WO1994024314A1 - Procede aleatoire de production de nouveaux composes chimiques - Google Patents

Procede aleatoire de production de nouveaux composes chimiques Download PDF

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Publication number
WO1994024314A1
WO1994024314A1 PCT/US1994/004314 US9404314W WO9424314A1 WO 1994024314 A1 WO1994024314 A1 WO 1994024314A1 US 9404314 W US9404314 W US 9404314W WO 9424314 A1 WO9424314 A1 WO 9424314A1
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Prior art keywords
reaction mixture
group
different
substrates
desired property
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PCT/US1994/004314
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English (en)
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Stuart A. Kauffman
Julius Rebek, Jr.
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Kauffman Stuart A
Rebek Julius Jr
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Application filed by Kauffman Stuart A, Rebek Julius Jr filed Critical Kauffman Stuart A
Priority to EP94916542A priority Critical patent/EP0695368A4/fr
Priority to AU68158/94A priority patent/AU6815894A/en
Priority to JP6523552A priority patent/JPH09500007A/ja
Publication of WO1994024314A1 publication Critical patent/WO1994024314A1/fr

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    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates generally to the generation of new compounds without predetermining a desired structure or composition, and the screening of such compounds for one or more desired properties.
  • This invention is more particularly related to the use of a random chemistry, with or without enzymes, to generate a variety of new compounds from which those with a desired property may be characterized or identified, e.g., for subsequent production in batch quantities by conventional methodologies or otherwise.
  • new compounds have been prepared synthetically, i.e., by the creation of compounds in the laboratory rather than through the isolation of naturally occurring compounds.
  • the synthetic generation of compounds utilizes the principles and methodologies of organic chemistry, especially reaction mechanisms.
  • a simple example is the sequential chemical synthesis of a desired peptide by cycles of protection and deprotection of the growing peptide chain as a succession of activated amino acids is added one by one.
  • a second major alternative strategy in the art is the synthesis of a desired chemical compound by the successive synthesis of increasingly complex sets of building blocks which are finally joined to make the desired target.
  • a simple example is the synthesis of a specific hexapeptide (ABCDEF) from the amino acid monomers A, B, C, D, E, F, by the synthesis of the dipeptides AB, CD, EF, then the joining of the dipeptides to form the hexapeptide.
  • ABCDEF specific hexapeptide
  • the same two general strategies are utilized in many areas of synthetic chemistry with a variety of different organic compounds. Both strategies are hindered by a variety of problems, including the necessity for knowledge about, and use of, prespecified reaction pathways.
  • the present invention eliminates the need to know the structure or chemical composition of the desired compound prior to its synthesis.
  • the disclosure of the present invention provides that a diversity of unknown compounds may be produced by "random" chemistry, and such a diversity may be screened for one or more desired properties to detect the presence of suitable compounds. It is central to the subject methods that one does not need to know in advance the structure or composition of the useful compound sought.
  • the present invention provides methods for the production of an organic molecule having a desired property, or for the generation and characterization of an organic molecule having a desired property.
  • the method comprises first providing a starting group of different organic molecules. At least one chemical reaction is caused to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group.
  • This step of causing at least one chemical reaction to take place is repeated at least once. Each repetition uses the reaction mixture of the previous step, and in the end produces a final reaction mixture as a result of the last repetition.
  • the final reaction mixture is screened for the presence of the organic molecule having the desired property.
  • the method for the production of an organic molecule having a desired property as described above is performed. If the screening step of this aspect is successful in detecting the organic molecule having the desired property in the final reaction mixture, then the following additional step* are performed.
  • the starting group of different organic molecules is divided into at least two subgroups, each containing less than all of the different organic molecules in the starting group.
  • the chemical reactions are performed on each of the subgroups in the same way as with the starting group to produce a final reaction submixture corresponding to each of the subgroups.
  • Each of the final reaction submixtures resulting from this step is screened for the presence of the organic molecule having the desired property.
  • the method comprises the steps of: (a) reacting a group of different substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture;
  • any subset of steps a-e above may be performed in any order prior to steps f and g. Further, steps a-e or any subset of these may be repeated in any order prior to steps f and g. Similarly, exposure to other reagents, singly, sequentially, or simultaneously, may be substituted for steps a-e, prior to steps e and f.
  • the method comprises the steps of:
  • steps a-e determining the structure or functional properties characterizing the organic molecule having the desired property. Any subset of steps a-e above may be performed in any order prior to steps f and g. Further, steps a-e or any subset of these may be repeated in any order prior to steps f and g. Similarly, exposure to other reagents, singly, sequentially, or simultaneously, may be substituted for steps a-e, prior to steps e and f.
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the method comprises first providing a starting group of different organic molecules. At least one chemical reaction is caused to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group. This step of causing at least one chemical reaction to take place is repeated at least once. Each repetition uses the reaction mixture of the previous step, and in the end produces a final reaction mixture as a result of the last repetition. The final reaction mixture is screened for the presence of the organic molecule having the desired property.
  • a diversity of compounds is generated from a group of substrates which are subjected to a group of enzymes representing a diversity of catalytic activities.
  • a diversity of compounds is generated from a group of substrates which are subjected to a variety of conditions, in the absence of enzymes.
  • An embodiment of either aspect utilizes a group of substrates with different core structures.
  • Another embodiment of either aspect utilizes a group of substrates with similar or identical core structures, but a variety of different functional groups as substituents.
  • the latter embodiment permits the creation of a diversity of compounds centered around a particular compound or a particular class of compounds.
  • the methods of the present invention are employed to generate new compounds having a desired property.
  • Examples of preferred desired properties include the ability to function as drugs, vaccines, liganding agents, catalysts, catalytic cofactors, structures of use, detector molecules, and building blocks for other compounds.
  • a liganding agent may bind, for example, to protein, DNA, RNA, carbohydrate, enzyme, receptor, or membrane.
  • Liganding agents include agonists and antagonists, such as competitive inhibitors of enzymes or hormones.
  • Structures of use include low energy structures (e.g., structures capable of self assembly) and material structures, like silk.
  • Detector molecules include compounds having optical reporter properties of interest. A new compound may mimic, modulate, enhance, antagonize, modify, or simulate a substance.
  • Specific molecules of interest include molecules: (1) able to bind to a helper T cell receptor of specific clones of helper T cells (e.g., such binding leads to amplification or deletion of specific helper T cell clones); (2) able to be incorporated into DNA or RNA in place of normal nucleotides (e.g., such incorporation alters biological activity); and (3) able to act as a substrate for an enzyme or modify the activity of an enzyme (e.g., may modify the binding activity of a biological molecule).
  • Such molecules are useful for a variety of diagnostic and therapeutic purposes.
  • Substrates for the processes described herein include all organic compounds.
  • a preferred group of substrates includes alkanes, alkenes, alkynes, arenes, alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds, organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleotides.
  • a more preferred group of substrates includes acids, amines, alcohols, amino acids, nucleotides, and unsaturated compounds, such as alkenes and alkynes.
  • substrates are amino acid-based compounds (e.g., amino acids, peptides and polypeptides), nucleotide-based compounds (e.g., nucleotides and nucleosides), and combinations thereof. These substrates may include additional functional groups as substituents and may be acyclic, cyclic, and heterocyclic in nature.
  • the acids, amines and alcohols can be primary, secondary, carboxylic, phosphoric, sulfonic, aromatic, heterocyclic, aliphatic, etc. For increased reactivity, primary amines and alcohols are preferred.
  • substrates that include compounds which are different but share one or more common structural features with a molecule of interest or a class of molecules of interest.
  • the diversity of compounds to be generated would be created around a molecule of interest or a class of molecules of interest.
  • a ringed compound such as a steroid, may be selected and then a variety of different derivatives obtained.
  • Derivatives include the addition and/or deletion of functional groups, and acyclic compounds with ringed substituents similar to a portion of the original cyclic compound.
  • Such derivatives are subjected to the random chemistry processes described herein to generate a greater diversity, from which a compound having a desired property may be detected for further characterization, with or without isolation.
  • a group of substrates consists of related compounds, which are then subjected to the methods without enzymes as described herein.
  • a group of substrates consists of related compounds plus reagents, which are then subjected to the methods with enzymes as described herein.
  • a variation upon these embodiments of the present invention is to generate derivatives using the random chemistry processes described herein, and then subject such derivatives to these processes to generate a greater diversity, from which a compound having a desired property may be detected for further characterization, with or without isolation.
  • Classes of molecules which are preferred focal points from which to obtain derivatives to serve as substrates, include heterocycles, steroids, alkaloids, and peptides/mimetics (including constrained molecules, e.g., constrained by S-S disulfide bonds).
  • heterocycles include purines, pyrimidines, benzodiazepins, beta-lactams, tetracyclines, cephalosporins, and carbohydrates.
  • steroids include estrogens, androgens, cortisone, and ecdysone.
  • alkaloids include ergots, vinca, curare, pyrollizidine, and mitomycines.
  • Examples of peptides/mimetics include insulin, oxytocin, bradykinin, captopril, enalapril, and neurotoxins (e.g., from snails, snakes, etc.).
  • the present invention provides methods for generation of new compounds wherein a group of substrates are acted upon by a group of "enzymes,” such that a diversity of product molecules are formed.
