The New Medicine Man

Courtesy of Zymark Combinatorial Chemistry Products and Services Finding cures for cancer, AIDS, and the plethora of other ailments that plague humans conjures up images from the 1992 movie Medicine Man. In it, Sean Connery as Dr. Robert Campbell treks through the tropical Amazon forests searching for an enigmatic plant that seems to produce a cancer-curing compound. Although scientists may never approach Mother Nature's ability to create novel compounds, new combinatorial chemistry techniques make a very respectable effort. Multitudes of new compounds can be synthesized rapidly, and high-throughput screening technology provides a mechanism to test them for beneficial properties. In fact, it can be argued that combinatorial chemistry provides the first real opportunity to capitalize on high-throughput screening. In some ways, combinatorial chemistry is to modern drug development what PCR was to molecular biology. In the past, drug development entailed the design and synthesis of individual compounds to target biological processes. Unfortunately, compounds that looked promising in vitro didn't always remain so in vivo, often due to bioavailability or toxicity problems. To develop new compounds with biochemical properties similar to known therapeutics', chemists resorted to one-by-one synthesis of variants of the parent molecule and testing of each analogue--a time-consuming and expensive process yielding very few, if any, successes. To overcome this technological hurdle, chemists looked to independent research from the mid-'80s on the solid-phase synthesis of oligonucleotides and peptides, and modern combinatorial chemistry was born.1,2,3 In simple terms, combinatorial chemistry is the process whereby thousands of compounds (a library), systematic variants of a parent chemical structure, are synthesized in parallel. Although combinatorial chemistry generally refers to a single process, in reality it comprises several integrated intellectual and technological processes: computer-aided drug design and combinatorial chemistry library design, automated solid/solution-phase organic synthesis, and high-throughput screening techniques. Alone each technology is an intellectual step forward in science, but their synergy has been a major leap for drug development and basic medical research. Pharmaceutical researchers can create and test libraries of thousands, if not millions, of compounds for pharmacological properties relatively quickly. Basic science researchers can use these libraries of compounds--potential new inhibitors, activators, pseudosubstrates--to explore the biological activity of enzymes and receptors. Combinatorial chemistry now plays an important role in polymer chemistry and the development of new pigments, superconducting materials, catalysts, and agrochemicals. This profile will focus on the technologies that contribute to combinatorial chemistry and the development of novel compounds for disease treatment and the probing of biological function. Automation looms large in the synthesis and screening of immense combinatorial libraries. Computer-Aided Drug Design In drug discovery, combinatorial chemistry is generally preceded by extensive biochemical and structural studies on the target macromolecule (enzyme, receptor, carrier, structural protein) and compounds known to interact with it. Computer-aided drug design (CADD) integrates the knowledge of the biochemical and structural properties of the target and ligand and the molecular details of their interaction to model new compounds. In most cases, detailed structural information about the target protein (e.g., ligand binding sites) guides these efforts. This type of information is available from 3-D structural databases such as the Protein Databank for macromolecules (rcsb.sdsc.edu) or any number of small-molecule databases (www.sdsc.edu/ResTools/biotools/biotools8.html). New molecules are usually designed by modeling the target-ligand interaction, but may also be fabricated based on a specific set of structural features that impart a desired pharmacophoric property. In the former case, conformational modeling of ligand docking to the target binding/active site using known 3-D structures helps in constructing a new molecule. Then molecular mechanics and dynamics are used to refine the binding interaction. When no structural information on the binding interaction is available, one may draw information from similar, defined binding interactions. These processes yield the structures for compounds that are likely to be the best candidates for drug discovery. The compounds may be analogues of the parent molecule or completely new creations, and will usually be the starting point in combinatorial synthesis. After initial synthesis and screening, candidate compounds may undergo another round of redesign using combinatorial chemistry library design applications. For further information on the bioinformatics and modeling tools used in computer-aided drug design, please see technology profile articles in recent issues of The Scientist.4,5 In just a few weeks, combinatorial chemists can achieve a degree of molecular diversity that would have taken Mother Nature eons to create. To put the results of the process in perspective, consider that the possible number of products is defined by the number of different reagents raised to the number of synthesis steps. Hence, for a three-step process involving 10 different reagents, the theoretical yield is 103 or 10,000 unique chemical combinations. For a simple pentamer peptide there are 205 or 3,200,000 possible combinations, which in theory could be synthesized in parallel in five reaction steps. To facilitate combinatorial synthesis, another set of computer applications has arisen, defined here as combinatorial chemistry library design (CCLD) tools. These applications are an extension of molecular modeling, augmented with classical computational