Current Topics in Medicinal Chemistry

ISSN: 1568-0266

Current Topics in Medicinal Chemistry
Volume 6, Number 11, 2006

Contents

Purine Binding Proteins as Anti-Cancer Targets
Guest Editor: Dr. Zhenhai Gao


Editorial Pp. 1069


Recent Advances in the Research and Development of RAF Kinase Inhibitors Pp. 1071-1089
Roger A. Smith, Jacques Dumas, Lila Adnane and Scott M. Wilhelm
[Abstract]


Inhibitors of the HSP90 Molecular Chaperone: Attacking the Master Regulator in Cancer Pp. 1091-1107
Edward McDonald and Paul Workmanand Keith Jones
[Abstract]


Structure-Function Based Design of Small Molecule Inhibitors Targeting Rho Family GTPases Pp. 1109-1116
Nicolas Nassar, Jose Cancelas, Jie Zheng, David A. Williams and Yi Zheng
[Abstract]


The Purinome, a Complex Mix of Drug and Toxicity Targets Pp. 1117-1127
Timothy A. J. Haystead
[Abstract]


Targeting Cancer: The Challenges and Successes of Structure Based Drug Design Against the Human Purinome Pp. 1129-1159
Mark Knapp, Cornelia Bellamacina, Jeremy M. Murray and Dirksen E. Bussiere
[Abstract]


Molecular Chaperones as Targets in Medicinal Chemistry and Drug Discovery
Guest Editor: Dr. Gabriela Chiosis


Editorial Pp. 1161


Using Natural Product Inhibitors to Validate Hsp90 as a Molecular Target in Cancer Pp. 1163-1171
Len Neckers
[Abstract]


Geldanamycin, Radicicol, and Chimeric Inhibitors of the Hsp90 N Terminal ATP Binding Site Pp. 1173-1182
M. Kyle Hadden, Donna J. Lubbers and Brian S. J. Blagg
[Abstract]


Discovery and Development of Purine-Scaffold Hsp90 Inhibitors Pp. 1183-1191
Gabriela Chiosis
[Abstract]


Discovery and Development of Pyrazole-Scaffold Hsp90 Inhibitors Pp. 1193-1203
Edward McDonald, Keith Jones, Paul A. Brough, Martin J. Drysdale and Paul Workman
[Abstract]


Hsp90: A Novel Target for Cancer Therapy Pp. 1205-1214
David B. Solit and Neal Rosen
[Abstract]


Hsp70 Molecular Chaperones: Emerging Roles in Human Disease and Identification of Small Molecule Modulators Pp. 1215-1225
Jeffrey L. Brodsky and Gabriela Chiosis
[Abstract]




Abstracts
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Editorial

The explosion of genomic information has led to the identification of a plethora of genes that are responsible for the malignant phenotypes of human cancers. Over the past two decades, the new paradigm and strategy in cancer therapy has been to develop agents that specifically target key molecules and signaling pathways involved in tumor growth and progression. The molecularly targeted therapy is widely expected to hold the potential of providing a much improved risk/benefit ratio, compared with conventional cytotoxic chemotherapy.

A prominent class of such targets, perhaps one of the most abundant target sources for oncology drug discovery, is the protein kinase family. ATP binding kinases play pivotal roles in oncogenic signal transduction. Indeed, the recent approval of several kinase inhibitors including Gleevec, Iressa, Tarceva, Nexavar and Sutent has firmly established the eminent drugability of protein kinases. We know now that the human genome might encode as many as 518 protein kinases, and about 20-30% of pharmaceutical discovery programs are currently focused on various kinases.

However, kinases are only part of a larger family of purine binding proteins in human genome, collectively called “purinome”. Structural analysis has indicated that the non-kinase purine binding proteins bind purines in a similar orientation as observed in kinases. Therefore, the utility of purinome as drug targets can be expanded beyond kinases to include many non-kinase purine binding proteins. Undoubtedly, this will further increase the diversity and broaden our repertoire of drugable targets. In fact, several non-kinase purine binding proteins, e.g. heat shock protein 90 (hsp90), Eg-5, and small G protein Rac, have recently emerged as very promising and attractive anti-cancer targets.

