Current Topics in Medicinal Chemistry - Volume 9, Issue 15, 2009

Through the transcriptional/translational process, linear DNA is transformed into biologically active, three-dimensional proteins that provide the machinery necessary for cellular life. Elucidation of the processes involved in replicating and transcribing DNA led to some of the most important scientific discoveries during the 20th Century, however, the conformational maturation of linear information into tertiary and quaternary structures has only recently become the focus of scientific intensity. The conformational maturation of nascent polypeptides as well as the rematuration of denatured proteins is most often mediated by molecular chaperones. While many of these protein folders are constituitively active, a large class of proteins is induced upon sensing environmental stress such as elevated temperature, which leads to induction of the heat shock response and increased levels of heat shock proteins (Hsp) Hsp27, Hsp40, Hsp70 and Hsp90. As a consequence of the cellular stresses encountered by a growing tumor, Hsp are also required for the survival of transformed cells and provide a mechanism by which mutant proteins can be folded into physiologically active structures. As the master regulator of the heat shock response and the facilitator of a large number of signaling proteins, Hsp90 has emerged as the primary chaperone target for the development of new anti-tumor agents. In this special issue of Current Topics in Medicinal Chemistry, entitled Inhibition and Function of Heat Shock Proteins 70 and 90, nine articles have been assembled to represent a summary of cutting-edge research aimed at further understanding Hsp inhibition and consequences thereof. Through the use of structural and computational biology, traditional and structure-based drug design strategies have been employed to produce a large number of inhibitory scaffolds that disrupt the protein folding machinery. These small molecules act through a variety of mechanisms, which necessitated the development of new biochemical assays before commencement of clinical trials for these compounds. In this issue, a summary of these studies is provided along with emerging paradigms that are likely to result in additional clinical candidates. Jason Gestwicki produces a succinct account of the structure, function, and inhibition of the 70 kDa heat shock protein, Hsp70. Although required for the assembly of many Hsp90 heteroprotein complexes, Hsp70 has recently and independently garnered the attention as a new target for chaperone inhibition and the potential treatment of cancer and/or neurodegenerative disorders. Some of the most significant advances made toward chaperone inhibition have resulted from solution of their threedimensional structures. Chris Prodromou provides a thorough account of these advances and explains how these structures implicate potential new targets within the protein that can be focused upon to achieve selective inhibition/disruption of heteroprotein complex formation. The result of which may provide small molecules that manifest greater differential selectivity, or perhaps those that exhibit minimal toxicity. Gennady Verkhiver provides an exemplary overview of computational approaches used to develop inhibitors of the Hsp90 protein folding machinery. In addition, the Verkhiver review extends upon the solid state structures and sheds light on the potential role intermediate complexes may play in the protein folding process mediated by Hsp90 and its cohorts. The first inhibitor of the Hsp90 N-terminal ATP-binding pocket identified was geldanamycin, of which many derivatives have been produced, and few have entered clinical trials. James Porter gives a historical perspective on the geldanamycin scaffold and its utility for the development of improved Hsp90 inhibitors, ranging from the discovery of 17-AAG through the most recent derivative to enter clinical trials, IPI-504. Another class of natural products investigated for inhibition of the Nterminal nucleotide binding site is based on the resorcinolic acid lactones, as carefully reviewed by Nicolas Winssinger and colleagues. In an effort to circumvent undesired effects manifested by both geldanamycin and radicicol, several new series of compounds have been examined and developed that contain the resorcinol moiety as a key mediator of essential hydrogenbonding interactions. Various conformational constraints placed into the macrocyclic lactone have resulted in promising new scaffolds that exhibit potent Hsp90 inhibitory activities. The first structure-based drug design approach toward inhibition of Hsp90 resulted in the production of the purine-scaffold class of inhibitors. As detailed by Gabriela Chiosis, this molecular scaffold can be easily diversified, while maintaining selective inhibition of Hsp90. In fact, a member of the purine class of Hsp90 inhibitors was placed into clinical trials for the treatment of cancer. A summary of these developments is provided in this article. In contrast to inhibition of the Hsp90 N-terminal ATP-binding site, a number of alternative binding motifs have emerged in recent years. As summarized by Gary Brandt and Brian Blagg, the Hsp90 C-terminal nucleotide binding site and the Cdc37 binding motif have become particularly attractive regions for the development of small molecules that inhibit Hsp90, but through mechanisms of action that vary greatly from N-terminal inhibition. Several high-throughput screening (HTS) assays and biochemical techniques were required to identify and develop new scaffolds for Hsp90 inhibition. Robert Matts provides a thorough overview and detailed summary of these HTS assays as well as an in depth analysis of the biochemical tools that were developed in an effort to differentiate the mechanisms by which various Hsp90 inhibitors function. Without doubt, the most important outcome for any anti-cancer research program is the clinical result. Len Neckers, who pioneered many of the Hsp90 investigations and discovered the first Hsp90 inhibitor to enter clinical trials, 17-AAG, provides a detailed account of the molecules undergoing clinical evaluation. Furthermore, issues and complications arising from these studies are outlined as considerations for future clinical investigations. As one peruses this issue, it is hoped that the importance of chaperone inhibition is clearly stated as a new and attractive target not only for the development of anti-cancer agents, but also, neurodegenerative diseases. Significant advances toward the understanding of Hsp90 function have led to the identification of various disease states that could benefit from Hsp modulation. Although much of the research to date has focused on cancer, new disease states have been identified and some of which have already been sought after in clinical trials with the use of Hsp90 inhibitors. Thus, in spite of the significant advances made during the past 20 years of Hsp research, the field remains in its infancy.

