797-801), column chromatography, Southern and Western blotting, and electrophoretic techniques.
  • the advantage of physically connecting the displayed peptide to the nucleic acid coding for it is that, at least in principle, the isolation of even a single particle that binds to the ligand is adequate for detection, since the genetic package on which it is displayed can be used to grow up large amounts of identical particles for characterization. Therefore, isolation of clones with particular affinity for the chosen ligand provides the opportunity to determine the sequences of the peptides or proteins displaying that affinity.
  • Once genetic packages displaying peptides with the desired binding properties are isolated their sequences can be obtained by sequencing the corresponding nucleic acid within the genetic package. The sequences thus identified may correspond to sequences within proteins that bind to the ligand. To optimize the probability of obtaining useful results, several libraries should be used.
  • the number of positives that a particular sequence will identify can be estimated.
  • a given pentapeptide sequence will occur roughly once in every 3.2 million amino acids. Given an average protein size of 500 amino acids and 75- 100,000 proteins in the human genome, one can expect about 16 occurrences of a particular pentapeptide. Since some sequences are far more common than others, as proteins have not evolved in a random fashion but as repetitions and modifications of pre-existing genetic units or elements, the number of proteins containing a consensus sequence could easily be twice that or substantially less. Still, even once the entire human genome is sequenced, screening by a random peptide library will rarely result in an unwieldy list of human proteins containing the sequence.
  • the maximum average number of contiguous amino acids does not exceed eight, even under the 5 angstrom standard.
  • the number saturates at between 7 and 10; larger binding sites are most commonly made up of more loops of discontinuous amino acids, not larger loops.
  • Example 1 that the ratio of specific to non-specific binding is adequately strong to be detectable relative to other peptides in a library.
  • the investigator may preferably utilize one of the many public-domain search engines (for instance, a BLAST search of GenBank) to find sequences of proteins which contain the consensus sequence or one similar to it. This technique was applied when analyzing the consensus sequences of examples 1 (SEQ. ID NO. 3) and 3 (SEQ. ID NO. 5). However, in the absence of a clear consensus sequence, the selected peptides still contain information useful for the identification of potential binding sites in proteins.
  • Utilization of this information may preferably be effected by programming a computer to perform the following algorithm to score the homology between the selected peptides and sequences of proteins that are suspected of being involved in a particular molecular activity (toxic or therapeutic, as the case might be). For instance, the taxol-selected peptides were scanned against all proteins known to be involved in apoptosis. Even removing the two peptides containing HTPHP (Seq. ID No. 3) from the set of taxol selected peptides from example 1, this algorithm can identify the flexible loop of Bcl-2 as the site of taxol binding because it has higher homology to the selected peptides than other places in other proteins known to be involved in apoptosis.
  • HTPHP Seq. ID No. 3
  • the algorithm is a simple brute force comparison of the sequence of a protein with all the selected peptides AND a control set of random (non-selected) peptides from the same peptide library.
  • the randomly selected peptide control is needed because most libraries have preferences for amino acid pairs and triplets that will lead to false positives if a control group of peptides is not used.
  • the NEB 12mer library used in example 1 has a preference for pairs of prolines (PP).
  • the pairs of prolines found in the taxol-selected peptides are therefore, not necessarily due to taxol selection, but rather to their preference in the library. If by some coincidence, a ligand had high affinity to the sequence PP, then the selected peptides would have a frequency of PP that would be greater than that of the random (control) peptides.
  • An important consideration in programming the algorithm for comparison is the determination of the means of comparing the sequences of the peptides with the sequences of proteins. This requires selection of (i) the length of the segment on which a comparison will take place (how many amino acids will be considered at one time); (ii) the means by which a homology score will be calculated (this usually involves selection of an amino acid similarity matrix); and (iii) the homology below which a peptide or peptide fragment will be considered to be unrelated to the protein sequence to which it is being compared.
  • the comparison is preferably made over a peptide length relevant to the binding of a ligand.