This special issue of “Current Topics in Medicinal Chemistry” is devoted to the review and highlight of the most recent achievements in pre-clinical and clinical development of small molecule drugs targeting purine binding proteins in cancer. The excellent papers from five research groups will cover the unique aspects of different classes of purine utilizing enzymes as drug targets, including Raf-1 (kinase), Hsp90 (ATPase) and Rac (GTPase), as well as discuss the state-of-art approaches such as structure based drug design (SBDD) and chemoproteomic technology (proteome mining) that are currently being explored in purine binding protein based drug discovery. It is my hope that these comprehensive and stimulating review articles will be beneficial to many scientists who are actively engaged in this very promising and exciting area of drug development research.

I would like to express my gratitude to Dr. Allen B. Reitz for inviting me to be the Guest Editor for this special issue. I am also very thankful to the following colleagues who have served as the reviewers of this issue: Drs. David Duhl, Paul Renhowe, Timothy Machajewski and Guoying (Karen) Yu. Finally, I would like to offer my special thanks to all authors for their enthusiasm and dedication, which have made this special issue a reality.

Dr. Zhenhai Gao
Department of Applied Biochemistry,
Chiron Corporation
Emeryville, CA
USA
E-mail: zhenhai_gao@chiron.com


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Recent Advances in the Research and Development of RAF Kinase Inhibitors
Roger A. Smith, Jacques Dumas, Lila Adnane and Scott M. Wilhelm

The RAS-RAF-MEK-ERK signaling pathway (ERK pathway) plays a key role in tumorigenesis and cancer progression. Mutations of RAS or B-RAF lead to a constitutive activation of the ERK pathway, which ultimately results in increased cell division, and cell survival. This review article focuses on the recent literature related to ERK pathway inhibitors, with a particular emphasis on RAF kinase inhibitors. Preclinical and clinical data for the RAF kinase inhibitor sorafenib (BAY 43-9006 tosylate), that was recently approved in the US for the treatment of advanced renal cell carcinoma, are also outlined.


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Inhibitors of the HSP90 Molecular Chaperone: Attacking the Master Regulator in Cancer
Edward McDonald and Paul Workmanand Keith Jones

The heat shock protein 90 (HSP90) chaperones represent some 1-2% of all cellular protein and are key players in protein quality control in cells. They are over expressed in many human cancers and the fact that many oncogenic proteins are clients has prompted much recent research on HSP90 inhibitors as new cancer therapeutics. A brief introduction is followed by a detailed review of the various classes of inhibitors, both natural product-based and synthetic, that have emerged over the last decade. The natural products geldanamycin, radicicol and novobiocin have provided the start points for new drugs in this area and their medicinal chemistry is reviewed, including the exciting recent results emerging from clinical trials using geldanamycin analogues. The detailed understanding of the binding mode of these compounds to HSP90 has been significantly enhanced by X-ray crystallography of HSP90 constructs co-crystallised with various ligands. Efforts to replace the natural product inhibitors with more drug-like synthetic compounds have mushroomed over the last 4 years. The purines and the 3,4-diarylpyrazoles have proven to be the most successful and their medicinal chemistry is reviewed with particular emphasis on structure-based design. Protein/ligand co-crystal structures have shown that conserved water molecules in the active site are a vital part of the hydrogen-bonding network established on binding both natural product and synthetic inhibitors. Medicinal chemists have used this information to develop high affinity lead compounds. Recent research provides the platform for exciting developments in the area of HSP90 inhibition over the next few years.


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Structure-Function Based Design of Small Molecule Inhibitors Targeting Rho Family GTPases
Nicolas Nassar, Jose Cancelas, Jie Zheng, David A. Williams and Yi Zheng

