Combinatorial Chemistry & High Throughput Screening - Volume 6, Issue 7, 2003

Both the completion of the Human Genome Project and the sequencing of the genetic codes of microorganisms are providing the unique opportunity to understand Life better and consequently are producing a vast number of potential targets for therapeutic intervention. There is a great need for novel, intelligent strategies for identifying valid targets and discovering druglike leads against them. This Special Issue provides an overview of bead technologies in the everyday practice of drug research. Bead technologies are defined here as a broad term covering combinatorial chemistry approaches based on polymer beads (e.g. solidphase synthesis, polymer supported reagents, on bead assays, etc.). Drug discovery proceeds as a multi-stage process from the identification of a potential therapeutic target through lead generation, lead optimization to clinical testing before a drug product is marketed. The first series of reviews are focused on the application of bead technologies at the very early stage of drug discovery, namely genomics and proteomics. A single polymer bead can be considered as a miniaturized reactor which is carrying a chemical entity and in which a biological assay can be performed. The application of a multiplexed bead-based assay is described in the first review of this collection by Yingyongnarongkul, How, Diaz-Mochon, Muzerelle and Bradley. They provide a comprehensive review of the role of beadbased assays for high-throughput screening, gene expression, single nucleotide polymorphism and genomic analysis. The review of Groth, Renil and Meinjohanns focuses on techniques based on biocompatible polymers and combining both high throughput organic synthesis and high throughput screening in one process. In the fields of chemical genetics and chemogenomics, chemical entities are exploited to identify and characterize proteins and to understand the cellular processes in which they operate. In groundbreaking work in this area, Eguchi, McMillan, Nguyen, Teo, Chi, Henderson and Kahn describe the concept of peptide secondary structural mimetics within chemogenomics. In a broader review of the subject, Thorpe summarizes the application of compound arrays generated from solid-phase chemistries to the field of forward and reverse chemical genetics. The selection of a therapeutic target is followed by the identification and optimization of lead structures. The synthesis of combinatorial libraries is today well recognized as a successful approach to generate novel chemical entities for the identification of new leads. The next series of review articles demonstrate the importance of solid-phase organic synthesis to generate libraries. The review of Goodnow, Guba and Haap focuses on design approaches to deliver small molecule libraries that will increase the success of lead optimization. The concept of chemical biology applied to a target family is explored in the review of Park and Kimmich, in which chemical libraries directed for protein kinases are described. Natural products have been one of the major sources of leads and drugs. In their review Knepper, Gil and Braese discuss the synthesis of complex molecules derived from natural products using solid-phase synthesis or chemistries assisted by polymer-bound reagents (one other component of bead technologies). Pulici, Cervi, Martina and Quartieri provide comprehensive coverage of a relatively new area of organic chemistry - multicomponent and domino reactions performed on solid-phase. In another variation of solidphase organic synthesis, Michalek, Horn, Tzschucke and Bannwarth describe the use of catalysts non-covalently bound to the polymer. The mechanisms of organic reactions on a solid support are not well understood, and the difficulties of transferring chemistry from solution to solid phase have discouraged many chemists. The final review by Schroeder provides perspectives on the application of NMR methods to monitor reaction steps and describe the interpretation and validation of the structures of complex molecules while attached to polymer beads. I would like to acknowledge the scientists for their excellent contributions and the experts that helped review the manuscripts for this issue. This collection of reviews, mini-reviews and research articles was assembled to illustrate the critical significance of bead technologies along the value chain of the drug discovery process. Considering the many uses of such techniques, we can expect the development of exciting new approaches and applications in the coming years.