chemistry methods (quantum/molecular mechanics and dynamics, electrostatics); pharmaceutical descriptors (elements that define compounds with ideal pharmaceutical ADME [absorption, distribution, metabolism, excretion] physiological properties); information on current chemical synthesis techniques; and available synthesis reagents (building blocks). Chemical Computing Group Inc., Molecular Simulations Inc., Oxford Molecular Group Inc., Pharmacopeia Inc., and Tripos Inc. are among the major providers of CCLD and CADD applications. Molecular Simulations Inc. (MSI) of San Diego, a Pharmacopeia subsidiary, is well known for its molecular modeling and visualization application and ligand design and simulation tools (Cerius2(r), WebLab(r), Insight II(r), and Catalyst(r)). MSI has enhanced many of these tools by adding new modules to existent programs and extending capabilities for rational and combinatorial drug design. Another well-known modeling application provider, Tripos of St. Louis, has integrated combinatorial chemistry and library design functions into its product line. Tripos provides comprehensive tools for molecular design and visualization (SYBYL, MOLCAD), conformational analysis (Advanced Computation), protein and biopolymer modeling (Genefold, Composer, MatchMaker, Biopolymer, SiteID), pharmacophore recognition (DISCO, GASP, FlexS, RECEPTOR), quantitative structure-activity relationship determination (QSAR with CoMFA, Charisma, HQSAR), and chemical information management and database searching (UNITY, ChemEnlighten). Relatively new modules for combinatorial library design (Legion/CombiLibMaker, DiverseSolutions, Selector) and virtual screening (Cscore, FlexX) complete a set of modeling applications that maximize the efficiency of combinatorial chemistry technology. Oxford Molecular Group of Hunt Valley, Md., has been a major developer of bioinformatics (OMIGA, MacVector, SeqWeb, GCG Wisconsin Package, etc.) and molecular/chemical modeling (CAChe, Tsar, TOPKAT) applications. The company has met the cheminformatics and combinatorial chemistry technologies head-on with an impressive array of applications: RS3 for chemical information storage, indexing, and retrieval; DIVA for information management and display; OMMM for HTS data management; and a comprehensive suite of programs, packaged as Diamond Discovery, for combinatorial library design and analysis. A relatively new player in the molecular modeling and combinatorial chemistry software arena is Chemical Computing Group of Montreal. Its initial software offering included powerful tools for protein modeling and structural bioinformatics, including MOE-Homology, MOE-Contact Analyzer, MOE-ProEval, MOE-SearchPDB, and MOE-Align. Its selection of applications has grown to include tools for molecular modeling/simulation, including MOE-Mechanics for large-scale energy minimization and conformational analysis, MOE-Dock for modeling binding conformations of ligand and target structures, and MOE-FlexAlign for alignment of small molecules that share similar biological activity. In addition, it now features a broad array of applications for what it refers to as "High-Throughput Discovery": QuaSAR-Binary (for HTS data analysis), QuaSAR-Reagent (for design of combinatorial libraries), QuaSAR-Descriptor (to define descriptors for the ideal pharmacophore), QuaSAR-Classify (classification tree for activity prediction), and QuaSAR-PLS (to build quantitative structure activity relationships). The flagship combinatorial chemistry software product from Daylight Chemical Information Systems Inc. of Mission Viejo, Calif., is appropriately named "Daylight' (v4.62). This product is actually a suite of applications (modules) for small-molecule structure analysis and comparison and combinatorial chemistry library design, integrated with a respectable collection of small-molecule databases and information resources. Among the most relevant modules for the combinatorial chemist are the CombiChem, Reaction, and Clustering Packages and the Monomer, SMARTS, Fingerprint, and Reaction Toolkits. MDL Information Systems Inc. of San Leandro, Calif., has been a long-term provider of biological and chemical information databases and data retrieval and management systems. Its ISIS (Integrated Scientific Information System) serves as an information management framework for scientific data. MDL's Reagent Selector enables researchers to quickly and efficiently identify, locate, organize, and share libraries of reagents and shop for databases of commercial suppliers. Central Library(tm) supports enterprisewide information management, and Project Library(tm) provides desktop information management for parallel and combinatorial synthesis. Another well-known product is Chemscape, which provides Web-based access to information. MDL also features a fair array of bioactivity (e.g., Metabolite, Toxicity) and synthesis planning information (e.g., ChemInform Reaction Library, SPORE), an integrated part of computational analyses in combinatorial chemistry. Argonaut Technologies' chemist using the Trident(tm) Automated Library Synthesizer Synthesizing a Medicine Chest Early combinatorial syntheses were manually performed using crude laboratory instrumentation. The advent of computer technology combined with microelectronics and robotics has spurred the development of self-contained, fully automated chemical, peptide, and nucleotide oligomer synthesizers. Researchers can now synthesize custom combinatorial libraries using their own instruments or contract services. Companies that produce software (drug design, database), instrumentation (synthesizers), reagents (including novel, proprietary synthesis products), and custom or premade compound libraries are listed in the accompanying table. Advanced ChemTech, Anachem, Argonaut Technologies, Molecular Devices, PE Biosystems, Spyder Instruments, and Zymark are among some of the providers of synthesizers and assorted