The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein's life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery.

Hsp90 is involved in the maturation and activation of client proteins. Often these are key proteins involved in signal transduction and regulatory pathways that in a mutated and/or deregulated form sustain an oncogenic cellular state. Consequently, the malignancy is maintained with the aid of Hsp90 upon which the mutated proteins have become particularly dependent for their activity. The requirement for the Hsp90 chaperone machine to drive the malignancy makes Hsp90 a prime anticancer target, an ‘axle in a wheel’ that when disrupted has been shown to be effective in killing cancerous cells. This review aims to identify potential drug targets, based on the current structural knowledge of the Hsp90-chaperone machine, that could be targeted with the aim of disrupting its functioning and promoting an anti-cancer activity.

The molecular chaperone Hsp90 (90 kDa heat-shock protein) mediates many fundamental cellular pathways involved in cell proliferation, cell survival, and cellular stress response. Hsp90 is responsible for the correct conformational development, stability and function in crowded cell environments. Structural and computational biology studies have recently provided important insights into underlying molecular mechanisms of Hsp90 function. These developments have revealed a critical role of Hsp90 structure, conformational dynamics and interdomain communication in promoting the binding and release of ligands and its interaction with client proteins. By disabling multiple signal transduction pathways, Hsp90 inhibition provides a powerful therapeutic strategy in cancer research, which is selective for specific cancer mechanisms, yet broadly applicable to disparate tumors with different genetic signatures. Herein, we review the recent developments in structural and computational studies of Hsp90 function and binding, with the emphasis on progress towards computational structure-based discovery and design of Hsp90 inhibitors. We also review the emerging insights from computational and structure-based approaches to develop anticancer therapies that can target novel allosteric binding sites and Hsp90 interactions with co-chaperones and client proteins. Structural and computational biology studies can provide a foundation for the design of Hsp90 modulators capable of regulating functional protein motions linked to biological activities. We highlight current challenges in translating molecular mechanisms of the molecular chaperone into therapeutic strategies and outline future directions for the computer-based design of Hsp90 inhibitors.

The ansamycin class of natural products is well known for its anti-tumor effects and has been extensively studied by cancer researchers for nearly four decades. The first description of geldanamycin in the scientific literature appeared in 1970 and nearly thirty years later the semi-synthetic derivative 17-AAG, or tanespimycin, entered Phase 1 clinical trials. In the subsequent years, three additional geldanamycin derivatives have entered clinical evaluation. Kosan Biosciences developed 17-DMAG or alvespimycin hydrochloride for clinical evaluation as both an intravenous and oral product. Infinity Pharmaceuticals is developing IPI-504 or retaspimycin hydrochloride as an intravenous product, which is in several Phase 2 clinical trials; IPI-504 is the hydroquinone hydrochloride salt of 17-AAG. More recently, Infinity Pharmaceuticals initiated a Phase 1 clinical trial with an oral formulation of 17-AG (IPI-493), the major metabolite of 17- AAG and IPI-504. Since a vast amount of scientific literature exists regarding the ansamycin field and Hsp90 inhibition, this review will survey key milestones in the development of the natural product class as anti-cancer drugs including discovery of the compounds and their anti-tumor effects, identification of Hsp90 as their biological target, the structureactivity relationships that have been identified in this interesting class of compounds, and development of clinical candidates for the treatment of cancer patients. A brief overview of important pre-clinical development data from each clinical lead is also provided.

