Proteases as Drug Targets: Technologies and Opportunities for Drug Discovery

Proteases as Drug Targets: Technologies and Opportunities for Drug Discovery

Publisher:	D&MD Publications Inc
Year of publication:	2004
Type of publication:	Management report
Publisher's reference (if any):	D&MD9158
Author(s):	Sreten Bogdanovic and Beata Langlands
Approximate page count:	250
Price when published:	$4,950
Remarks: Page numbers, where given, refer to the draft manuscript (which may differ from the published version). The copyright in this report is owned by the publisher, to whom any requests for copies should be addressed. The price shown is for a single copy of the print version. Multiple copies and electronic copies usually have different prices.
PROTEASES AS DRUG TARGETS: TECHNOLOGIES AND OPPORTUNITIES FOR DRUG DISCOVERY TABLE OF CONTENTS CHAPTER 1 INTRODUCTION TO THE PROTEASES 1.1 Overview of proteases 1.1.1 Catalytic function 1.1.2 Categories of proteolysis 1.1.3 Proteases, proteinases and peptidases 1.1.4 EC classification of proteases 1.1.5 The serine endopeptidases 1.1.6 The cysteine endopeptidases 1.1.7 The aspartic endopeptidases 1.1.8 The metalloendopeptidases 1.1.9 The threonine endopeptidases 1.2 Overview of protease inhibitors 1.2.1 Low-molecular weight inhibitors 1.2.2 Natural (protein) protease inhibitors 1.3 Regulation of proteolysis 1.3.1 Proteolytic cascades 1.3.2 Proteases as signaling molecules 1.3.3 Compartmentalization of proteolysis CHAPTER 2 STRATEGIES FOR PROTEASE INHIBITOR DISCOVERY 2.1 Overview of therapeutic protease inhibitors 2.2 Discovery of small molecule protease inhibitors 2.2.1 Proteomics-based approaches 2.2.2 Role of high-throughput screening 2.2.3 Trends in lead optimization 2.2.4 Integrated use of technologies 2.3 Mechanisms of protease inhibition 2.3.1 Competitive reversible inhibitors 2.3.2 Competitive irreversible inhibitors 2.3.3 Allosteric inhibitors 2.3.4 Inspiration from nature 2.4 Determination of 3D protease structures 2.4.1 X-ray crystal diffraction 2.4.2 Solution NMR 2.4.3 Homology modeling 2.5 Structure-driven drug discovery 2.5.1 Use of structures to generate leads 2.5.2 Fragment-based methods 2.5.3 Recent successes in the protease field 2.6 Identifying protease substrates CHAPTER 3 PROTEASE INHIBITORS IN INFECTIOUS DISEASE 3.1 Introduction 3.2 HIV/AIDS 3.2.1 Overview of HIV protease inhibitors 3.2.2 First-generation peptidomimetic inhibitors 3.2.2.1 Limitations 3.2.3 Improved peptidomimetic inhibitors 3.2.4 Non-peptidomimetic inhibitors 3.2.5 Strategies to enhance inhibition 3.2.6 Shape of protease inhibitors to come 3.3 HCV 3.4 Rhinovirus 3.5 SARS coronavirus 3.6 Bacteria 3.7 Candida 3.8 Plasmodium and other parasites CHAPTER 4 PROTEASE INHIBITORS IN INFLAMMATORY DISORDERS 4.1 Inflammation and tissue injury 4.1.1 Osteo- and rheumatoid arthritis 4.1.2 Chronic obstructive pulmonary disease 4.1.3 Miscellaneous 4.2 Protease targets 4.2.1 Serine endopeptidases 4.2.1.1 HNE 4.2.1.2 Kallikrein 4.2.1.3 Trypsins and tryptases 4.2.2 Cysteine endopeptidases 4.2.2.1 Cathepsin S 4.2.2.2 ICE 4.2.2.3 Caspases 4.2.3 Metalloendopeptidases 4.2.3.1 Matrix metalloproteinases 4.2.3.2 ADAM and ADAM-TS families 4.2.3.3 Procollagen C-proteinase 4.2.4 Proteasomes 4.2.5 Miscellaneous CHAPTER 5 PROTEASE INHIBITORS IN CANCER 5.1 Overview of cancer 5.1.1 Established therapies 5.1.2 Molecular targeted therapies 5.2 Proteolytic cascades in cancer 5.2.1 Matrix remodeling cascades 5.2.2 Fibrinolysis cascade 5.2.3 Apoptosis cascades 5.2.4 Cell cycle cascade 5.3 Protease targets 5.3.1 Serine endopeptidases 5.3.1.1 uPA 5.3.1.2 Transmembrane proteases 5.3.2 Cysteine endopeptidases 5.3.2.1 Caspases 5.3.2.2 Cathepsin B 5.3.3 Metalloendopeptidases 