  • a peptide length relevant to the binding of a ligand In investigation of the binding of a peptide to a small ligand, only 4-8 amino acids are likely to be involved in binding. Use of less than 4 amino acids would lead to too many false positives - a very noisy output; use of more than 8 amino acids would lead to too many false negatives. In most cases 6 amino acids were used at a time, although 5 or 7 may be preferable for other applications.
  • the off-diagonal elements of the matrix show similarity scores. For instance, alanine and serine have a low positive score, but tryptophan and serine have a significant negative score.
  • Blossum62 Henikoff, S. et al., 1992
  • the preferable noise level has been determined entirely empirically using Bcl-2 as an example and other apoptosis-related proteins as controls. The absolute number depends on the similarity matrix being used.
  • the algorithm may be characterized by the following steps: (a) Choose the first block of the protein sequence (e.g. the first 6 amino acids).
  • (c) Use the similarity matrix to calculate the homology score between the block of the protein and the block of the peptide. This is done by summing the matrix elements that correspond to the pairs of amino acids made up of one from the protein and the corresponding amino acid in the peptide.
  • Taxol is an anti-cancer drug with proven efficacy against a wide variety of malignancies, with those of most clinical interest being human breast and ovarian carcinomas (Rowinsky and Donehower, 1991). Its only known molecular target is tubulin which it induces to polymerize, disrupting the dynamic instability of microtubules in the mitotic apparatus and halting mitosis at the metaphase/anaphase transition (Jordan et al., 1993). A wide variety of other responses to taxol have been reported, including the induction of programmed cell death or apoptosis, an increased level of a number of intracellular messengers and growth factors including p53 (Roth, W.
  • a random phage-displayed peptide library was searched for peptides with high affinity for taxol. It was reasoned that a peptide with high affinity for taxol when exposed on the surface of a phage particle might also have high affinity for taxol when present on the surface of a cellular protein.
  • a random dodecamer library displayed at the N-terminus of p3 of bacteriophage Ml 3 (Ph.D.-12 library, available from New England Biolabs, Beverly, Massachusetts) was screened for members with high affinity for taxol.
  • a taxol derivative biotinylated at the C 7 position was synthesized as described in Example 2.
  • This taxol derivative was immobilized on the surface of streptavidin coated plates and standard techniques of biopanning were used to select for members with high affinity for the biotinylated taxol, with the following modifications.
  • a ten-fold higher molar quantity of ligand i.e. biotinylated taxol
  • These taxol-binding phage were designated Round I. Twenty individual phage particles were selected at random from this Round I pool and subjected to nucleic acid sequence analysis.
  • Amplified Round I clones were subjected to a second round of screening using a forty-fold higher amount of input phage onto the culture dish containing the conjugated taxol (to enrich for low K D or 'tight' binding phage particles versus non-specific binding phage particles), producing an enrichment of 5.1 X 10 4 fold.
  • Amplification of these Round II phage particles, followed by a third round of screening with taxol (Round III) gave an enrichment value of 2.3 X 10 4 fold. Seventy members of the Round II and twenty three members of the Round III bacteriophage were selected at random and similarly subjected to nucleic acid sequencing.
  • Characterization of the sequence properties of the original library was accomplished by calculating a Poisson distribution from the incidence of individual amino acids at each of the twelve positions for the random inserts.
  • the information content of each clone was defined as minus the natural log of the inverse of its probability of occurrence within the library. This information content value is a convenient measure of probability, with larger information contents being associated with rarer sequences.
  • the amino acid sequences of peptides isolated by affinity selection have relatively high associated information content compared to those from the parent library.
  • SEQ. ID NO. 3 match is a less likely statistical occurrence than the SEQ. ID NO. 4 match by a factor of about 10,000.
  • SEQ. ID NO. 3 was identified as most likely corresponding to a taxol binding motif.
  • SEQ. ID NO. 3 does not appear in any known tubulin and is found in only two known human proteins, Bcl- 2 (residues 55-59) and ataxin-2 (SCA2) (Pulst, et al., Nat. Genet. 1996 Nov.;14(3):269-276; Imbert et al., Nat. Genet 1996 Nov;14(3):285-291).
