Current Medicinal Chemistry - Volume 10, Issue 9, 2003

The recent use of chemical genomics to identify bioactive small molecules that interact with specific proteins has had a tremendous impact on both the functional analysis of genes and drug development. Accordingly, the current review focuses on the utilization of this new research engine in the target identification and validation of antiangiogenic agents capable of regulating the growth and spread of cancer cells. In addition, the use of chemical genomics to discover novel anti-angiogenic agent targets and to validate their biological relevancy is providing new insights into the biological role of targets in angiogenesis as well as advancing the development of new antiangiogenic agents.

In this review, I have mainly described the cell cycle inhibitors isolated from microbial metabolites. Once the molecular target of the inhibitor is determined, the inhibitor can be used as bioprobe to dissect the diverse aspect of biological functions in chemical biology research. Reveromycin A and phosmidosine inhibited the protein synthesis of mammalian cells and arrested the cell cycle at G1 phase. Lucilactaene arrested cells at G1 phase through restoration of mutant p53. Tryprostatin A inhibited the microtubule polymerization by interfering with the interaction between tubulin and microtubule associating protein. On the contrary, cyclotryprostatin D, structurally related to tryprostatin A, enhanced the tubulin polymerization. Terpendole E inhibited the motor activity of mitotic kinesin, Eg5 and induced monoastral spindle in M phase.

Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for stability and function of multiple mutated, chimeric, and over-expressed signaling proteins that promote cancer cell growth and / or survival. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, HER2 / Neu (ErbB2), and HIF-1α. Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the destabilization and eventual degradation of Hsp90 client proteins, and they have also shown promising anti-tumor activity in preclinical model systems. One Hsp90 inhibitor, 17-AAG, is currently in Phase I clinical trial. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple signaling pathways on which cancer cells depend for growth and survival. Benzoquinone ansamycin binding to Hsp90 led to the identification of radicicol as an additional Hsp90 inhibitor. Additional target-based screening uncovered novobiocin as a third structurally distinct small molecule with Hsp90 inhibitory properties. Use of novobiocin, in turn, led to identification of a previously uncharacterized C-terminal ATP binding site in the chaperone. Small molecule inhibitors of Hsp90 have been very useful in understanding Hsp90 biology and in validating this protein as a molecular target for anti-cancer drug development.

Recruitment of cytoplasmic signaling proteins into the nucleus is an essential step in the activation of gene expression in response to an extracellular signal. Nucleocytoplasmic transport of macromolecules is mediated by the transport receptors of an importin β family. Post-translational modifications and masking / unmasking of specific signal sequences responsible for nuclear import and export are important for the coordinated control of the nucleo-cytoplasmic transport. Malfunctioning of the nucleocytoplasmic transport is profoundly involved in a number of diseases including cancer. Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of fission yeast and mammalian cells. The target molecule of LMB has been shown by genetic and biochemical analyses to be CRM1, a highly conserved protein in eukaryotes. CRM1 was shown to be a member of the importin β family and a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells. LMB binds directly to CRM1, which results in dissociation of the NES from the nuclear export machinery containing CRM1. Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins and a potential therapeutic drug for diseases caused by mislocalization of regulatory proteins.

Cancer is the leading cause of death in United States and World wide. Drug discovery and development for cancer therapeutics takes several years before a patient is benefited from a new drug. The average time length from start to finish is approximately 15 years. This time length includes 4-5 years of basic research, discovery, preclinical development and validation studies. Next, it takes approximately 7-10 years for a drug to go through human clinical trials. This time length is too long and need to be shortened to benefit patients quickly from new technologies and product development ideas. Furthermore, with the recent explosion of genomics and proteomics information, it is now becoming difficult to make rapid and logical decisions on hundreds of potential drug targets available. Thus, there is immediate need to develop and integrate tools and technologies that will not only reduce the time length but also the risk of late clinical drug failure. Chemical Genomics is an emerging field in which tools and technologies from biology and chemistry are utilized in a parallel and cyclic fashion very early in the development process. In addition chemical genomics proposes to integrate latest developments in tools and technologies from a variety of modern fields such as combinatorial chemistry, informatics, synthesis chemistries, cell based assays, microarrays, genomics and proteomics tools to accelerate drug discovery and development. Thus, in cancer therapeutics the aim of chemical genomics is not only to reduce the time length of pre-clinical development but also the risk of late clinical failure by making smart decisions early in the process.