5.3.3.1 Broad-spectrum inhibitors 5.3.3.2 Next-generation MMP inhibitors 5.3.3.3 ADAMs family 5.3.4 Proteasomes 5.3.5 Exploiting tumor proteases CHAPTER 6 PROTEASE DRUGS IN OTHER CONDITIONS 6.1 Introduction 6.2 Cardiovascular diseases (main focus) 6.2.1 Background 6.2.2 Protease targets for inhibition 6.2.2.1 ACE in cardiovascular diseases 6.2.2.2 ACE in miscellaneous diseases 6.2.2.3 ACE2 6.2.2.4 Renin 6.2.2.5 ACE/NEP, NEP/ECE 6.3 Anticoagulation 6.3.1 Background 6.3.2 Protease targets for inhibition 6.3.2.1 Thrombin 6.3.2.2 Factor Xa 6.3.2.3 FactorXa/thrombin 6.3.2.4 Factor VIIa 6.4 Thrombolysis 6.4.1 Background 6.4.2 Thrombolytic proteases 6.5 Neurodegenerative diseases 6.5.1 Background 6.5.1.1 Secretases in Alzheimer's disease 6.5.2 Protease targets for inhibition 6.5.2.1 Gamma-secretase 6.5.2.2 Beta-secretase 6.5.2.3 Caspases 6.5.2.4 Miscellaneous 6.6 Osteoporosis 6.6.1 Background 6.6.2 Cathepsin K inhibitors 6.7 Type 2 diabetes 6.7.1 Background 6.7.2 DPP-IV inhibitors 6.8 Acute diarrhea 6.8.1 Background 6.8.2 Encephalinase inhibitors CHAPTER 7 MARKET CONSIDERATIONS AND FORECASTS 7.1 Introduction 7.2 Infectious Disease 7.3 Inflammation/tissue injury 7.4 Cancer 7.5 Cardiovascular conditions 7.6 Neurodegenerative diseases 7.7 Other conditions 7.8 National Market Forecasts CHAPTER 8 COMPANY PROFILES 8.1 Abbott Laboratories 8.2 Amedis Pharmaceuticals Ltd 8.3 AngioDesign Inc 8.4 Arriva Pharmaceuticals Inc 8.5 Astex Technology 8.6 Bayer AG 8.7 Biopharmacopae Design International Inc. 8.8 Boehringer Ingelheim GmbH 8.9 Celera Genomics 8.10 Cellzome 8.11 Collagenex Pharmaceuticals Inc 8.12 Curacyte AG 8.13 De Novo Pharmaceuticals Ltd 8.14 Dendreon 8.15 Gilead Sciences Inc 8.16 GlaxoSmithKline plc 8.17 Incyte Corporation 8.18 Locus Technology Inc 8.19 Medivir 8.20 Millennium Pharmaceuticals Inc 8.21 Myriad Genetics Inc 8.22 NeoGenesis Pharmaceuticals 8.23 Nereus Pharmaceuticals Inc 8.24 Novartis Pharma AG 8.25 Ono Pharmaceuticals Co. Ltd. 8.26 SIGA Technologies Inc 8.27 Syrrx Inc 8.28 Torii Pharmaceutical Co. Ltd. 8.29 Vertex Pharmaceuticals 8.30 Wilex AG CHAPTER 9 TRENDS AND OPPORTUNITIES 9.1 Building on successes 9.2 Learning from clinical failures 9.3 Exopeptidases as drug targets 9.4 Serine endopeptidases as drug targets 9.5 Cysteine endopeptidases as drug targets 9.6 Aspartic endopeptidases as drug targets 9.7 Metalloendopeptidases as drug targets 9.8 Threonine endopeptidases as drug targets 9.9 Prospects in acute diseases 9.10 Prospects in chronic diseases 9.11 Exploring allosteric inhibitors 9.12 Exploring irreversible inhibitors 9.13 Challenges for future PI discovery 9.14 Towards systems biology approaches APPENDIX 1 GLOSSARY EXECUTIVE SUMMARY Proteases have roles that extend far beyond a simple catalytic function in the hydrolysis of peptide bonds of proteins. It is now known that proteases control a wide variety of essential physiological processes, often by participating in complex proteolytic cascades. Many disease states manifest altered protease expression and substrate proteolysis. In particular, excessive protease activity is often observed, offering targets for therapeutic inhibition. There are at least 475 known and putative proteases and 103 homologs to known proteases in humans, and it is estimated that up to 1,200 human genes encode proteases. Proteases representative of all five known mechanistic classes have been implicated in human disease. Successful established protease inhibitor drugs on the market - ACE inhibitors and HIV protease inhibitors - act by inhibiting key proteases. Their success has led to proteases being increasingly viewed as valuable drug targets in disease treatment and a range of protease inhibitors are in various stages