Advances in high throughput screening (HTS), together with the rapid progress in combinatorial chemistry, genomic and proteomic sciences have dramatically stimulated the development of a variety tools to enable the drug discovery process to become more efficient. Major future challenges in HTS include obtaining high density and good quality data based on assays that are rapid, reliable, inexpensive, sensitive, simple and miniaturised. This paper reviews the development and role of bead-based assays for HTS including DNA and single nucleotide polymorphism (SNP) assays, particularly from a multiplex perspective and evaluating the recent advances in bead-based arrays. The encoding strategies that are commonly used in bead-based assays are highlighted, while the importance of magnetic beads in genomic and proteomic purifications is discussed. In conclusion, bead-based assays offer a powerful promising approach for many aspects of drug discovery.

This review will cover the entire hit identification process performed with biocompatible, aqueous solvated, poly[ethylene glycol] (PEG) based resins - from synthesis, through screening, to analysis. The different types of resins (including their preparation) will be discussed and compared individually. Examples of one-bead-one-compound substrate libraries will be presented, as will one-bead-two-compounds libraries used for the discovery of enzyme inhibitors. The review includes a section covering organic and bio-organic reactions performed on all-PEG resins and discusses on-bead screening of the libraries with biomolecules. Finally, analysis of compounds on single beads, either via investigation by on-bead NMR or by ladder-coding of the combinatorial compound is covered. In general, the review will focus on chemistry, libraries, synthesis, screening, and analysis, using all-PEG based resins.

There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R α) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.

Combinatorial chemistry is being applied to diverse problems in the biological and pharmaceutical sciences. This review will describe an emerging application called “chemical genetics” or “chemical genomics” - genetics and genomics are often used interchangeably in this context. In forward chemical genomics, chemical libraries are tested in living systems to discover compounds that cause a desirable effect. Subsequently, the protein target is identified using various biochemical and molecular biological tools. By this method, we gain insights into the inner workings of life, and indeed, in some forms this has been the path by which most of the pharmacopoeia was discovered. In reverse chemical genetics, proteins of interest are used to probe compound collections, and those compounds that bind the proteins of interest are used to treat living systems and observed for interesting biological responses. Plausible biological roles of these proteins are inferred from the effects of the compounds because they are assumed to generally inhibit, or more rarely, stimulate, the protein's functions. Interestingly, the reverse genetic approach is emerging as the leading model for drug discovery today. Different methods and cases will be described to illustrate forward and reverse paradigms, including those developed in the author's laboratory.

The generation of novel structures amenable to rapid and efficient lead optimization comprises an emerging strategy for success in modern drug discovery. Small molecule libraries of sufficient size and diversity to increase the chances of discovery of novel structures make the high throughput synthesis approach the method of choice for lead generation. Despite an industry trend for smaller, more focused libraries, the need to generate novel lead structures makes larger libraries a necessary strategy. For libraries of a several thousand or more members, solid phase synthesis approaches are the most suitable. While the technology and chemistry necessary for small molecule library synthesis continue to advance, success in lead generation requires rigorous consideration in the library design process to ensure the synthesis of molecules possessing the proper characteristics for subsequent lead optimization. Without proper selection of library templates and building blocks, solid phase synthesis methods often generate molecules which are too heavy, too lipophilic and too complex to be useful for lead optimization. The appropriate filtering of virtual library designs with multiple computational tools allows the generation of information-rich libraries within a druglike molecular property space. An understanding of the hit-to-lead process provides a practical guide to molecular design characteristics. Examples of leads generated from library approaches also provide a benchmarking of successes as well as aspects for continued development of library design practices.

Over 500 human protein kinases identified to date are susceptible to play crucial roles in the regulation of many signal transduction pathways, making them significant drug discovery targets. However, their active sites share a high level of similarity, which constitutes a major challenge in the finding of selective and safe inhibitors. In order to meet this challenge, whether via traditional or alternative approaches, the use of chemical libraries to find either unknown natural ligands or specific inhibitors of particular kinases is more important than ever. This review briefly summarizes the recent literature on such libraries of peptides, natural product analogues, and small molecules. Significant chemical scaffolds, some synthetic routes particularly on solid-phase support, and computational tools employed for the efficient design of both selective and bioavailable inhibitors are highlighted.