  • Bcl-2 it appears in the middle of a highly flexible, 60 amino acid loop identified by comparison with the Bcl-2 homologue, Bcl-X L , for which both x-ray crystallographic and NMR structures have been obtained (Muchmore et al., 1996).
  • This loop acts as a regulatory domain in both Bcl-x L and Bcl-2 (Chang et al., 1997) and is unnecessary for its anti-apoptotic activity (Muchmore et al., 1996). Because Bcl-2 function is known to be regulated by taxol, it represents a highly plausible molecular target for taxol.
  • ELISA binding assays were used to determine if the Bcl-2/GST fusion protein binds directly to immobilized biotinylated taxol.
  • Anti-Bcl-2 antibodies were used to detect Bcl-2 binding. These results demonstrate that Bcl-2 binds to the biotinylated taxol derivative with a K d of approximately 0.4 ⁇ M (see FIG. 2).
  • the graph shows the ELISA data for the binding of the Bcl-2/GST fusion protein to taxol (open triangles), biotin (shaded triangles), and biotinylated dioxin (closed squares), as well as the binding of Bcl-X L to taxol (open circles) and biotin (shaded circles).
  • the x-axis is the log nM concentration of protein in solution (Bcl-2 or Bcl-X L ); the y-axis is the uncorrected optical density at 490 nm.
  • the insert is the chemical structure of the biotinylated taxol used in the selection of peptides.
  • Bcl-2 also binds to biotinylated taxotere, at an approximately ten to fifty fold reduced affinity.
  • the binding of taxol to the Bcl-2 portion of the Bcl-2/GST fusion protein was corroborated by circular dichroism spectroscopy with the CD results demonstrating that the Bcl-2/GST construct undergoes a substantial conformational change upon binding taxol.
  • the spectra of the fusion protein with and without taxol added are significantly different (see FIG. 3).
  • the graph shows the circular dichroism spectrum of human Bcl/GST fusion protein with (solid line) and without (dotted line) taxol. Each spectrum is the averaged result of five spectra and subtracted baselines with standard deviations as indicated. These differences are much larger than the error bars (standard deviations) in the measurements, and involve a change in both the shape of the curve (ratio of the 220/210 nm peaks) and the peak positions.
  • the secondary structures calculated for the Bcl-2 fusion protein with and without taxol differ by approximately 4%, and appear to involve a change in a region of the molecule that is found in a beta-turn conformation.
  • 4 a-c were treated with biotin N- hydroxylsuccinimide ester 7 at room temperature in DMF to give 2'-TES'7- biotinamido-carbamate 5a-c. Removal of the 2'-TES groups from 5a-c with HF/Py/MeCN (1:10:10 v/v/v) at room temperature afforded the desired 7- biotinamidocarbamated 6a-c after chromatography.
  • Dioxin is a pervasive environmental toxin that exhibits toxic effects at very low doses. For the most part, toxicologists believe that its effect is due to the interaction of dioxin with a single molecular target, the aryl hydrocarbon receptor (AhR) (Birnbaum, 1994). However, the effects of dioxin are so pleiotropic that the possibility of other molecular targets cannot be ruled out.
  • Dr. Valery Petrenko U. Missouri, Columbia
  • EPFP SEQ. ID NO.
  • p8 libraries are significantly different from p3 libraries used by the applicants in example 1 in that there are -3000 copies of p8 on a virion, and only 5 p3 proteins. Therefore, the avidity of the interaction becomes more important and weaker interactions may lead to binding of phage to the target.
  • Another weakness of the p8 libraries is that the fact that there are so many inserts per virion that the synthesis of the inserts produces an observable effect on the host metabolism (Rodi et al. (1997) Proceedings of the 22nd Tanaguchi International Symposium. (Nov. 18-21, 1996)).
  • LIF binds to human and murine LIF with a K D of approximately 10-100 ⁇ M, indicating an affinity approximately 10,000 fold less than the anti-dioxin antibodies used as a positive control.