instrumentation for solid- and solution-phase syntheses. Argonaut Technologies of San Carlos, Calif., focuses on technology enabling parallel organic synthesis--from high-throughput library synthesis to downstream structure-activity relationship studies and faster process development. It offers synthesizer instruments, proprietary reagents (chemical resins and reagents), custom syntheses, and technology partnerships for those who wish to capitalize on Argonaut's in-house chemistry and engineering expertise. Another major player providing instrumentation and software solutions for drug development is Advanced ChemTech of Louisville, Ky. It provides a complete line of peptide synthesizers and solid- and solution-phase organic synthesizers ideal for combinatorial library generation. Its synthesizers run the gamut from a small-scale benchtop LabTech synthesizer to the ultrahigh-throughput Venture synthesizer. For researchers who lack the expertise, time, or budget to invest in combinatorial chemistry methods, many of the companies providing combinatorial chemistry solutions for drug development offer collaborative research partnerships. These opportunities bring varied problem-solving expertise together, reduce the time from development to drug discovery, and cultivate novel intellectual relationships. Because the scope of a problem or expense of a project can sometimes exceed the capacity of one or two research teams, multigroup collaborations have also arisen. One example is the Combinatorial Chemistry Consortium (CCC) sponsored by MSI. This consortium is one of six that brings together groups of industrial researchers, academic experts, and MSI scientists to focus on developing, validating, and applying simulation to specific industrial research areas. High-Throughput Screening The power of combinatorial chemistry to generate thousands or millions of new compounds would be lost without a feasible method to screen these leads for beneficial properties. Recent advances in high-throughput technology have come to the rescue. These days, high-throughput screening has become nearly synonymous with microarray technology. Developed to hasten gene identification, microarrays have been around for many years, but were relatively underused for drug development until the advent of combinatorial chemistry. This technology affords simultaneous screening of hundreds or thousands of target molecules (protein, DNA) or ligands mounted to an inert material such as a microtiter plate or microetched silico/glass wafer. The physical testing process has been largely automated, and photo-optic systems read results directly into a computer. The data are archived and available for a multitude of quantitative computational analyses. Microarray high-throughput screening speeds testing, consumes small quantities of test material and reagents, and affords precise and accurate means of data analysis. Biological Applications Combinatorial chemistry has certainly been a bonanza for drug development, but it also has benefited basic science researchers. Combinatorial chemistry has provided novel avenues to probe molecular structure, biochemical activity (enzyme kinetics, ligand binding), and biological function. In 1999 alone, more than 600 research studies reported using combinatorial chemistry, with more than 25 percent of these applied to basic research. Using combinatorial peptide libraries, investigators have found new ligands for somatostatin receptors,6 and another research team has found an inhibitor to a protease from Trypanosoma cruzi, the causative agent for African sleeping sickness.7 The substrate specificity of enzymes such as S-farnesyl transferase has also been examined in detail using peptides.8 Lew Cantley's research group at Harvard University puts a twist on the use of peptide libraries to define "functional" sequence motifs for protein kinases and Src-homology domains SH2 and SH3. They have been able to predict, based on primary sequence alone, which SH2 domains in a protein will interact with downstream signaling molecules.9 In most cases, combinatorial chemistry technology providers can be a key source of expertise and help in finding solutions to your combinatorial needs. In addition, a wealth of books, journals, and meetings provide detailed information on technology, methods, and applications, and serve as up-to-date sources of information as this technology grows (See Additonal resources). Christopher M. Smith can be contacted at csmith@sdsc.edu.< References 1. R. Frank et al., "A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports," Nucleic Acids Research, 11:4365-77, 1983. 2. H.M. Geysen et al., "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid," Proceedings of the National Academy of Sciences, 81:3998-4002, 1984. 3. R.A. Houghten, "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids," Proceedings of the National Academy of Sciences, 82:5131-5, 1985. 4. C. Smith, "Computational gold," The Scientist, 13[19]:21-23, April 26, 1999. 5. C. Smith. "Molecular modeling," The Scientist, 12[17]:17-19, August 31, 1998. 6. A.J. Souers et al., "Identification of a potent heterocyclic ligand to somatostatin receptor subtype 5 by the synthesis and screening of beta-turn mimetic libraries," Journal of the American Chemical Society, 121:1817-25, March 10, 1999. 7. S. Vendeville et al., "Identification of inhibitors of an 80 kDa protease from Trypanosoma cruzi through the screening of a combinatorial peptide library," Chemical and Pharmaceutical Bulletin, 47:194-8, February 1999. 8. J.A. Boutin et al., "Investigation of S-farnesyl transferase substrate specificity with combinatorial tetrapeptide libraries," Cellular Signalling, 11:59-69, January 1999. 9. S. Zhou, L.C. Cantley. "SH2 domain specificity determination using oriented phosphopeptide library," Methods in Enzymology, 254:523-35, 1995.

The New Medicine Man

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