of commercial development. At present, an estimated 5-10% of all pharmaceutical targets are proteases. This report reviews progress made in the field so far, commenting on positive findings as well as setbacks encountered. Although the number of proteases currently targeted represents only a small fraction of all potential protease targets, the investigations are generating valuable clues about the validity of targeting specific proteases and protease families as well as general approaches to inhibition. We forecast that the world market for protease-related drugs (mostly inhibitors, except for thrombolytics which are native or modified proteases) will increase from $11.2 billion in 2003 to $23.1 billion in 2009 in real terms, a compound annual growth rate (CAGR) of 12.9% at constant exchange rates. Currently the major applications by sales volume are antihypertensives and HIV protease inhibitors, with small contributions from the inflammation, cancer, and vascular therapeutic areas. At present all protease-related drugs for use in hypertension are ACE inhibitors, but other - possibly more potent and better tolerated - antagonists of the RAS pathway will be introduced. These five segments will all increase as new products are introduced, and they will be joined by newly introduced agents for the treatment of other infectious diseases, osteoporosis, type II diabetes, and (possibly) Alzheimer's disease. Much of the increase in this market segment beyond 2009 will be due to these new applications. Most therapeutic protease inhibitors currently under development are small molecule drugs. Originally, protease inhibitors were derived from the peptide substrates of the proteases, which led to the development of peptidomimetic competitive inhibitors. There has been a gradual move to design inhibitors less related to the enzyme substrate with improved pharmacokinetic properties. Strategies to design peptidomimetic and non-peptidomimetic competitive inhibitors, as well as other types of inhibitors are discussed in the report. Naturally occurring protease inhibitors are also considered, as they provide a basis for protein-based therapeutics, for which improved methods of delivery are being developed. Current efforts to design therapeutic protease inhibitors are being accelerated by the increased availability of structural information. Target protease structures are increasingly used at an early stage to generate leads of high quality, thereby saving costs associated with drug discovery. The determination of the HIV protease 3D structure in 1989 and subsequent development of HIV-specific protease inhibitors launched the modern era of structure-based drug design. The availability of high quality protease structures is increasing, throwing fresh light on old targets. For example, in 2003 the structure of ACE was finally solved, revealing an unexpected similarity to two proteases with which ACE does not share amino acid sequence similarity. New structural information is being used to design more selective small molecule inhibitors. In the infectious disease field, protease inhibitor approaches take advantage of the fact that most viruses contain at least one protease, while all other microbes possess several. We review the prospects for HIV protease inhibitors in epidemiologically significant infectious diseases. In HIV/AIDS, the challenge is to develop improved peptidomimetic and non-peptidomimetic inhibitors, enhance protease inhibition and overcome the growing menace of viral resistance. Earlier stage protease inhibitor programs are underway in other viral infections, in particular HCV, as well as in infections due to bacteria, candida and parasites. Most companies