  • enzyme includes enzymes (e.g., naturally or non-naturally occurring or produced), catalysts (e.g., catalytic surfaces), candidate catalysts and candidate enzymes (e.g., antibodies, RNA, DNA or random peptides/polypeptides).
  • the method comprises the steps of: (a) reacting a group of different enzymes representing a diversity of catalytic activities under suitable conditions with a group of different substrates, thereby producing one or more organic molecules different from the enzymes and substrates in the reaction mixture; (b) screening the reaction mixture for the presence of an organic molecule having a desired property; and (c) isolating from the reaction mixture the organic molecule having the desired property.
  • the method comprises the steps of: (a) reacting a group of different enzymes representing a diversity of catalytic activities under suitable conditions with a group of different substrates, thereby producing one or more organic molecules different from enzymes and substrates in the reaction mixture; (b) screening the reaction mixture for the presence of an organic molecule having the desired property; and (c) determining the structure or functional properties characterizing the organic molecule have the desired property. From a library of product molecules produced by the methods provided herein, those of practical interest are characterized. As noted above, it is central that, in the present procedures, one does not need to have prior knowledge of the structure or composition of the useful molecule sought.
  • This aspect of the present invention rests on catalysis of, or otherwise causing, a sufficient diversity of reactions among a group of initial substrates, such that a diversity of further products are formed.
  • a statistical analysis of the average properties of reaction graphs among a set of molecules, as well as the average properties of the catalyzed reaction subgraph among these molecules which is formed when the molecules are incubated in the presence of candidate enzymes or catalysts which may catalyze one or more of the reactions it may be helpful to consider a statistical analysis of the average properties of reaction graphs among a set of molecules, as well as the average properties of the catalyzed reaction subgraph among these molecules which is formed when the molecules are incubated in the presence of candidate enzymes or catalysts which may catalyze one or more of the reactions.
  • a reaction graph is the proper mathematical description of a set of organic molecules and all the reactions that those molecules can undergo.
  • Organic reactions can be categorized into classes by the number of substrate and number of product molecule species.
  • a first class transforms a single substrate into a single product.
  • An isomerization reaction, catalyzed by an isomerase, is an example.
  • a second class joins two substrates to form one product.
  • a dehydration reaction joining two nucleotides by an ester bond is an example.
  • Such reactions are commonly catalyzed by ligases.
  • a third broad class cleaves one substrate into two products. Cleavage of a polynucleotide by a phosphodiesterase is a familiar example, as are many steps in intermediate metabolism.
  • a fourth class transforms two substrates into two products. Often this occurs by transfer of a reactive group from one of the two initial substrates to the second substrate.
  • a convenient representation of a reaction graph denotes each organic molecule species as a point in three dimensional space.
  • One or two lines lead from the one or two substrate molecules derived from the reaction of the substrates. Arrows on the lines leaving the substrates point into a box denoting the reaction. Arrows leaving the reaction box for the products point toward the products. Since reactions are reversible, the arrows merely indicate one possible direction of the reaction.
  • the set of all such arrows and boxes, representing all the reactions among all the organic molecules in the system, comprises the reaction graph.
  • reaction graphs An important feature of reaction graphs is that, for almost any initial set of organic molecules, the reaction graph in which that set is considered as substrates will also require addition of new organic molecules ⁇ i.e., molecules not in the initial set of substrates) where those new organic molecules are the products of one or more of the possible reactions among the initial set of substrates.
  • the reaction graph In a mathematical process, called the "growth of the reaction graph", the reaction graph "grows" by iterations.
  • a set of initial substrate molecules is listed.
  • the reaction graph among those substrates is formed mathematically, and a second iterate of the reaction graph is formed by listing both the initial substrates plus any new organic molecule products of the possible reactions.
  • This new reaction graph may indicate that still further novel organic molecules are products of the reactions now possible.
  • the set of organic molecules included in the graph may increase enormously compared to the initial set of substrates. This successive increase is called "supracritical behavior.”
  • Another possible mathematical behavior of the reaction graph growth processes is that a few new products may be formed on the first graph growth cycle, and successively fewer on the successive graph growth cycles, until no further new product molecules are generated.
  • the latter accounting of enzymes and the reactions catalyzed comprises the catalyzed reaction subgraph of the reaction graph.
  • This mathematic representation is formed by noting, for each candidate enzyme, which reactions if any it catalyzes. An arrow may then be drawn from that enzyme to the reaction box representing the reaction catalyzed, and the arrows into and out of the box representing transformations of substrate(s) into product(s) can be noted in a convenient way, e.g., by coloring those arrows "red.”
  • the set of all red arrows represents the reactions which are catalyzed by one or more of the candidate enzymes present in the system.
  • reaction graph itself may be subcritical or supracritical in its behavior, so too may the catalyzed reaction subgraph among the organic molecules.
  • the catalyzed reactions lead to products not in the founder set of substrates. These new products are available, together with the initial founder set of substrates, to allow further reactions, some of which might be catalyzed by the set of candidate enzymes present in the system. Over a succession of iterations, this process of catalyzed reaction growth may increase vastly in diversity, in a supracritical mode.
  • the set of novel molecules formed via catalyzed reactions may dwindle over successive iterations of the growth of the catalyzed reaction graph. This is a form of subcritical behavior.
  • the total behavior of the system is represented by the behavior of the reaction graph plus the catalyzed reaction subgraph, over iterations.
  • the uncatalyzed reactions represent reactions that occur spontaneously. Whether a reaction graph behaves subcritically or supracritically depends upon the diversity of founder substrates and the diversity of candidate enzymes present in the system. In addition, the behavior is dependent upon factors including concentrations of all reagents, solubility of the organic molecules, and directions of deviation from equilibrium across each reaction.
  • a phase transition from subcritical to supracritical behavior of a reaction system is governed by the diversity of organic molecules and the diversity of enzymes in the system.
  • Systems with low diversity of both organic molecules and enzymes are typically subcritical.
  • Systems with high diversities of either organic molecules alone, low diversities of organic compounds together with high diversities of enzymes, or high diversities of both are typically supracritical.
  • Systems of organic molecules alone without addition of exogenous enzymes can be supracritical, because the spontaneous reaction graph is supracritical, or because some of the substrates or products are enzymes themselves in the sense defined above.
  • the present invention takes advantage of the mathematical phase transition between subcritical and supracritical behavior to choose reaction conditions which yield high diversity libraries of organic molecules from a founder set of organic molecules.
  • phase transition from subcritical to supracritical behavior can be illustrated, by way of a non- limiting example, based on the preferred use of a cloned library of antibody molecules as the candidate set of enzymes, and a set of substrates which, without loss of generality, can be taken to be peptides containing mixtures of D and L amin ⁇ acids and nonnatural amino acids, or can be taken to be small polynucleotides, or a wide variety of other organic molecules.
  • To illustrate the general character of the phase transition it is useful to estimate the number of reactions in a reaction graph with a given number of small organic molecules. In general, the number of reactions is not known. However, minimum realistic estimates are obtainable.
  • a founder set of peptides made of D and L amino acids and non-natural amino acids, each with 10 amino acids, may be used as substrates.
  • the number of possible substrates is very large, and given by the number of kinds of amino acids raised to the tenth power. Any two peptides length ten can undergo transpeptidation reactions cleaving and exchanging the terminal amino acid(s) subsequences at any of the internal peptide bonds of each of the decapeptides. Since there are 9 internal bonds in each, any pair of decapeptides can undergo 81 such transpeptidation reactions. In each case, two substrates yield two products.
  • N squared is a conservative estimate of the diversity of reactions in a reaction graph with N kinds of organic molecules.
  • a set of 100,000,000 cloned human antibody molecules is used as the set of candidate enzymes. Based on the statistics of generating catalytic antibodies (as described below), the probability that a randomly chosen antibody molecule is able to catalyze a randomly chosen reaction is between 10 "5 and 10 "8 (Pollack et al., Science 234:1570, 1986; Tramontano et al., Science 234:1566, 1986; Tramontano et al., Proc.
  • each reaction might be catalyzed by any one of the 1,000,000 candidate antibody enzymes, and each antibody has a chance of one in a hundred million of being able to act as a catalyst for each reaction.
  • 10,000 reactions among the million possible should be catalyzed by one or more of the antibodies present. Therefore, as these catalyzed reactions occur, the products of the 10,000 reactions will be formed. Most of these will differ from the 1 ,000 substrate molecules initially present. Thus, the diversity of the set of organic molecules has increased.
  • the new system After sufficient time has elapsed for the concentrations of these novel molecules to increase sufficiently, the new system has a diversity of substrates on the order of 10,000 rather than 1,000, hence, now a diversity of 10,000 squared reactions are possible among the enlarged set of substrates.
  • the expected number of reactions which now find catalysts among the antibody molecules is thus 10 8 x 10 6 /10 ⁇ or
  • the human antibody repertoire is used herein as a non- limiting example of a set of candidate enzyme molecules. As is known in the art, the combinatorial diversity of human antibody molecules due to genomic rearrangement is on the order of 100,000,000 (prior to the onset of somatic mutation during maturation of the immune response which further increases potential diversity).
  • antibody molecules can function to catalyze a wide variety of reactions with a rate (Vmax) acceleration of three to eight orders of magnitude compared to the spontaneous reaction.
  • Vmax rate of three to eight orders of magnitude compared to the spontaneous reaction.