Rho GTPases of the Ras superfamily are involved in the regulation of multiple cell functions and have been implicated in the pathology of various human diseases including cancer. They are attractive drug targets in future targeted therapy. A wealth of structure-function information made available by high resolution structures and mutagenesis studies has laid out the foundation for the derivation of a mechanism-based targeting strategy. Here we describe the rational design and characterizations of a first generation Rac-specific small molecule inhibitor. Based on the structure-function information of Rac interaction with GEFs, in a computer based Virtual Screening we have identified NSC23766, a highly soluble and membrane permeable compound, as a specific inhibitor of a subset of GEF binding to Rac and therefore Rac activation. In fibroblast cells NSC23766 inhibited Rac1 GTP-loading without affecting Cdc42 or RhoA activity and suppressed the Rac-GEF, Tiam1, and oncogenic Ras induced cell growth and transformation. NSC23766 also potently inhibited the prostate PC-3 cancer cell proliferation and invasion induced by Rac hyperactivation. Intraperitoneal administration of NSC23766 to laboratory mice resulted in effective Rac GTPase suppression and hematopoietic stem cell mobilization from the bone marrow to the peripheral blood, similar to the effects of genetically targeted disruption of Rac GTPases in the animals. A co-crystal structure of NSC23766 bound to Rac1 provided further insight for future medicinal chemistry modification and improvement of this lead Rac-specific inhibitor. Thus, structure-function based rational design may represent a new avenue for generating lead small molecule inhibitors of Ras superfamily GTPases that are useful for modulating pathological conditions in which the small GTPase deregulation may play a role.


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The Purinome, a Complex Mix of Drug and Toxicity Targets
Timothy A. J. Haystead

Much attention has focused on the development of protein kinases as drug targets to treat a variety of human diseases including diabetes, cancer, hypertension and arthritis. To date, Gleevec is one example of a drug targeting protein that has successfully treated human cancer. Several other protein kinase inhibitors are in clinical development. However, protein kinases are in fact part of a larger collection of some 2000 distinct proteins expressed by the genome that like the protein kinases also bind purines (the purinome), either to be utilized as substrates or as co-factors in the form of NAD, NADP and co-enzyme A. The solution structures of many representative gene family members within the purinome show these proteins bind purines in a similar orientations to that observed in all protein kinases. Several non-protein kinase purine utilizing proteins are established drug targets such as HMG CoA reductase, dihydrofolate reductase, phosphodiesterase and HSP90. Searches of OMIM identifies many purine utilizing enzymes that are associated with inborn errors in metabolism. Inhibition of any one of which by a drug could lead to an undesirable side effect. The purinome is therefore somewhat of a drug discovery mixed blessing. It is a rich source of therapeutic targets, but also contains a large collection of diverse proteins whose inhibition could result in an adverse outcome. Drug discovery within the purinome should therefore encompass strategies that enable broad assessment of selectivity across the entire purinome at the earliest stages of the discovery process. In this article we review the purinome within the context of drug discovery and discuss approaches for avoiding off target binding during the discovery/lead optimization process with particular emphasis on use of proteome mining technology.


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Targeting Cancer: The Challenges and Successes of Structure Based Drug Design Against the Human Purinome
Mark Knapp, Cornelia Bellamacina, Jeremy M. Murray and Dirksen E. Bussiere

Purine-binding proteins are of critical importance to all living organisms. Approximately 13% of the human genome is devoted to coding for purine-binding proteins. Given their importance, purine-binding proteins are attractive targets for chemotherapeutic intervention against a variety of disease states, particularly cancer. Modern computational and biophysical techniques, combined together in a structure-based drug design approach, aid immensely in the discovery of inhibitors of these targets. This review covers the process of modern structure-based drug design and gives examples of its use in discovery and development of drugs that target purine-binding proteins. The targets reviewed are human purine nucleoside phosphorylase, human epidermal growth factor receptor kinase, and human kinesin spindle protein.


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Editorial

Contained in this issue of CTMC are several papers that highlight recent contributions to the development of small molecules that target the molecular chaperones Hsp90 and Hsp70, the use of these molecules to understand the clinical significance of modulating these chaperones’ function, and finally, progress in the translation of such agents to clinic.