currently developing protease inhibitors have chosen to pursue applications in chronic diseases, reflecting a general trend in the industry. Excessive protease activity has long been believed to play a role in the development of many chronic inflammatory conditions, in particular osteo- and rheumatoid arthritis and chronic obstructive pulmonary disease. The clinical progression of the first generation of MMP inhibitors has been hampered by side effects, but inhibitors targeting diverse protease targets remain under investigation and their progress is assessed in the report. Protease inhibition programs in cancer have produced some disappointments and some unexpected successes in recent years, which we evaluate. While the much-hyped MMP inhibitors failed in late-stage clinical trials, the proteasome inhibitor Velcade progressed rapidly through clinical trials and was approved in 2003, becoming the first new treatment in more than a decade for multiple myeloma. The growing recognition of the role of proteases and proteolytic cascades in both the growth and metastasis of tumors is enabling the development of more targeted protease inhibitors. This fits in with the general trend in cancer treatment towards molecular targeted therapies. We consider the rationale for the current choices of protease targets in cancer. In the cardiovascular area, despite high levels of competition and high cost of development of drugs, the need for more specific protease inhibitors is driving further developments. Other active areas include anticoagulation, neurodegenerative conditions, osteoporosis, and type 2 diabetes, where inhibitors in development focus on high-profile disease-associated protease targets such as secretases in Alzheimer's disease, cathepsin K in osteoporosis and DPP-IV in type 2 diabetes. The report profiles 30 key companies active in the protease drug discovery, which includes small specialist companies, commercial-stage biotech companies, and large pharmaceutical companies. Information is provided on promising proprietary protease inhibitor drug discovery technologies, and recent acquisitions and collaborations in this area.

EXECUTIVE SUMMARY
Proteases have roles that extend far beyond a simple catalytic function in the hydrolysis of peptide bonds of proteins. It is now known that proteases control a wide variety of essential physiological processes, often by participating in complex proteolytic cascades. Many disease states manifest altered protease expression and substrate proteolysis. In particular, excessive protease activity is often observed, offering targets for therapeutic inhibition.
There are at least 475 known and putative proteases and 103 homologs to known proteases in humans, and it is estimated that up to 1,200 human genes encode proteases. Proteases representative of all five known mechanistic classes have been implicated in human disease.
Successful established protease inhibitor drugs on the market - ACE inhibitors and HIV protease inhibitors - act by inhibiting key proteases. Their success has led to proteases being increasingly viewed as valuable drug targets in disease treatment and a range of protease inhibitors are in various stages of commercial development. At present, an estimated 5-10% of all pharmaceutical targets are proteases. This report reviews progress made in the field so far, commenting on positive findings as well as setbacks encountered. Although the number of proteases currently targeted represents only a small fraction of all potential protease targets, the investigations are generating valuable clues about the validity of targeting specific proteases and protease families as well as general approaches to inhibition.