  • Such catalytic antibodies are commonly generated by immunization of an immune competent animal with a molecule that is a stable analogue of the transition state of the desired reaction. Monoclonal antibodies are generated from this immunization, and each is tested for its capacity to catalyze the desired reaction. Because the stable analogue is similar chemically to the transition state of the reaction, typically on the order of 5% to 10% of the monoclonal antibodies tried are able to catalyze the desired reaction.
  • the catalysis reflects high affinity for the transition state and lower affinity for substrates and products.
  • the fraction of B cells which respond to immunization with an arbitrary epitope bearing antigen is on the order of one in a hundred thousand.
  • B cells which respond to an antigen typically have modestly high affinities for the antigen to be triggered to divide.
  • the probability that a randomly chosen antibody molecule can bind with modest affinity, 10 4 M " ⁇ to an arbitrary antigen is about one in a hundred thousand.
  • the monoclonal antibodies used to create catalytic antibodies may have undergone further somatic mutation that increased affinity for the antigen. It is reasonable to estimate the probability that a randomly chosen antibody has high affinity for an arbitrary antigen is about 10 to 10 " *.
  • a cloned high diversity library (10 8 or more) of antibody molecules can be and has been created in a variety of ways.
  • such antibody libraries are a non-limiting example of a high diversity set of candidate enzymes.
  • libraries can be cloned in prokaryotic or eukaryotic hosts to amplify the polynucleotide sequences and obtain protein products which constitute the candidate enzyme library.
  • the polynucleotide sequences can be amplified in vitro and translated in vitro to obtain the candidate enzyme library.
  • the candidate protein library can be isolated from other molecular components by means known in the art. For example, an advantageous means to do so uses libraries of fusion proteins with stochastic peptides, polypeptides, or proteins fused adjacent to, for example, ubiquitin. Antibodies to ubiquitin allow affinity purification of the library of fusion proteins which then serves as the set of candidate enzymes.
  • a library of antibody molecules can be derivatized by cloning partially stochastic DNA sequences into the hypervariable region of the antibody molecules. A refinement of this involves cloning such stochastic sequences into one or more of the complement determining regions (CDRs), of the antibody molecule. Each CDR has on the order of 5 to 10 amino acids.
  • This modified library is a set of candidate enzymes.
  • RNA sequences can be cloned into a gene encoding any protein, e.g., histone 1 or any other protein, to create a fusion protein with the novel
  • DNA or RNA at one end, or in the middle of the host protein sequence.
  • the well folded host protein serves as a framework to aid folding and stability of the cloned sequences.
  • the set of such proteins is a library of candidate enzymes.
  • Libraries of DNA sequences in themselves, or RNA sequences in themselves, constitute libraries of candidate enzymes.
  • the existence of ribozymes and of DNA sequences able to bind arbitrary ligands, such as thrombin, show that both kinds of polymers are strong candidates to bind transition states and catalyze reactions.
  • Other libraries of combinatorial molecular diversity, linear sequences or otherwise, as known in the art, may be used as candidate catalysts.
  • the substrates of interest in generating a library of molecules may be D and L amino acids, including nonnatural amino acids, and some small dipeptides, tripeptides, and tetrapeptides formed of these building blocks. It is known in the art that larger peptides can be synthesized from amino acids and small peptides using proteases, peptidases, lipases, hydrolases, and esterases
  • enzymes include oxidoreductases; transferases; hydrolases; lyases; isomerases; and ligases.
  • Oxidoreductases catalyze oxidation and reduction reactions. Examples of oxidoreductases include dehydrogenases; reductases; oxidases (monooxygenases and dioxygenases); and peroxidases. Transferases catalyze the transfer of functional groups. Examples of transferases include aminotransferases (transaminases); phosphotransferases; pyrophosphokinases; and nucleotidyltransferases (RNA and DNA polymerases). Hydrolases catalyze the hydrolytic cleavage of bonds, such as ester, glycosyl, and peptide bonds.
  • hydrolases examples include phosphodiesterases; amylases; proteases (peptidases, proteinases); nucleases (exo- and endo-; ribo- and deoxyribonucleases); and phosphatases.
  • Lyases catalyze double bond formation by non- hydrolytic removal of groups from substrates. Examples of lyases include decarboxylases; anhydrases; and synthases.
  • Isomerases catalyze geometric or structural changes within one molecule. Examples of isomerases include racemases; epimerases; tautomerases; and mutases.
  • Ligases catalyze the joining together of two molecules coupled with the hydrolysis of pyrophosphate bond. Examples of ligases include synthetases.
  • the substrates be soluble in the solvent, that the candidate enzymes be soluble in the solvent, that the volume be sufficiently small and concentrations sufficiently high that substrates and enzymes encounter one another rapidly, and at high enough concentrations to occupy a sufficient fraction of enzymatic sites to enhance reaction velocities, and that the high diversity product library be present in high enough concentrations that useful molecules can be detected. All these requirements have been considered for the present invention.
  • enzymes typically can tolerate some percentage of organic solvents such as ethanol, methanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or combinations thereof, in aqueous (water based) solutions (Gupia, Eur. J. Biochem. 205:25, 1991).
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • Substrates of the types indicated vary in solubility. In general, it is reasonable to obtain millimolar concentrations of on the order of 1 ,000 substrate species in small reaction volumes, on the order of 1 to 100 microliters. Under reaction conditions such that the diversity of these 1 ,000 substrates increases by a factor of 1 ,000,000, yielding 1,000,000,000, or a library of small molecules with a diversity of 10 9 , the average concentration will have fallen by a factor of 10 "6 , hence have fallen from millimolar to nanomolar, 10 "9 M.
  • the detection methodologies discussed below to identify a molecule of interest are able to detect readily in the nanomolar range, and typically are able to detect in the picomolar, 10 '12 M, range. Thus, even with a 1, 000-fold decrease in concentrations of some products below the mean when diversity is one billion, the detection means can detect molecules of interest. Other detection procedures allow detection at 10 '15 to 10 '20 molar.
  • the diversity of candidate enzymes in a reaction mixture is limited by the solubility of the enzymes. For example, for proteins in aqueous media, a 10mg/ml concentration is typically attainable. For candidate enzymes with 200 amino acids, on the order of 10 80 protein molecules can be in solution in 1,000 microliters. Thus, if a diversity of candidate enzymes in a reaction mixture is limited by the solubility of the enzymes. For example, for proteins in aqueous media, a 10mg/ml concentration is typically attainable. For candidate enzymes with 200 amino acids, on the order of 10 80 protein molecules can be in solution in 1,000 microliters. Thus, if a diversity of candidate enzymes in a reaction mixture is limited by the solubility of the enzymes. For example, for proteins in aqueous media, a 10mg/ml concentration is typically attainable. For candidate enzymes with 200 amino acids, on the order of 10 80 protein molecules can be in solution in 1,000 microliters. Thus, if a diversity of candidate enzymes in a reaction mixture
  • a group of enzymes representing a diversity of catalytic activity are separated in part or in entirety from one another and the substrates contacted sequentially.
  • a group of enzymes are separated by membranes, such as dialysis bags, or by immobilizing different enzymes (representing different catalytic activities) on solid supports, such as resins.
  • Candidate enzymes can be localized on phage, using phage display libraries as is known in the art, or other means to generate and display combinatorially diverse libraries of molecules, such as peptides or other molecules on beads, or surfaces, or polysome trapped peptide libraries.
  • candidate enzymes may be contained within or displayed upon one or more types of eukaryotic or prokaryotic cells, the cells and the substrates being brought into contact.
  • a group of substrates is circulated (e.g., by peristaltic pump) through the separated enzymes.
  • substrates are circulated in and out of dialysis bags with pore sizes which prevent escape of the enzymes.
  • Substrates are bound by the first set of enzymes, modified, released and circulated to the next set of enzymes.
  • a group of substrates may be confined and enzymes having one or more catalytic activities circulated through sequentially. In general, the reactions are conducted over a period of several hours at temperatures of about 37°C or below.
  • Cofactors such as ATP, NADH, 0 2 and CoA are added where appropriate. Many of the cofactors may either be added directly or generated in situ.
  • 0 2 may be introduced by injecting the gas or air directly into the reaction mixture or by use of an electrode to generate 0 2 .
  • An electrode need not directly contact a reaction mixture, but rather may be introduced into a compartment from which 0 2 may pass to the reaction mixture.
  • an electrode may be placed inside of a dialysis bag which in turn is surrounded by a dialysis bag containing a set of enzymes. It will be readily appreciated by those of ordinary skill in the art that a group of substrates may be subjected to the various separated enzymes in a variety of orders.
  • a combinatorial library of organic molecules or other molecules which are similar to an initial molecule of interest, are generated by derivatization of the initial molecule in a very large number of possible ways to produce a high diversity library of "local" mimics of the initial molecule of interest.
  • two ways are provided for generating such a library, one which does not use enzymes, but uses a variety of possible adducts or other molecules which may undergo reactions with the initial molecule of interest, and also uses a variety of chemical reagents and physical conditions to drive the synthesis of a library of derivatized products of the initial molecule.
  • the core initial molecule plus a set of candidate adducts and other molecules which may react with the initial molecule are used, but also included is a set of enzymes which may increase the rate of formation of the local high diversity library of derivatized forms of the initial compound.