Hsp90 is a chaperone with important roles in maintaining transformation and in elevating the survival and growth potential of cancer cells. However, in addition to its roles in facilitating the transformed phenotype, Hsp90 serves several functions in normal cell homeostasis. Even under non-stressed conditions, Hsp90 comprises as much as 1–2% of total cellular protein content, and this amount increases only about two to three fold under stress. In eukaryotes, constitutive genetic knockout of Hsp90 is lethal. Thus, until the discovery of pharmacological agents that inhibit Hsp90 function, it has not become apparent that the chaperone may be a target in disease. Len Neckers begins this CTMC issue by providing a description of the biology of Hsp90 and how natural product derivatives such as geldanamycin, radicicol and novobiocin have been used to understand both the structure and biological significance of Hsp90 in cancer. Further, Brian Blagg and his colleagues discuss structure-activity relationship investigations in the natural product Hsp90 inhibitor classes and present several synthetic efforts directed at both improving the selectivity and pharmacological profiles of these agents. In spite of the usefulness of these natural products as proof-of-principle compounds, their clinical use has been encumbered by their less than desirable pharmacological profiles. These drawbacks have prompted for the discovery of novel Hsp90 inhibitors. Several synthetic second generation Hsp90 inhibitors are now in late preclinical development, while others are going through early stages of discovery. These new compounds hold the promise of better drug-like properties and of improved pharmacological profiles. Two such classes are discussed in this issue. First, Gabriela Chiosis provides the rationale behind the discovery and development of the first synthetic class of Hsp90 inhibitors, the purine-scaffold series. The biological activities, selectivity for tumor cells and tumor retention profiles of these inhibitors are further discussed in this review. Ted McDonald and his colleagues from the Institute of Cancer Research, UK and from Vernalis Ltd. describe the identification by high-throughput screening of pyrazole-based Hsp90 inhibitors and discuss the optimization of compounds based on the pyrazole scaffold by structure-based design, emphasizing in particular the value of X-ray crystallography. Further, David Solit and Neal Rosen update the reader on efforts directed at the rational translation of Hsp90 inhibitors in cancer treatment and on progress in the ongoing clinical trials with these agents. Finally, Jeffrey Brodsky and Gabriela Chiosis will introduce another chaperone with important biological functions, Hsp70. In this review, the authors will summarize the structural and functional characteristics of Hsp70 chaperones, and discuss their roles in cellular physiology. They will also review the recent discovery of small molecules that alter Hsp70 expression and function, and further discuss their possible application as treatments of specific cancers, infections, and protein conformational diseases.


Dr. Gabriela Chiosis
Department of Medicine,
Program in Molecular Pharmacology Chemistry,
Memorial Sloan-Kettering Cancer Center,
1275 York Ave.,
New York,
NY 10021,
USA


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Using Natural Product Inhibitors to Validate Hsp90 as a Molecular Target in Cancer
Len Neckers

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 telomerase, mutated p53, Bcr-Abl, Raf-1, Akt, HER2/Neu (ErbB2), mutated B-Raf, mutated EGF receptor, and HIF-1α. Hsp90 inhibitors, by interacting specifically with a single molecular target, cause inactivation, destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising anti-tumor activity in various preclinical tumor models. One Hsp90 inhibitor, 17-AAG, is currently in Phase II clinical trial and other inhibitors will shortly be entering the clinic. 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. Identification of benzoquinone ansamycins as the first Hsp90 inhibitors allowed investigators to determine the biologic effects, at first in vitro and then in vivo, of pharmacologic inhibition of Hsp90. These studies rapidly enhanced our understanding of Hsp90 function and led to the identification of radicicol as a structurally distinct 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.


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Geldanamycin, Radicicol, and Chimeric Inhibitors of the Hsp90 N Terminal ATP Binding Site
M. Kyle Hadden, Donna J. Lubbers and Brian S. J. Blagg

Natural products have continued to drive the development of new chemotherapeutics and elucidation of new biological targets for the treatment of disease. Since Whitesell and Neckers’ original discovery that geldanamycin does not directly inhibit v-Src, but instead manifests its biological activity through inhibition of the Hsp90 molecular chaperone, additional natural products and natural product derivatives have been identified and developed to inhibit the Hsp90 protein folding machinery. 17-AAG, a geldanamycin analogue, is currently in clinical trials for the treatment of several types of cancer. Recent work has produced improved radicicol analogues that show promising Hsp90 inhibitory activity in vitro. In addition, chimeric molecules of these two natural products are active in vitro and represent a novel class of Hsp90 inhibitors for cancer treatment. In addition to their chemotherapeutic uses, natural product inhibitors and their derivatives have been utilized to probe the biological mechanisms by which Hsp90 inhibition regulates tumor cell growth. As a consequence of these studies, the molecular chaperones have emerged as an exciting new class of therapeutic targets. This review will highlight the utility of the natural products, geldanamycin and radicicol, as well as improved analogues and the activities exhibited by these compounds against various cancer cell lines.