We forecast that the world market for protease-related drugs (mostly inhibitors, except for thrombolytics which are native or modified proteases) will increase from $11.2 billion in 2003 to $23.1 billion in 2009 in real terms, a compound annual growth rate (CAGR) of 12.9% at constant exchange rates. Currently the major applications by sales volume are antihypertensives and HIV protease inhibitors, with small contributions from the inflammation, cancer, and vascular therapeutic areas. At present all protease-related drugs for use in hypertension are ACE inhibitors, but other - possibly more potent and better tolerated - antagonists of the RAS pathway will be introduced. These five segments will all increase as new products are introduced, and they will be joined by newly introduced agents for the treatment of other infectious diseases, osteoporosis, type II diabetes, and (possibly) Alzheimer's disease. Much of the increase in this market segment beyond 2009 will be due to these new applications.
Most therapeutic protease inhibitors currently under development are small molecule drugs. Originally, protease inhibitors were derived from the peptide substrates of the proteases, which led to the development of peptidomimetic competitive inhibitors. There has been a gradual move to design inhibitors less related to the enzyme substrate with improved pharmacokinetic properties. Strategies to design peptidomimetic and non-peptidomimetic competitive inhibitors, as well as other types of inhibitors are discussed in the report. Naturally occurring protease inhibitors are also considered, as they provide a basis for protein-based therapeutics, for which improved methods of delivery are being developed.
Current efforts to design therapeutic protease inhibitors are being accelerated by the increased availability of structural information. Target protease structures are increasingly used at an early stage to generate leads of high quality, thereby saving costs associated with drug discovery. The determination of the HIV protease 3D structure in 1989 and subsequent development of HIV-specific protease inhibitors launched the modern era of structure-based drug design. The availability of high quality protease structures is increasing, throwing fresh light on old targets. For example, in 2003 the structure of ACE was finally solved, revealing an unexpected similarity to two proteases with which ACE does not share amino acid sequence similarity. New structural information is being used to design more selective small molecule inhibitors.
In the infectious disease field, protease inhibitor approaches take advantage of the fact that most viruses contain at least one protease, while all other microbes possess several. We review the prospects for HIV protease inhibitors in epidemiologically significant infectious diseases. In HIV/AIDS, the challenge is to develop improved peptidomimetic and non-peptidomimetic inhibitors, enhance protease inhibition and overcome the growing menace of viral resistance. Earlier stage protease inhibitor programs are underway in other viral infections, in particular HCV, as well as in infections due to bacteria, candida and parasites.
Most companies currently developing protease inhibitors have chosen to pursue applications in chronic diseases, reflecting a general trend in the industry. Excessive protease activity has long been believed to play a role in the development of many chronic inflammatory conditions, in particular osteo- and rheumatoid arthritis and chronic obstructive pulmonary disease. The clinical progression of the first generation of MMP inhibitors has been hampered by side effects, but inhibitors targeting diverse protease targets remain under investigation and their progress is assessed in the report.
Protease inhibition programs in cancer have produced some disappointments and some unexpected successes in recent years, which we evaluate. While the much-hyped MMP inhibitors failed in late-stage clinical trials, the proteasome inhibitor Velcade progressed rapidly through clinical trials and was approved in 2003, becoming the first new treatment in more than a decade for multiple myeloma. The growing recognition of the role of proteases and proteolytic cascades in both the growth and metastasis of tumors is enabling the development of more targeted protease inhibitors. This fits in with the general trend in cancer treatment towards molecular targeted therapies. We consider the rationale for the current choices of protease targets in cancer.
In the cardiovascular area, despite high levels of competition and high cost of development of drugs, the need for more specific protease inhibitors is driving further developments. Other active areas include anticoagulation, neurodegenerative conditions, osteoporosis, and type 2 diabetes, where inhibitors in development focus on high-profile disease-associated protease targets such as secretases in Alzheimer's disease, cathepsin K in osteoporosis and DPP-IV in type 2 diabetes.
The report profiles 30 key companies active in the protease drug discovery, which includes small specialist companies, commercial-stage biotech companies, and large pharmaceutical companies. Information is provided on promising proprietary protease inhibitor drug discovery technologies, and recent acquisitions and collaborations in this area.