  • a high diversity library of derivatized forms of a steroid hormone core such as estrogen
  • a set of reactants including estrogen and a variety of other small molecules which are candidates to react with estrogen to form new product molecules partially or entirely containing the steroid core, are utilized in a common reaction milieu. These are reacted in the presence of a set of enzymes to catalyze the reactions afforded by the system. Enzymes can be chosen by a number of means, some known in the art, others specified herein. The formation of a library of derivatized molecules under these reaction conditions can be assessed by a number of means known in the art. For example, the steroid core may be radioactively labeled at a variety of positions.
  • the reaction mixture can be subjected to HPLC analysis, mass spectrograph analysis, or other modes of analysis to test for the diversity of molecules which are labeled.
  • All radioactively labeled molecules contain atoms derived from the steroid core, hence the new molecule species are at least partially comprised of the steroid core. If it is desirable to assure that a large part of the steroid core is contained in the novel species, then two or more distinct radioactive labels can be used to label distinct and distant atoms in the core. Simultaneous presence of all labels suggests strongly that those portions of the steroid core are intact.
  • Alternatives to radioactive labels include isotope labels and other means known in the art.
  • the high diversity library is tested (e.g., by means described herein) to determine if it contains molecules of interest. If such molecules are detected, they may then be isolated by a variety of means, including sib selection as described herein.
  • the detection of molecules which are candidates to act as antagonists of estrogen is discussed first as a non-limiting example of detection of one or more molecules of interest in the library of this estrogen example. Detection of molecules which have higher affinity than estrogen for the estrogen receptor (and hence which may be of use in hormone replacement therapy at lower concentrations and thus lower side effects than estrogen itself) is discussed as a second non-limiting example. Detection of candidate antagonists in the reaction mixture may be accomplished by use of very high specific activity radioactive estrogen bound to receptors by means known in the art. Unlabeled competitors in the library will displace the labeled estrogen, and this competitive interaction can be detected by loss of label.
  • Detection of candidate high affinity agonists for replacement therapy may be carried out by use of appropriate cell assays similar to the frog melanocyte assay or the use of pH changes described in detail herein. Presence of a high affinity agonist in the reaction mixture is demonstrated because a very low concentration of the agonist compared to estrogen suffices to trigger the cell response. Such assays may be carried out in the absence of estrogen, or in the presence of increasing concentrations of estrogen. In the latter case, cell response at lower concentrations of estrogen than would elicit a response with estrogen alone, detects the presence of an agonist in the high diversity library. If the agonist can act alone to trigger the cell response, then during the sib selection winnowing procedure, as its concentration increases the threshold level of estrogen required for triggering a cell response will dwindle.
  • the creation of a set of candidate enzymes able to catalyze reactions derivatizing the core molecule, e.g., estrogen, is carried out by selecting from a large set of enzymes (for example the mouse or human immune repertoire), a subset of candidates which bind to the initial set of substrates, the core molecule plus the candidate substrates which are to react with the core and derivatize it.
  • This set of enzymes may then advantageously be enlarged by generating a mutant variant spectrum of each.
  • the purpose of this step is the following: The enzymes have been selected because they bind to the substrates of the potential reactions, rather than selectively binding the transition states of the reaction.
  • a succession of candidate enzymes can advantageously be selected as candidates to catalyze the succession of reactions steps leading away from core molecule, for example the steroid core, and the initial adducts, to generate the high diversity library.
  • core molecule for example the steroid core, and the initial adducts
  • candidate enzymes leading from the initial core and initial adducts can be advantageously eliminated in later iterative steps, since they have already acted to catalyze formation of their products.
  • one means to identify such further enzyme candidates at each iterative step consists in labeling the substrate and the product molecules in the reaction mixture, each at a variety of positions, with radioactive iodine.
  • the purpose of labeling a variety of positions on each compound with iodine is to assure that the iodine labeling of at least some members of that species of compounds will not prevent binding of candidate enzyme molecules at almost any compound site unhindered by the iodine label.
  • These labeled molecules are then reacted with the high diversity of candidate library enzymes, for example with human antibody molecules, to detect which antibody molecules bind the labeled molecules from the reaction mixture.
  • This set includes antibody molecules which bind the novel product molecules created in the reaction system.
  • the antibody molecules plus their mutant variants are then used to enlarge the set of candidate enzymes.
  • a variety of means are known in the art to identify the antibody molecules which bind iodine labeled molecules in the reaction mixture. Among these, it is advantageous to use plaque assays or cell assays expressing the antibody library to test which plaques or cells bind iodine labeled material. If a fluorescent label is used instead of iodine, it is advantageous to make use of the natural display of antibody molecules on cell surfaces of immortalized B cells, where each such monoclonal antibody producing cell displays its unique antibody. It is then advantageous to expose the population of cells to the fluorescent labeled molecules in the reaction mixture, then sort the B cells. Those immortalized cells which are labeled generate antibody molecules which bind the labeled molecules from the reaction mixture.
  • These immortalized cells can be grown to create a library of monoclonal antibodies which are the candidate enzymes.
  • a high diversity antibody library to find candidate enzymes
  • Another preferred means to create a set of candidate enzymes which may help derivatize a core molecule with a set of adducts or other substrates consists in using known enzymes involved in the normal biosynthetic pathway leading to the core, plus mutant variants of those enzymes. Similarly, known enzymes utilizing any of the adducts as substrates, plus mutant variants of those enzymes, may be used.
  • enzymes which bind substrates and products can be identified by means known in the art, including binding assays to cloned enzymes via plaque or other assays. It is also advantageous to use a set of candidate enzymes formed by the union of a set of known enzymes and their mutant spectra, as just described, plus a set of candidates derived from a high diversity library of candidates, such as the mouse antibody repertoire as described above.
  • a procedure to do so consists in creating sets of "shape-complements" to the "shape" of the desired target, then using the sets of shape- complements to bind and affinity select candidate substrates whose own “shapes" are similar to the target shape of the desired molecule.
  • the target molecule of interest is estrogen
  • DNA, RNA, or otherwise can be used to find shape-complements to the target molecule, here estrogen.
  • This "target shape" procedure can be advantageously extended in three ways.
  • the set of each such rank one antibodies can be used to affinity select candidate substrates with shapes similar to estrogen.
  • Second, the set of second rank antigens which compete with estrogen for binding sites on rank one antibodies can be used to affinity select candidate enzymes which will act on estrogen-like substrates to yield estrogen mimics.
  • this set of rank two antibodies can be used to generate "rank three" antibodies which can be used to affinity select a wide variety of estrogen-like substrates.
  • other sets of shape and shape-complement molecules including DNA, RNA and other complex molecules can be used. These can, as one non-limiting example, be selected from high diversity combinatorial libraries of molecules.
  • a group of molecules are used which contain autocatalytic sets, e.g., autocatalytic sets of catalytic polymers. Reaction mixtures comprise such organic molecules which are simultaneously substrates and catalysts. Reactions are carried out in a chemostat under flow conditions.
  • molecules A, B and C are present wherein molecule B catalyzes its own formation out of substrate molecule A, and molecule C catalyzes its own formation out of substrate molecule A.
  • This reaction is carried out in a chemostat where a receptor molecule, such as acetylcholine receptor is affixed to the walls of the chemostat and can bind any molecule that looks like acetylcholine.
  • molecule B but not C looks sufficiently like acetylcholine to bind to the receptor for acetylcholine that is on the chemostat walls. Under flow conditions, the B molecule will tend to be selectively retained within the chemostat and the C molecule will not be retained.
  • This provides selective conditions which leads to the selective amplification of the B autocatalytic set compared to the C autocatalytic set. For example, if B, even when bound to the receptor acts as a catalyst leading to its own formation, then its retention within the system is selectively favored, and B is amplified with respect to C. More generally, in a complex reaction mixture in which molecule B functions as a catalyst in its own formation out of the complex reaction mixture, then retention of B is selectively favored because it binds to the receptor for acetylcholine.
  • the method comprises the steps of (a) reacting a group of different substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture; (b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture; (c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture; (d) performing a condensation reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture; (e) exposing the fourth reaction mixture to light of wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents; (f) screening the exposed fourth reaction mixture for the presence of an organic molecule having a desired property; and (g) isolating from the exposed fourth reaction mixture the organic molecule having the desired property.
  • the method comprises the steps of: (a) reacting a group of different substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture; (b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture; (c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture; (d) performing a condensation reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture; (e) exposing the fourth reaction mixture to light of wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents; (f) screening the exposed fourth reaction mixture for the presence of an organic molecule having a desired property; and (g) determining the structure or functional properties characterizing the organic molecule having the desired property.
  • a group of different substrates are subjected to a series of reaction conditions from which one or more compounds having a desired property are produced without the use of enzymes. More specifically, a group of different substrates are reacted under suitable conditions with a dehydrating agent to yield a first reaction mixture.
  • Suitable dehydrating agents include carbodiimides, carbonyldiimidazole, sulfonyl halides, phosgene equivalents and activated phosphoramides, as well as other agents in common use for solid phase peptide synthesis and nucleotide synthesis, etc.
  • the most preferred solvent(s) are dependent upon the particular group of substrates selected. For example, if all the substrates are fairly polar in nature, a solvent such as methanol may be used.
  • Concentrated solutions of individual substrates are made and then the group of substrates prepared by mixing aliquots of each concentrated solution.