Chemical genomics represents a cooperation of biology and chemistry to identify and intervene the biological targets. Small molecules with diverse structural characteristics should be used to validate the target through interfering with the biological processes. Because of the limitation of existing chemical libraries, the diversity can be exploited using both the molecular design techniques; structure-based design and ligand-based design. These methods can guide the selection of small molecules with optimal binding properties to desired biological targets. Studies of potential molecular targets for novel anticancer drug discovery including in silico screening, QSAR, and de novo design demonstrated the importance of chemical genomics strategy to find the chemical probes and drug lead compounds.

In recent years there has been a growing interest in computer-based screening. One of the driving forces has been the increased efficiency of protein crystallography leading to the real possibility of using structure-based design as a significant contributor to the discovery of novel ligands. In 1957 after 22 years of work the first protein structure, determined by x-ray crystallography was produced [1]. Now the process has become increasingly automated and nearly 20,000 protein structures are available in the Protein Data Bank (PDB) [2]. Equally, progress in genomics will result in a great expansion of validated targets for cancer therapy. The understanding of the relationships between structure and function of gene products will be one of the key routes to new therapeutic advances.The challenge now is to use this data in the discovery of novel therapeutics. One approach is obviously to synthesize molecules and co-crystallize or soak them into the protein crystal and so determine the position and interaction of the molecule with the protein. The structural information obtained (where does the molecule bind; what are the ligand / protein / solvent interactions?) can be invaluable in the generation of novel molecules or in the re-design of existing molecules whose drug properties are not optimal. However, when dealing with large numbers (millions) of molecules, when crystallization is difficult or in testing hypotheses, a significant contribution can be made using computer based screening methods.In order to use the structural information derived from x-ray crystallography (or other sources, for example NMR or homology modelling) when evaluating the utility of a novel ligand, we need to understand where in the protein (or other macromolecule such as RNA) the ligand is likely to bind and also if possible, the strength of the binding interactions. This problem is known as the ‘docking problem’. There have been many approaches to the solution of this problem over the last ten years. For example, some methods rely on complex molecular dynamics simulations while others use less costly graph matching approaches. There is generally a compromise between speed and accuracy, with some methods giving much more information and insight into the nature of the protein / ligand interactions and other methods optimised for speed of docking thousands of putative ligands. We will describe some of the more common methods and algorithms used to solve the docking problem and in particular, we will review recent applications in cancer research.

Ribonucleases (RNases) have proven to be excellent model systems for the study of protein structure, folding and stability, and enzyme catalysis, resulting in four Nobel Prize lectures in chemistry. Beside this ‘academic’ success, RNases are also relevant from a medical point of view. The RNA population in cells is controlled post-transcriptionally by ribonucleases (RNases) of varying specificity. Other therapeutic proteins like angiogenin, neurotoxins, and plant allergens have RNase activity or significant structural homology to known RNases. Also, RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. To date, no clinical drugs are available that target this important class of enzymes. Small-molecule RNase inhibitors derived from mono- or dinucleotides, as well as pentavalent oxyvanadate transition state analogs are found to be rather marginal inhibitors. These compounds bind their target RNase with dissociation constants in the micromolar range, whereas transition state theory predicts picomolar values for genuine transition states. The rational design for new transition state analog inhibitors requires knowledge of the precise nature of the transition state and of the occurring intermolecular enzyme-substrate interactions. This review focuses on these chemical and structural features of RNase A and RNase T1, the best characterized members of two separate classes of ribonucleases.

Vacuolar-ATPase (V-ATPase) has been proposed as a drug target in osteoporosis due to its involvement in bone resorption, and as a target in cancer due to potential involvement in tumor invasion and metastasis. The classical selective inhibitors of V-ATPase are microbial macrolides of the bafilomycin and concanamycin class. These inhibitors have proven to be too toxic for therapeutic use, however recent structure-activity studies on bafilomycins, and the isolation of novel macrolide structures from marine sources, have provided new avenues for development of potentially less toxic V-ATPase inhibitors. The novel salicylihalamide and lobatamide series of compounds were predicted to share a common mechanism of action based on the patterns of cytotoxicity produced in the NCI 60-cell cancer screen. They have subsequently been shown to selectively interact with mammalian V-ATPases, but not with fungal V-ATPases. With the recent achievement of total syntheses of salicylihalamide, lobatamide, and related compounds, the elaboration of congeners with specificity for particular enzyme isoforms may provide drug candidates which are less toxic. This review summarizes recent advances in V-ATPase inhibition and the prospects for further progress.