Heat shock protein 90 (Hsp90) is an ATP-dependent chaperone which is involved in the post-translational maturation and stabilization of over one hundred proteins (“its clients”). In the absence of Hsp90's chaperoning, its clients are misfolded and degraded via ubiquitin-proteasome pathway. HSP90 has become the focus of intense drug discovery efforts as its activity has been implicated in diverse pathologies ranging from oncology to neurodegenerative and infectious diseases. The most promising inhibitors reported to date inhibit the ATPase activity by binding to the Nterminal ATP pocket. Radicicol, a member of the resorcylic acid lactones (RALs), represents an important pharmacophore to this end. Efforts towards the development of this pharmacophore and its SAR are reviewed herein.

Hsp90 is a molecular chaperone with important roles in regulating the function of several proteins with potential pathogenic activity. Because many of these proteins are involved in cancer and neurodegenerative promoting pathways, Hsp90 has emerged as an attractive therapeutic target in these diseases. Molecules that bind to the N-terminal nucleotide pocket of Hsp90 inhibit its activity, and consequently, disrupt client protein function. A number of these inhibitors from several chemical classes are now known, and some are already in clinical trials. This review focuses on the purine class of Hsp90 inhibitors, their discovery through rational design, and on efforts aimed towards their optimization and development into clinically viable drugs for the treatment of cancer. Their potential towards neurodegenerative diseases will also be touched upon.

The 90 kDa heat shock protein (Hsp90) has become a validated target for the development of anti-cancer agents. Several Hsp90 inhibitors are currently under clinical trial investigation for the treatment of cancer. All of these agents inhibit Hsp90's protein folding activity by binding to the N-terminal ATP binding site of the Hsp90 molecular chaperone. Administration of these investigational drugs elicits induction of the heat shock response, or the overexpression of several Hsps, which exhibit antiapoptotic and pro-survival effects that may complicate the application of these inhibitors. To circumvent this issue, alternate mechanisms for Hsp90 inhibition that do not elicit the heat shock response have been identified and pursued. After providing background on the structure, function, and mechanism of the Hsp90 protein folding machinery, this review describes several mechanisms of Hsp90 modulation via small molecules that do not induce the heat shock response.

The Hsp90-dependence of many oncogenic proteins has precipitated a great deal of interest in Hsp90 as a drug target, as evidence mounts that Hsp90 inhibitors may be effective chemotherapeutic agents for the treatment of cancer. In addition, Hsp90-inhibitors have shown promise for the treatment of neurodegenerative diseases characterized by the accumulation of toxic denatured protein aggregates. The development of assays for the identification of novel Hsp90 inhibitors began in earnest when it became apparent that the Hsp90 inhibitors available at the time had less than ideal pharmacological properties. This review summarizes what is known about Hsp90's structure and function, its ATPase cycle, its interactions with co-chaperones and clients, and the effect of Hsp90-inhibitors on these processes. It further summarizes various high throughput assays (and secondary confirmatory assays) developed to identify new Hsp90 inhibitors from chemical libraries based on the inhibitors ability to: inhibit Hsp90's ATPase activity; compete for ligand binding to Hsp90 and its N-terminal ATP-binding domain; inhibit Hsp90-dependent refolding of denatured luciferase; and deplete culture cells of Hsp90-dependnet client protein or induce Hsp70 expression. In addition, in vitro assays are described that help determine the site of inhibitor binding to Hsp90 (N- or C-terminal domain) and there mechanism of action based on effects of inhibitors on Hsp90/ co-chaperone and client interactions, and Hsp90 conformation characterized by proteolytic fingerprinting.

Twenty-five years ago the first small molecule inhibitors of Hsp90 were identified. In the intervening years there has been dramatic progress in basic scientific understanding of the Hsp90 chaperone machinery and in the role of Hsp90 in malignancy. The first-in-class Hsp90 inhibitor 17-AAG entered into Phase I clinical trials in 1999. There are now 13 Hsp90 inhibitors in clinical trial, representing multiple drug classes, and hundreds of patients have been treated in adult oncology and pediatric oncology trials. This review will provide an overview of the clinical trial results thus far. In addition, pivotal issues in further development of Hsp90 inhibitors as anticancer drugs will be discussed.