  • Mixtures of solvents which are miscible with one another ⁇ i.e., do not form two phases) are appropriate where all the substrates are not soluble in a single solvent.
  • solvent mixtures are acetone and water, dimethyl formamide and water, or ethanol and water. Reaction conditions may be varied, but generally the reaction will be performed from about one hour to overnight at a temperature from about room temperature to the boiling point of the solvent.
  • the first reaction mixture such as that described above, is reacted under suitable conditions with a reducing agent to yield a second reaction mixture.
  • Suitable reducing agents include dissolving metals, hydride reagents, molecular hydrogen with suitable metal catalysts (e.g., platinum, palladium, nickel or rhodium), etc.
  • suitable metal catalysts e.g., platinum, palladium, nickel or rhodium
  • reducing metals include sodium, lithium, potassium, various amalgams, calcium, iron, and tin.
  • hydride reagents include sodium borohydride, lithium aluminum hydride, and borane. Reaction conditions may be varied, but generally the reaction will be performed from about one hour to overnight at a temperature from about room temperature or below (e.g., in an ice bath). It will be evident that certain reducing agents perform best in certain solvents.
  • oxidizing agents include ozone, peroxides, chromate, permanganate, osmium tetroxide, chlorine, bromine, and air in the presence of suitable metal catalysts (such as ruthenium tetroxide). Reaction conditions may be varied, but generally the reaction will be performed from about 1-2 hours to overnight at a temperature from about room temperature or below (e.g., in an ice bath).
  • oxidizing agents function best in certain solvents.
  • a mixture of water and alcohol may be used with hydrogen peroxide, but water only with permanganate, and hexane (or petroleum ether) with halogens such as chlorine or bromine.
  • a condensation reaction is performed under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture.
  • the third reaction mixture may be subjected to condensation by dehydrating agents or heat.
  • Suitable dehydrating agents include molecular sieves, carbodiimides, azeotropic distillation (to remove water), etc.
  • toluene may be added and then azeotropic distillation performed to remove water. It will be evident that reaction conditions vary depending upon the type of dehydration agent used.
  • the fourth reaction mixture is exposed to light.
  • the light generally is within a range of about 220 nanometers to 600 nanometers, which includes portions thereof or discrete wavelengths if desired.
  • Reaction conditions may be varied, but generally the irradiation of a reaction mixture will be performed from about 15 minutes to 2 hours at a temperature from about room temperature or below (e.g., in an ice bath).
  • reagents used singly, or in mixtures, or used sequentially, in addition to the above examples, or with the above examples where practical, can be utilized.
  • the repetition of steps need not be in the order initially performed and additional substrates may be introduced at any step if desired.
  • one or more of the substrates used initially, or introduced at a subsequent reaction step may be generated by any of the methods provided herein, i.e., by random chemistry with or without enzymes.
  • the group of substrates is provided by derivatization of an initial molecule or a class of molecules.
  • Such a group of substrates is subjected to the above-described reactions without enzymes to generate a high product diversity which is centered around the initial molecule or a class of molecules.
  • a variety of means are available which allow detection of low concentrations of one or more species of a desired molecule in a mixture of molecules generated by the methods provided herein.
  • a variety of cell systems are well known to those of ordinary skill in the art which allow detection of low concentration ligands, e.g., ligands binding a hormone receptor.
  • a system which clones human G peptide hormone receptors into frog melanocytes (Lerner, Proc. Natl. Acad. Sci. USA).
  • the hormone receptors typically located in the cell membrane, respond to binding of the corresponding hormone, but trigger a cell response releasing or reabsorbing melanophores.
  • cells darken dramatically, then can be induced to lighten in color again.
  • Response of the cell depends upon the affinity of the hormone for the receptor. Typical responses occur in the nanomolar to 100 picomolar hormone concentration range. For some hormone receptor- hormone pairs, where affinity is higher, response occurs in the picomolar hormone concentration range.
  • This cell system is an example of an assay system which allows detection, in a mixture of molecules, of one or more species of ligands able to bind to the receptor.
  • the set of molecule ligands able to bind the receptor are then the ligands of interest, for they are candidates to act as drugs by antagonizing, agonizing, substituting for, or modifying the effects of the natural hormone.
  • a second example of a cell assay is that available commercially from Molecular Devices (Palo Alto, CA). It consists of an array of chemfets which respond to very small changes in local pH. In turn, these small pH changes reflect the altered metabolic activity of a population of cells upon receipt of some molecular signal, such as a hormone binding its receptor.
  • cell assays in which a hormone binds a receptor are known to those of ordinary skill in the art and allow nanomolar or subnanomolar concentrations of the hormone ligand to be detected.
  • a preferred means of using the present invention consists in exposing such cells to a high diversity library of molecules generated by the methods provided herein, to detect the presence of one or more species of molecules able to trigger the cell response.
  • That set of small molecules, each of which is highly likely to bind the hormone receptor, are the molecules of interest which may serve as drugs.
  • Another example is to use blast B cells, which on their surface express antibodies directed to a molecule of interest, to detect in a high diversity library the presence of molecules which sufficiently mimic the molecule of interest to be able to bind to its antibody on a B cell.
  • a high diversity library of molecules generated by the methods provided herein is screened using the population of B cells. For example, binding may stimulate cell cycling or division by the last B cell bound. Cell cycling or division may be detected by means known in the art.
  • a receptor for a hormone can be used directly to detect binding of a radioactivity labeled ligand.
  • Other means, known in the art, to accomplish this include the following: (i) The estrogen receptor is used as a non-limiting example. The cloned receptor can be affixed to a flat surface, for example, a filter. Very high specific activity estrogen is prepared, and bound to the receptor population. This set of bound receptors is then used in a competitive assay. The bound receptors are exposed to a library of compounds generated by the methods of the present invention.
  • the library contains ligands which also bind the estrogen receptor, those ligands will compete with the radioactively labeled estrogen itself for the receptors. Hence the radioactively labeled estrogen will be competitively displaced from the receptor, and can readily be detected by means known in the art.
  • this assay allows detection of one or more species of ligands in the mixture which compete with estrogen for the estrogen receptor.
  • This set of ligands is the set of interest, as they are candidates to be drugs mimicking or antagonizing estrogen.
  • the estrogen receptor is again used as a non- limiting example. By means known in the art, one raises antibody molecules which are able to bind the receptor when the receptor is not bound by estrogen, but not bind the receptor when occupied by estrogen.
  • These antibody molecules can then be decorated with reporter groups by a variety of means known in the art, and used to detect the presence of one or more ligand species in a library of high diversity, which bind to the estrogen receptor.
  • reporter groups by a variety of means known in the art, and used to detect the presence of one or more ligand species in a library of high diversity, which bind to the estrogen receptor.
  • antibodies which only bind the receptor if the receptor is itself unbound by estrogens, one tests for loss of antibody binding in the presence of the library of compounds and in the simultaneous absence of estrogen.
  • antibodies which bind the receptor only if the receptor is bound by estrogen one tests for an increase in binding of the antibody in the presence of the receptor and high diversity library.
  • An advantage of this procedure is that a receptor for the target molecule need not be available.
  • Use of a set of monoclonal antibodies is advantageous because, a priori, it is not certain which molecular feature, or epitope, of the target molecule mediates its biological action.
  • Means are established in the art to detect protein- protein binding based on plasmon resonance and detection of a shift in refractive index.
  • a monoclonal antibody, or a hormone receptor is layered onto a gold chip. Binding of hormone, or other ligands to a receptor, is detected in very low concentrations (e.g., in the nanogram range or less).
  • any receptor, or antibody, or other "shape complement" of a target molecule of interest can be placed on the gold chip, the latter can be exposed to a high diversity library, and the presence of liganding species can be detected.
  • RNA aptomer is the shape- complement which binds estrogen
  • fluorescent labeled versions of that RNA aptomer can be used in Rigler's system.
  • An estrogen-mimic which binds the fluorescently labeled RNA will slow its diffusion as detected in the laser system.
  • estrogen-mimics at very low, 10 '15 M or femtomolar, concentrations can be detected.
  • a further means to detect ligands of interest at very low concentrations consists in seeking ligands which block a DNA polymerase. By blocking the DNA polymerase chain reaction (PCR) enzyme, amplification of the DNA can be blocked.
  • PCR DNA polymerase chain reaction
  • PCR amplification can yield billions or more copies of the initial DNA sequence
  • blocking PCR amplification yields a readily detectable signal of a ligand which blocks the polymerase.
  • this method generalizes to other means to amplify DNA, RNA, or DNA- or RNA-like molecules such as ligation amplification, and extends to general means to block polymerases directly or indirectly with ligands of interest.
  • compounds of interest in the high diversity library may act as catalysts for a desired reaction, or as cofactors with other molecules to form an active catalyst.
  • Other molecules may act as inhibitors of enzymes.
  • the latter set of enzymes can be quantitatively removed from the high diversity library by affinity columns bearing molecules directed to a constant part of each of the set of enzymes, or other means known in the art.
  • the resulting high diversity library itself is then assayed for candidates of interest.