  • the negative control CNTF exhibited no binding to LIF.
  • a Phe-156-Ala mutant exhibited a reduction of about three-fold in binding (in the three dimensional structure of LIF Phe 156 is adjacent to the EPFP (SEQ. ID NO. 5) dioxin binding motif; and a Pro-51-Ala mutant showed no change in binding.
  • dioxin binds to LIF at detectable affinities and at affinities greater than other proteins that are similar in sequence and structure but lack the EPFP motif; (ii) alteration of the EPFP motif may lead to a decrease in the observed dioxin levels; and (iii) other residues in the immediate vicinity of the EPFP motif (i.e. Phe 156) also are involved in dioxin binding.
  • Haldar, S., Basu, A. and Croce, CM. Bcl2 is the guardian of microtubule integrity. Cancer Research. 57, 229-233 (1997).

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  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'identification des cibles moléculaires d'un médicament ou d'une toxine constitue la première étape pour comprendre le fonctionnement du médicament ou de la toxine et, par conséquent, une avancée importante dans l'étude de l'amélioration d'un médicament ou l'évaluation des risques dus à une toxine. L'action primaire d'un médicament consiste habituellement à se lier à une protéine; les actions secondaires peuvent se manifester sous forme d'effets secondaires et, dans certains cas, peuvent être dus à une liaison à d'autres protéines. Par conséquent, il est utile d'identifier tous les sites d'action, physiologiquement appropriés, d'un médicament ou d'une toxine. Un moyen simple d'obtenir une liste des cibles potentielles d'un médicament, d'une toxine ou d'une autre substance biologiquement active (génériquement appelés ligands) consiste en un procédé multi-étapes. La première étape consiste à cribler une banque de protéines ou de peptides pour identifier les éléments de la banque qui présentent une forte affinité vis-à-vis d'un ligand particulier. La deuxième étape consiste à rechercher dans des bases de données de séquences des protéines qui contiennent les séquences des éléments de la banque qui s'avèrent présenter une forte affinité vis-à-vis du ligand. Les protéines ainsi identifiées constituent une liste de cibles potentielles du ligand. Si l'on a utilisé des banques de peptides aléatoires, la position des séquences consensus identifiées à l'intérieur de la protéine identifiée constitue une identification du site de liaison potentiel du ligand sur la cible.
EP98945752A 1997-07-07 1998-07-07 Identification de cibles moleculaires Withdrawn EP0990051A1 (fr)

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US5178097P 1997-07-07 1997-07-07
US5178197P 1997-07-07 1997-07-07
US51780P 1997-07-07
US51781P 1997-07-07
PCT/US1998/014082 WO1999002733A1 (fr) 1997-07-07 1998-07-07 Identification de cibles moleculaires

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US6576636B2 (en) 1996-05-22 2003-06-10 Protarga, Inc. Method of treating a liver disorder with fatty acid-antiviral agent conjugates
AU2002303164A1 (en) * 2001-03-23 2002-10-08 Protarga, Inc. Fatty amine drug conjugates
CA2658334C (fr) 2003-01-16 2012-07-10 Caprotec Bioanalytics Gmbh Composes de capture, collections associees et methodes d'analyse du proteome et de compositions de complexes
KR101692275B1 (ko) * 2010-02-11 2017-01-04 노오쓰웨스턴 유니버시티 ２차 구조 안정화된 ｎｍｄａ 수용체 조절제 및 그의 용도
SG10201811584RA (en) 2010-02-11 2019-01-30 Univ Northwestern Secondary Structure Stabilized NMDA Receptor Modulators And Uses Thereof
CN111566261A (zh) * 2017-08-18 2020-08-21 诺迪勒思生物科技公司 选择结合试剂的方法

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US5723286A (en) * 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems
US5635182A (en) * 1994-06-16 1997-06-03 Genetics Institute, Inc. Method of detecting ligand interactions

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WO1999002733A1 (fr) 1999-01-21

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