High-throughput technologies allow the selection of new biological targets for drug discovery in the post-genomic era. These tools increase the need of new methods to rapidly obtain potent small molecules and natural products to discover new lead structures. In particular, the solid-phase synthesis offers a great potential to obtain large compound sets.

The availability of small organic molecules covering as much chemical space as possible is seen as the only means that guarantees potential modulation of the many biological targets that are ultimately being unveiled by genomics. Therefore diversity oriented organic synthesis is rapidly becoming one of the paradigms in the process of modern drug discovery. This has spurred research in those fields of chemical investigation that lead to the rapid assembly of not only molecular diversity, but also molecular complexity. As a consequence multi-component as well as domino or related reactions are witnessing a new spring. Coupling these one-pot processes with solid-phase synthesis offers new perspectives for the preparation of both primary and thematic libraries. The progresses recently made in this field that perfectly suits the needs of modern drug discovery are the subject of the present review.

Supported catalysts have become valuable tools for simplified product isolation and catalyst recycling. The common method is covalent attachment to a solid support. An alternative strategy is to immobilize catalysts by non-covalent bonding through hydrogen bridges, ionic, hydrophobic or fluorous interactions. Compared to covalent attachment, such non-covalent approaches increase the flexibility in the choice of the support-material, reaction conditions and work-up strategies. Numerous catalytic reactions employing one of these non-covalent fixation strategies have meanwhile appeared in the literature.

Solid phase synthesis has become a routine technique in combinatorial chemistry. The need in analytical methods to characterize nondestructively resin bound molecules has been fulfilled by the introduction of High Resolution Magic Angle Spinning (HR MAS) NMR of solvent swollen beads. HR MAS NMR can give solution like proton NMR spectra and one- and two-dimensional NMR techniques are amenable, allowing detailed structure analysis. Recent developments are the application of a diffusion filter to suppress solvent signals and dipolar recoupling techniques to gain spatial information. HR MAS NMR has been applied to monitor reactions and elucidate reaction products.

Gerard Rosse grew up in Chatillon, a small town located in the French speaking region of Switzerland. He received his B.S. and Ph.D. in Chemistry from the University of Basle, Switzerland. His undergraduate studies, completed in 1991, encompassed a Diploma work on natural products total synthesis under the supervision of Prof. Christoph Tamm. Dr. Rosse's doctoral thesis on the design, synthesis and biological characterization of multisubstrate-complex analogues as inhibitors of the EGF-Receptor protein tyrosine kinase was completed in 1995 with Prof. Urs Sequin and in collaboration with Dr. Heinz Fretz and Dr. Peter Traxler from Novartis (formerly Ciba-Geigy), Basle, Switzerland. He then studied as a post-doctoral fellow at Stanford University under Prof. John Griffin and working on combinatorial libraries of vancomycin derivatives. In 1997, Dr. Rosse moved back to Basle, where he was hired as a research chemist by F. Hoffmann-La Roche Inc. While at Roche, he worked within the combinatorial chemistry group of Dr. Lutz Weber on the synthesis of libraries small molecules. He also was involved in a variety of other projects including the development of antiinfective agents, high throughput NMR analysis and the investigation and application of bead-based methods for deorphaning genomically derived proteases. Since 1999, Dr. Rossé is working with Aventis, at the Aventis Combinatorial Technologies Center (ACTC), Tucson, Arizona. He is a Chemistry Group Leader and is leading the ACTC purification group as well. The research interests of Dr. Rossé include many aspects of drug discovery, with special emphasis on understanding the chemical biology of GPCRs and Kinases by designing and synthesizing focused libraries. He is also involved in the optimization of hits to leads, in the development of information technologies and instrumentations for parallel synthesis and purification. He has published many articles and patents. Dr. Rosse currently lives with his wife, Frederique, and two daughters, Alyssa and Noanne, in Oro Valley, AZ.