  • Detectionof molecules able to inhibit an enzyme may proceed by detecting ligands able to bind the enzyme, as described above. Identifying molecules which are candidates to catalyze a reaction alone or as a cofactor, may proceed by testing high diversity libraries alone, or in the presence of a helper molecule, say a protein, for which a desired molecule will be a cofactor. The system is tested for the presence of ligands able to bind a stable analogue of the transition state of the reaction. Such binding molecules are the candidate catalysts or cofactors sought, for they are candidates to catalyze the reaction itself.
  • a variety of means are known in the art which allow detection of the products of a catalyzed reaction itself.
  • chromogenic or fiuorogenic substrates for a variety of reactions of interest are available. Catalysis of the reaction increases the rate of formation of the colored or fluorescent product.
  • assay systems are available or readily prepared which detect the presence of a product molecule because that product molecule binds a receptor, an antibody molecule, or other shape complement. Thus, detection of higher rates of formation of that product molecule demonstrates that the reaction itself was catalyzed.
  • Characterization and/or isolation depend upon the information desired, and can be carried out at different mole abundances of the target molecule of interest. Thus, using modern mass spectrograph analysis, about 10 '15 to 10 "18 moles can be assayed for mass and charge, then fragmented in a variety of ways known in the art and the fragments assayed for mass and charge. Using this data, it is possible to derive the structure of the molecule of interest. For example, ligands of interest may be isolated by binding to a given hormone receptor, or monoclonal antibody, then the liganding molecules released by means known in the art, and finally characterized analytically. One means comprises attaching a target receptor or antibody to a solid support.
  • a reaction mixture or subset thereof is contacted with the solid support. Those molecules that are bound will be retained, while the non-bound molecules are readily separated from the solid support. The molecules of unknown structure which have been retained, are then eluted. The freed molecules are characterized analytically, e.g., by mass spectroscopy, NMR, IR, UV, and may be synthesized in batch quantities. Examples of analytic techniques involving mass spectrometry include gas chromatography-mass spectrometry (GC-MS), HPLC-mass spectrometry (LC-MS), and field desorption mass spectrometry (FD-MS).
  • GC-MS gas chromatography-mass spectrometry
  • LC-MS HPLC-mass spectrometry
  • FD-MS field desorption mass spectrometry
  • concentrations of molecules of interest in the high diversity library will allow detection of their presence, but may be too low for further isolation or characterization.
  • a preferred procedure called “sib selection” allows ready winnowing of the set of candidate enzymes, the set of founder substrates, and the set of reaction conditions and chemical reagents, to smaller sets. This winnowing simultaneously reduces the side products generated in the high diversity library, increases the concentration of the target molecule of interest, and identifies the subset of candidate enzymes which catalyze the pathway leading to synthesis of the target molecule, and identifies the set of founder substrates required for synthesis of the desired target.
  • this sib selection procedure is a means to generate a previously unknown molecule of interest, as well as identify both that molecule and the substrates and enzymes needed to form that molecule.
  • a library where the target of interest is a molecule which binds the estrogen receptor, is used as a non-limiting example.
  • a high diversity library derived from D and L amino acids, including nonnatural amino acids, and small peptides which may be composed thereof is provided by the methods described herein.
  • Such a library will contain linear, branched, cyclic and other singly or multiply constrained forms due to formation of disulfide (S-S) intramolecular bonds.
  • the presence of one or more ligands for the estrogen receptor is detected in the high diversity library of this example by any of the means described above, or any other means.
  • the set of candidate enzymes and set of founder substrates suffice to lead to reactions which generate the desired ligands.
  • a set of four reaction steps, using seven of the initial substrates at different reaction steps, may lead to the desired target molecule.
  • the target molecule may be synthesized in high concentrations. High concentrations may be achieved because, given the solubility limits, higher concentrations of the seven critical substrates may be attained than when 1 ,000 initial substrates were used, and because only the four critical enzymes would be present.
  • Sib selection achieves this winnowing.
  • the set of candidate enzymes can be derived, for example, from a cloned polynucleotide library. Thirty- two aliquots are created, each of which contain a random half of the initial diversity of the candidate enzyme library. Thus, if the initial enzyme library diversity was 1 ,000,000, thirty-two aliquots are created, each containing a diversity of
  • the set of candidate enzymes may be winnowed down to the four needed to catalyze the synthesis of the target molecule. In the present case about 18 iterations are required.
  • This winnowing procedure therefore, allows the isolation of a set of enzymes needed to synthesize a target molecule of interest. Thereafter, mutation, recombination and selection can be used on this set of enzymes to increase their efficiency and specificity in producing the target molecule. Thus, this procedure yields an efficient set of enzymes for later synthesis of the target molecule from its progenitor substrates.
  • mutant forms of these enzymes can be utilized to catalyze a related family of reaction steps leading to variant forms of the target molecules. Those variants may be more useful than the initial molecules.
  • the set of substrates may also be winnowed to the seven needed. This winnowing can occur either before or after the set of enzymes is winnowed.
  • the process is the same. Thirty-two aliquots are created, each containing a random 80% of the 1,000 initial substrates. The chance that any aliquot contains the seven critical substrates is .8 7 . Thus, on average one or more of the aliquots contains the requisite set of 7 substrates. Each aliquot is tested for the presence of the target molecule of interest that binds the estrogen receptor. A positive aliquot is chosen. Thirty-two aliquots are again generated, each containing a random 80% of the remaining, now reduced substrate diversity. The aliquots are again tested for those which contain the target molecule of interest. In a logarithmic number of steps it is possible again to winnow to the seven critical initial substrates. The number of steps is modest.
  • fraction of the candidate enzymes or initial substrates used in each aliquot at the first winnowing step, and each step thereafter can be chosen such that the expected number of aliquots which form the desired molecule is one or greater than one at each step of the winnowing process.
  • the set of initial substrates may be winnowed using the sib selection procedure described above. This increases the concentration of the target molecule because the diversity of molecules present and resulting side reactions is sharply reduced. In addition, in advantageous cases it may be possible to winnow out those reagents or physical conditions not needed to synthesize the target molecule.
  • One aim of the sib selection procedure is to obtain a sufficient abundance of the target molecule for its characterization and synthesis by independent means known in the art. Typically, microgram or milligram quantities are sufficient for such analysis by standard techniques. As noted, it may often be possible to deduce structure and composition from far smaller quantities by mass spectrographic analysis or other means known in the art.
  • a r compound in a reaction mixture may be characterized functionally (e.g., it is defined by the set of molecules with which it is capable of interacting).
  • a compound in a reaction mixture may interact with a particular amino acid or small sequence of a polypeptide, resulting in enhanced or diminished function of the polypeptide.
  • the compound might be a suicide substrate which covalently links to a polymer near the catalytic site.
  • Such a bound suicide substrate may be used to identify catalysts with a desired activity, or to characterize features of the active site of such a polymer.
  • the site of interaction on the polypeptide may be detected by analytic techniques which are capable of detecting perturbations to individual amino acids or regions of the polypeptides.
  • This information regarding the locus for alteration of the polypeptide's function ⁇ i.e., information about the target) may be equally or more important than the structure of the compound in the reaction mixture which interacted with the polypeptide. It will be evident that, based on this type of information, one may modify a particular amino acid or region of a polypeptide in a variety of ways.
  • the single-stranded DNA needed for 38, 71, and 104 amino acid polypeptide libraries is synthesized. The total diversity is on the order 10 15 .
  • PCR amplification is carried out by routine methodology.
  • An efficiency yielding of about 10,000,000 to 100,000,000 transformants per ug of plasmid DNA is attainable (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989).
  • 500,000 to 5,000,000 clones per transformation is achieved. Transformation may be optimized by (i) purifying the insert
  • each sequence among the 10 7 ligated is unique.
  • the diversity obtained is tested by counting total transformants created, sampling random ampicillin resistant clones, carrying out plasmid preparations, restriction mapping and screening for inserts. This allows calculation of the total number of transformed clones obtained, but, since any sequence might be present in multiple copies, the total alone does not yet specify the total diversity.
  • Clone redundancy in the library is tested using plasmid preparations of a pool of 5,000 plasmids. Redundancy among these distinct plasmids is tested via hybridization with the unique random DNA region from each of several specific plasmids among the 5,000.
  • 5,000 transformed colonies are grown on a single plate, lifted onto nylon filters (GeneScreen Plus, DuPont), the cells lysed, the DNA is UV- crosslinked to the filter, washed, the DNA denatured with NaOH, and then neutralized. Thereafter hybridization is carried out under stringent conditions with radiolabeled unique DNA probes purified from each of several plasmids among the 5,000.
  • Probe DNA is cut from the adjacent ubiquitin sequences and gel purified prior to labeling. Probe is labeled by random primer labeling (Prime-It, Stratagene Cloning Systems). Autoradiography of the resulting filters reveals if any insert DNA sequence occurs in an expected one, or many among the 5,000 colony diversity on the plate. Given the distribution of numbers of colonies bound for each of 10 to 20 probe insert DNA sequences, the expected diversity of the library may be calculated based on maximum likelihood methods.
  • the combinatorics of the libraries described in Example 1 are tested for the onset of catalyzed reactions as libraries of polymers act on one another.
  • the number of possible interactions is enormous.
  • the combinatorics admit of 10 21 possibilities of interactions in the DNA and peptide libraries of a 10,000,000 diversity.
  • the probability that an arbitrary polypeptide catalyzes a given ligation reaction is 10 '9 (an estimate based on the ease of finding catalytic antibodies), a very large number of distinct reactions are catalyzed.