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Discovery and Development of Purine-Scaffold Hsp90 Inhibitors
Gabriela Chiosis

Hsp90 allows cancer cells to tolerate the many components of dysregulated pathways in a transformation-specific manner by interacting with several client substrates, such as kinases, hormone receptors and transcription factors that are directly involved in driving multistep malignancy, and also with mutated oncogenic proteins required for the transformed phenotype. This distinctive broad involvement in maintaining the transformed phenotype has suggested Hsp90 as an important target in cancer therapy. Discovery of pharmacological agents that selectively inhibit its function have aided in probing the biological functions of Hsp90 at the molecular level and in validating it as a novel target for anticancer drug action. Two natural product derivatives, 17-allylamino-17-desmethoxy-geldanamycin (17AAG) and 17-dimethylaminoethylamino-17-desmethoxy-geldanamycin (17DMAG) have further entered clinical trials, proving that Hsp90 may be modulated pharmacologically without causing target related toxicities in humans. In spite of their usefulness as proof-of-principle compounds, the clinical use of these two agents has been encumbered with some limitations due to their structural characteristics and also to less than optimal pharmacological profiles. Thus, the identification of Hsp90 inhibitors with improved structural characteristics and better pharmacological profiles is a major focus of interest in the field. One such emerging class is the purine-scaffold series. This review intends to inform the reader on efforts ranging from the discovery to their clinical translation.


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Discovery and Development of Pyrazole-Scaffold Hsp90 Inhibitors
Edward McDonald, Keith Jones, Paul A. Brough, Martin J. Drysdale and Paul Workman

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.


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Hsp90: A Novel Target for Cancer Therapy
David B. Solit and Neal Rosen

Hsp90 is a molecular chaperone required for the stress-survival response, protein refolding, and the conformational maturation of a variety of signaling proteins. Natural products that bind selectively to Hsp90 and inhibit its function have been used to determine its biologic role. Experiments with these drugs have shown that Hsp90 is required for maintaining the malignant phenotype of cancer cells. Studies in vivo show that Hsp90 inhibitors have antitumor activity when given alone and in combination with cytotoxics. The basis for the therapeutic index (selective toxicity to cancer cells) of Hsp90 inhibitors is complex and may have to do with induction of degradation of mutant oncoproteins and other proteins necessary for their proliferation and survival as well as to an enhanced requirement of these cells for Hsp90 stress-survival functions. Based on these data, 17-AAG, an ansamycin antibiotic inhibitor of Hsp90, is being tested extensively in clinical trials in patients with advanced cancer. These trials demonstrate that the biologic function of Hsp90 can be inhibited in patients and antitumor activity has been noted in patients with breast cancer, multiple myeloma and other cancers. These data and the physicochemical properties of 17-AAG that limit its use as a drug, have led to broad efforts to develop improved and novel Hsp90 inhibitors. This article will review the preclinical data which supports the testing of Hsp90 inhibitors as cancer drugs and update the reader on the current status of the ongoing clinical trials of Hsp90 inhibitors.


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Hsp70 Molecular Chaperones: Emerging Roles in Human Disease and Identification of Small Molecule Modulators
Jeffrey L. Brodsky and Gabriela Chiosis

Molecular chaperones are best known for their ability to aid in the solubilization of mis-folded proteins, and as a result play essential roles in protein folding, degradation, and transport. However, many molecular chaperones also play essential roles in signal transduction cascades. For example, Hsp70 molecular chaperones are a highly conserved, abundant class of chaperones that are found in every species and in nearly every cellular compartment in eukaryotes. In addition to their well-established roles in facilitating protein folding and in the targeting of proteins to organelles and to proteolytic machines, Hsp70s are anti-apoptotic and inhibition of Hsp70 function in some cases is sufficient to induce tumor cell death. Hsp70 function is also vital for the replication of viruses. Based on these data, small molecule Hsp70 modulators might, in principle, be used for the treatment of specific cancers, infections, and protein conformational diseases. In this review, we summarize the structural and functional characteristics of Hsp70 chaperones, and then discuss their roles in cellular physiology. Finally, we will review the recent discovery of small molecules that alter Hsp70 expression and function.