  • the combinatorics favor the onset of catalyzed reactions, as the diversity of reactants increases, the concentration of any type of sequence decreases proportionally. For bimolecular reactions, the forward rate decreases as the square, for trimolecular reactions the rate decreases as the cube of the falling concentrations.
  • the desired product concentrations and catalyzed reactions may be achieved with diversities of 10 4 in both the DNA and polypeptide libraries.
  • diversities on the order of 10 5 to 10 ⁇ in both the substrate and catalyst library are needed.
  • a first set of experiments utilizes single stranded DNA sequences as substrates.
  • Subsequent experiments use polypeptides as substrates. This choice is made for three reasons. First, production of novel DNA sequences, whose length differs from the initial set of substrates, all of identical length, is easy to detect on sequencing gels.
  • single stranded DNA like RNA, is able to fold into complex structures (Lu et al., J. Mol. Biol. 223:781-789, 1991), hence afford a wider variety of sites for binding and catalysis than double stranded DNA sequences of the same total diversity.
  • single stranded DNA is easier to obtain from the libraries than the corresponding RNA, and somewhat more stable against degradation. Nevertheless, RNA of high diversity specified by the libraries may be purified.
  • DNA sequences may be modified to include RNA polymerase premier sites such as T7 to allow in vitro RNA transcription (Ellington and Szostak, Nature 355:850-852, 1992), and obtain high diversity RNA libraries for use as substrates.
  • protocols are stated in terms of single stranded
  • DNA substrates but single stranded RNA libraries may also be used.
  • the plastein reaction (Wang et al., Biochem. Biophys. Res. Commun. 57:865, 1974; Silver and James, Biochemistry 20:3177, 1981) is a general model for the experiments.
  • protein substrates are incubated with trypsin, which cleaves the substrates to smaller peptides. Since any enzyme catalyzes forward and reverse reactions, trypsin is capable of catalyzing ligation of larger polypeptides from the smaller peptide fragments.
  • single stranded DNA sequences of constant length from the libraries, end labeled after the reaction in one set of experiments, and uniformly labeled prior to the reaction in another set of experiments, are incubated with ⁇ P nucleotides.
  • the substrates are then incubated with affinity purified polypeptides from the libraries of Example 1 with length 38, 71, and 104 of tuned diversities in the ranges noted.
  • Divalent cations such as Mg++, Pb++, Mn++, as well as ATP as a potential energy source may be included.
  • concentrations of DNA substrates and polypeptides are tuned over a range sufficiently broad to include conditions under which biological polynucleotides are cleaved or are ligated in vitro.
  • the polypeptides in the library catalyze, for example, cleavage, ligation or transeste fication reactions among the single stranded DNA target molecules.
  • cleavage is energetically favored in an aqueous medium
  • transesterification reactions like transamination reactions among polypeptides in the plastein reaction, are nearly constant energetically in aqueous media, in addition, a variety of crosslinking reactions between two single stranded substrates may occur.
  • Transesterification reactions between two substrate sequences of length L can yield two product molecules, one of which is larger than either of the two substrate sequences.
  • the beginning library of DNA molecules are all of the identical length.
  • control reactions are carried out using a control library encoding ubiquitin alone.
  • affinity purified ubiquitin alone derived from the control library, catalyzes reactions among the DNA substrates, then this can be controlled for in two ways.
  • novel random peptides are cleared free from ubiquitin as noted above, the novel peptide fragments repurified by size under non-denaturing conditions, and retested for catalysis using these random peptides freed of ubiquitin.
  • the particular reaction substrates acted on by ubiquitin or cell background material can be identified by a logarithmic dilution technique, as described below, and eliminated from the DNA substrate library. A number of features of this system may be assessed.
  • the probability that a protein catalyzes a detectable reaction on DNA substrates may be estimated. At low diversities of the libraries, the appearance of a few distinct bands of lower or higher molecular weight than the initial DNA substrate library may be seen. Where these are the only reactions catalyzed, then as the incubation period increases, no further bands appear. Each cleavage reaction involving a single DNA substrate may give rise to two product sequences. Transesterification reactions between two substrates again give rise to two product sequences per reaction. Crosslinking and ligation reactions yield one new product sequence. Single crosslinking and end ligation reactions yield one new product sequence.
  • Single crosslinking and end ligation reactions among a uniform set of single stranded DNA sequences length L should all have a total length of 2L nucleotides. Therefore, for new bands corresponding to lengths less than 2L, the number of reactions is estimated as half the number of such new bands. Using this data, one may estimate the probability that an arbitrary polypeptide catalyzes a detectable reaction. (Some crosslinked DNA sequences with 2L nucleotides may have aberrant migration characteristics, perhaps leading to erroneously count them as products of transesterification reactions. This could cause a two-fold error in the estimated probability.) Second, this estimated probability may be confirmed by increasing the substrate and polypeptide diversity. Third, by tuning polymer length at constant diversity, the effective number of substrate sites and of catalytic sites may be measured as a function of polymer length.
  • DNA sequences of constant length derived from the libraries is incubated with ⁇ P labeled nucleotides or short oligonucleotides, acrylamide gels run, and the labeled material is tested for incorporation into large molecular weight DNA material.
  • a new general "logarithmic dilution" procedures is carried out to isolate both the specific polypeptide(s) catalyzing any specific reaction, and the specific substrates involved.
  • the procedure introduced here also serves to isolate both the specific set of substrates and the specific set of novel enzymes leading to the synthesis of a target molecule of interest.
  • the probability that any random half of the diversity of the polypeptide library has the requisite enzymatic polypeptide is 0.5.
  • two of the set of four random half-library aliquots contains the required polypeptide. If no random halved aliquot had the required polypeptide, a larger number of halved aliquots is tested.
  • Each new diminished library is incubated with the full set of single stranded DNA substrates, and the products analyzed on a long sequencing-type gel. On average, for two such gels, the desired product of the reaction continues to be present.
  • the corresponding half polypeptide library contains the polypeptide which catalyzes the reaction.
  • That now diminished library is again divided into four random halves in four aliquots. Each is incubated with the full set of DNA substrates, the gel run and the product identified if formed in at least one of the four aliquots. By a logarithmic number of halvings of the initial polypeptide library, the single polypeptide catalyzing a specific reaction is isolated. Simultaneously, the fusion gene encoding this polypeptide is isolated. Thus, if the polypeptide diversity is on the order of 10,000, then about 13 halvings suffice.
  • the specific substrates for the reaction in question are obtained.
  • eight random halves of the DNA substrate library are progressively formed.
  • the probability that any aliquot contains the two substrates is 0.25, hence on average two of the eight have the two substrates.
  • These aliquots with the now known catalytic polypeptide are incubated, gels run, which aliquot exhibits the desired reaction product confirmed, thereby concluding that the corresponding half of the substrate diversity contains the desired two substrates. Over a logarithmic number of successive rounds, the two substrates are thus isolated.
  • a main virtue of this approach is that it is possible to carry it out for any set of molecule substrates, and any set of polypeptide, RNA, or other potential catalysts.
  • a modest number of halving steps isolates both the substrates for and enzymes for the reaction leading to the product.
  • This approach generalizes to cases in which several enzymes carry out a succession of reactions from an initial set of substrates. It is merely necessary to alter the random fraction of the diversity in each aliquot, and number of aliquots at each step, to assure that at least one such aliquot contains the requisite set of substrates or enzymes.
  • a logarithmic number of steps is required to isolate both the set of substrates and the set of enzymes leading to synthesis of a desired novel target compound.
  • polypeptide libraries of tuned diversity may be permitted to act on themselves as substrates. Many of the same considerations apply to polypeptide and DNA sequences as substrates for reactions. Cleavage is energetically favored in aqueous medium, while transamination reactions are energetically neutral. Thus, as noted, in the plastein reaction, increasing the concentration of the peptide fragments by dehydration shifts the transamination reactions in favor of synthesis of large molecular weight polypeptides, and the reactions proceed without ATP hydrolysis (Neumann et al., Biochemistry 73:33, 1959). Thus, after incubation of a set of labeled polypeptides of a constant length and mean molecular weight, formation of novel lower and higher molecular weight sequences may be seen.
  • endoproteases may be used to drive the efficient synthesis of larger polypeptides from smaller peptide substrates.
  • Enzymes used include subtilisin, papain, thermolysin, chemotrypsin, and carboxypeptidase Y, in enzyme concentrations ranging from micromolar to millimolar, and substrate concentrations ranging from millimolar to molar (Wong and Wang, Experientia 47:1123-
  • each 71 amino acid fusion peptide is present at approximately 0.6 micromolar concentration. With a diversity of 1 ,000,000, each is present at 0.06 nanomolar concentration. In a volume of 10 ml, a diversity of 1,000,000 corresponds to 10 nanograms of each. These concentrations are detectable. For example, gold stained blots on Immobilon P filters can detect spots with 3.5 nanograms, and polyacrylamide gel staining can detect bands or spots of 2.0 nanograms (Pluskal et al, Bio/Techniques 4(3):272-282, 1986; Ausubel et al., eds., Current Protocols in Molecular
  • Two-dimensional gels may be used to confirm that unique bands on one-dimensional gels correspond to unique spots in two dimension, hence a single product polypeptide. This allows one to count the number of reaction products.
  • the total number of reactions catalyzed may be estimated. From this, the probability that a polypeptide catalyzes a reaction can be calculated. As the lengths of the polypeptides are altered, one may obtain measures of the scaling relation for numbers of types of reactions catalyzed as a function of polymer length of substrates and enzymes.
  • phase transitions afford the ability to catalyze an explosion of molecule diversity from a diverse founder set of organic molecules acted upon by a sufficient diversity of potential catalytic polymers.
  • target small molecules of interest are detected among the products of the catalyzed reactions, the logarithmic partitioning procedures above should allow the recovery of the specific substrates and novel enzymes leading to the molecule of interest.
  • polymers are all of length L, the maximum length polymer which can be formed by a single ligation reaction is of length 2L.
  • the maximum length which can be formed by use of two such newly formed polymers in a new ligation reaction where they are the substrates is 4L, then 8L and so forth.
  • visualization of an increasing maximum molecular weight among the product molecules is evidence favoring supracritical behavior of the reaction system.
  • the mean and variance in molecular weights among the product polymers increase over time and gives rise to a characteristic unimodal distribution.
  • the diversity of polymers of a given length present in the system can be plotted on the ordinate and the lengths of those polymers on the abscissa.
  • the resulting curve may rise steeply to a peak as length increases, then fall off with an exponential tail.
  • the diversity of new bands which appear on sequencing gels are analyzed as a function of time and as a function of the diversity of the polypeptide library catalyzing the reactions.
  • a modest number of new products may appear early, then not increase over time.
  • detection of a sustained increase in total diversity over time is strong evidence for supracritical behavior of the reaction system.
  • the closed reaction system allows one to test for the total increase in product diversity over time.
  • the flow chemostat environment allows one to test, as a function of flow and driving rates, whether the reaction system settles down to a sustained set of founder polymers and their direct and indirect reaction products.
  • Parallel experiments are carried out in which both the substrates and the catalysts are polypeptides. To do so, one may again begin with the minimal diversity 71 or 104 amino acid polypeptide libraries required to see the onset of catalysis of new molecular size products, then tune diversity upward several orders of magnitude.
  • Minimally complex polypeptide systems can form a small number of novel product polymers which does not increase further over incubation time.
  • a supracritical system shows an increasing diversity over time.
  • One-dimensional and two-dimensional gel electrophoresis are used to analyze the total increase in diversity over time. Unlike analysis of DNA sequences, however, use of two-dimensional gels may allow one to discriminate several novel product molecules with the same moiecular weight on SDS page analysis. A sustained increase in total diversity over time (as limited by the product concentrations detectable), and a sustained increase in the highest molecular weight classes seen, is strong evidence for supracritical behavior of the reaction system.
  • labeled amino acids and short peptides, up to hexamers are incubated with libraries of increasing diversity from the larger amino acid library plus the polypeptide library.
  • Supracritical behavior may be demonstrated in a particularly clean way: Theoretical work shows that a sufficiently low diversity founder set of amino acids and small peptides will be subcritical. However, if the concentrations of members of that founder set are maintained by exogenous addition, and the set is incubated with a high diversity of larger polypeptides added once only at the outset of the experiment, then the larger polypeptides can catalyze the formation of many polypeptides built up out of the founder set. Those novel polypeptides themselves come to play catalytic roles in sustaining the formation of themselves and yet further novel polypeptides. Indeed, such a system might include collectively autocatalytic sets of polypeptides. In short, the small peptides alone, in sustained concentrations, are subcritical, but transient exposure to a high diversity of larger polypeptides triggers supracritical behavior which is thereafter sustained without further addition of the larger polypeptides.
  • HPLC analysis appears to fulfill the requirements.
  • UV absorbance detection HPLC can detect concentrations down to the nanomolar range. For example, tryptophan can be detected down to about 10 nanomolar. It may be possible to increase the range of small molecules which are detectable using IR rather than UV spectra (Kemp and Vellaccio, Organic Chemistry, Worth Publishers, Inc., 1980).
  • a chosen set of fifty to a few hundred organic molecules gives rise to a discrete set of peaks which can be discriminated from a far more complex mixture containing a number of additional peaks due to the presence of new product molecules.
  • Evidence of reactions include both the appearance of new peaks and the disappearance of the initial substrate peaks.
  • founder organic molecules are first assembled with well-displaced peaks on HPLC analysis, followed by sequential addition of trial substrate compounds to solutions containing previously accepted members of the founder set.
  • Founder sets are created which optimize both founder concentrations and diversity, such that novel product molecules yield easily detectable peaks.
  • experiments are carried out with a fixed input of founder organic molecules, and under conditions which drive forward synthesis and hold the system displaced from equilibrium by continuous addition of the founder set of organic molecules to otherwise closed stirred reaction systems.
  • radioactively labeled founder set molecules are used to establish that radioactive atoms are incorporated into new product molecules.
  • concentrations of product molecules ultimately depends upon the ratio of the diversity of founder set to product set, the number of reaction steps from the founder set to a given product molecule, and the detailed forward and reverse kinetics along the reaction pathway(s) leading to and from the product species.
  • the foundation is provided by which to increase the diversity of the polypeptides to which the same founder set is exposed. For a sufficient diversity of polypeptides, a very large increase in the diversity of small organic product molecules, hence peaks, is seen in the system. As in our analysis of systems using DNA or polypeptides of fixed initial length, here too, as reactions proceed, ever larger molecular weight products can be formed.
  • This procedure is a minor modification of that described above and reflects the fact that several, e.g., 4, enzymes might be needed to catalyze a chain of reactions, and reflects the fact that several, e.g., 7, initial substrates may be required in those reactions.
  • the four enzymes may be logarithmically isolated as follows. At each step, the current polypeptide library diversity is randomly partitioned into ten aliquots each containing a random 0.7 of the total diversity. The probability that any aliquot contains the four requisitive polypeptides is .24, hence on average two of the aliquots have the four enzymes.
  • Reactions with the full diversity of initial substrates are carried out and the target of interest identified in one or two aliquots, thereby reducing the polypeptide library diversity by a factor 0.7. Successive cycles will, again in a logarithmic number of steps, isolate the four enzymes needed.
  • the substrate diversity is randomly assigned to 10 aliquots each containing a random 0.8 of the initial diversity. The probability that any aliquot has the seven critical substrates is .21 , thus on average two aliquots are successful.
  • a receptor for the normal agonist is already in hand and is used to screen for small molecule mimics of the agonist.
  • no receptor is yet available, but only the agonist itself.
  • inhibitors of an enzyme are sought.
  • the receptor for the agonist is not known, but the agonist is known.
  • a set of random polypeptides which bind to the agonist, hence are its shape complements, is sought.
  • This set of polypeptides then can be used, in place of the unknown receptor, to screen for novel organic molecules which compete with the agonist for binding to members of the set of shape complement polypeptides. While one would not yet know which polypeptides bound the agonist by groups of atoms which reflected the function of the agonist, some among the polypeptides presumably do bind the important agonist epitopes.
  • the set of organic molecules binding to the polypeptide set is a set of candidate drugs to mimic or modulate the activity of the agonist.
  • a third approach seeks a novel small molecule inhibitor of an enzyme such as HIV protease by slowing cleavage of the peptide substrate.
  • the cloned estrogen receptor which is immobilized on Immobilon P filters as dot blot arrays is utilized.
  • Competition assays are carried out with radioactively labeled estrogen and the molecules formed in the reaction mixtures.
  • Dot blot filters are incubated with decreasing concentrations of labeled estrogen and constant concentrations of the mixture of organic molecules. Control filters have no organic molecules added. As estrogen concentration decreases, tests are conducted to determine whether competitive displacement of the labeled estrogen occurs.
  • Tritium labeled estrogen and its analogues are available as 150 Ci per millimole.
  • a picomole of this probe is 0J5 microcuries.
  • 125 l labeled estrogen and its analogues labeled at over 2200 Ci per millimole are available.
  • a picomole is 2.2 microcuries.
  • novel products in the 100 to 1000 picomolar range are generated, even estrogen mimics with modest affinity for the receptor displace labeled estrogen present in picomole concentration, and thus are detectable.

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Abstract

Procédé de production de nouveaux composés éliminant la nécéssité de connaître à l'avance la structure ou la composition chimique d'un composé présentant une propriété voulue. Selon la présente invention, une variété de composés inconnus peut être produite par un procédé aléatoire, puis faire l'objet d'un criblage portant sur une ou plusieurs propriétés déterminées afin de détecter la présence des composés recherchés. Dans l'une des réalisations, on fait subir à un groupe de composés organiques de départ une suite de réactions chimiques donnant naissance à une variété de nouveaux composés organiques qui sont criblés pour détecter les composés organiques ayant la propriétés recherchée. Dans une autre réalisation, on produit une variété de composés à partir d'un groupe de substrats soumis à l'action d'un groupe d'enzimes exerçant différentes activités catalytiques.
PCT/US1994/004314 1993-04-19 1994-04-19 Procede aleatoire de production de nouveaux composes chimiques WO1994024314A1 (fr)

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CA2160457A1 (fr) 1994-10-27
EP0695368A1 (fr) 1996-02-07
EP0695368A4 (fr) 1996-05-08
AU6815894A (en) 1994-11-08
JPH09500007A (ja) 1997-01-07

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