Evaluation of Enzyme Inhibitors in Drug Discovery

A Guide for Medicinal Chemists and Pharmacologists

Robert A. Copeland

• Publisher: Wiley-Interscience
• Year: 2013
• Print ISBN:
  9781118488133
• eBook ISBN:
  9781118540282
• Edition: 2
• Topic: Scienze biologiche
• Subtopic: Biochimica

Offers essential guidance for discovering and optimizing novel drug therapies

Using detailed examples, Evaluation of Enzyme Inhibitors in Drug Discovery equips researchers with the tools needed to apply the science of enzymology and biochemistry to the discovery, optimization, and preclinical development of drugs that work by inhibiting specific enzyme targets. Readers will applaud this book for its clear and practical presentations, including its expert advice on best practices to follow and pitfalls to avoid.

This Second Edition brings the book thoroughly up to date with the latest research findings and practices. Updates explore additional forms of enzyme inhibition and special treatments for enzymes that act on macromolecular substrates. Readers will also find new discussions detailing the development and application of the concept of drug-target residence time.

Evaluation of Enzyme Inhibitors in Drug Discovery begins by explaining why enzymes are such important drug targets and then examines enzyme reaction mechanisms. The book covers:

• Reversible modes of inhibitor interactions with enzymes
• Assay considerations for compound library screening
• Lead optimization and structure-activity relationships for reversible inhibitors
• Slow binding and tight binding inhibitors
• Drug-target residence time
• Irreversible enzyme inactivators

The book ends with a new chapter exploring the application of quantitative biochemical principles to the pharmacologic evaluation of drug candidates during lead optimization and preclinical development.

The Second Edition of Evaluation of Enzyme Inhibitors in Drug Discovery continues to offer a treatment of enzymology applied to drug discovery that is quantitative and mathematically rigorous. At the same time, the clear and simple presentations demystify the complex science of enzymology, making the book accessible to many fields— from pharmacology to medicinal chemistry to biophysics to clinical medicine.

CHAPTER 1

Why Enzymes as Drug Targets?

Key Learning Points

• Enzymes are excellent targets for pharmacological intervention, owing to their essential roles in life processes and pathophysiology.
• The structures of enzyme active sites, and other ligand binding pockets on enzymes, are ideally suited for high-affinity interactions with drug-like inhibitors.

Medicine in the twenty-first century has largely become a molecular science in which drug molecules are directed toward specific macromolecular targets whose bioactivity is pathogenic or at least associated with disease. In most clinical situations the most desirable course of treatment is by oral administration of safe and effective drugs with a duration of action that allows for convenient dosing schedules (typically once or twice daily). These criteria are best met by small molecule drugs, as opposed to peptide, protein, gene, or many natural product-based therapeutics. Among the biological macromolecules that one can envisage as drug targets, enzymes hold a preeminent position because of the essentiality of their activity in many disease processes, and because the structural determinants of enzyme catalysis lend themselves well to inhibition by small molecular weight, drug-like molecules. Not surprisingly, enzyme inhibitors represent almost half the drugs in clinical use today. Recent surveys of the human genome suggest that the portion of the genome that encodes for disease-associated, “druggable” targets is dominated by enzymes. It is therefore a virtual certainty that specific enzyme inhibition will remain a major focus of pharmaceutical research for the foreseeable future. In this chapter we review the salient features of enzyme catalysis and of enzyme structure that make this class of biological macromolecules such attractive targets for chemotherapeutic intervention in human diseases.

1.1 Enzymes Are Essential for Life

In high school biology classes life is often defined as “a series of chemical reactions.” This popular aphorism reflects the fact that living cells, and in turn multicellular organisms, depend on chemical transformations for every essential life process. Synthesis of biomacromolecules (proteins, nucleic acids, polysaccarides, and lipids), all aspects of intermediate metabolism, intercellular communication in, for example, the immune response, and catabolic processes involved in tissue remodeling, all involve sequential series of chemical reactions (i.e., biological pathways) to maintain life’s critical functions. The vast majority of these essential biochemical reactions, however, proceed at uncatalyzed rates that are too slow to sustain life. For example, pyrimidines nucleotides, together with purine nucleotides, make up the building blocks of all nucleic acids. The de novo biosynthesis of pyrimidines requires the formation of uridine monophosphate (UMP) via the decarboxylation of orotidine monophosphate (OMP). Measurements of the rate of OMP decarboxylation have estimated the half-life of this chemical reaction to be approximately 78 million years! Obviously a reaction this slow cannot sustain life on earth without some very significant rate enhancement. The enzyme OMP decarboxylase (EC 4.1.1.23) fulfills this life-critical function, enhancing the rate of OMP decarboxylation by some 10¹⁷-fold, so that the reaction half-life of the enzyme-catalyzed reaction (0.018 seconds) displays the rapidity necessary for living organisms (Radzicka and Wolfenden, 1995).

Enyzme catalysis is thus essential for all life. Hence the selective inhibition of critical enzymes of infectious organisms (i.e., viruses, bacteria, and multicellular parasites) is an attractive means of chemotherapeutic intervention for infectious diseases. This strategy is well represented in modern medicine, with a significant portion of antiviral, antibiotic, and antiparasitic drugs in clinical use today deriving their therapeutic efficacy through selective enzyme inhibition (see Table 1.1 for some examples).

TABLE 1.1 Selected Enzyme Inhibitors in Clinical Use or Trials

Source: Adapted and expanded from Copeland (2000).

Compound	Target Enzyme	Clinical Use
Acetazolamide	Carbonic anhydrase	Glaucoma
Acyclovir	Viral DNA polymerase	Herpes
Amprenavir, indinavir, nelfinavir, ritonavir, saquinavir	HIV protease	AIDS
Allopurinol	Xanthine oxidase	Gout
Argatroban	Thrombin	Heart disease
Aspirin	Cyclooxygenases	Inflammation, pain, fever
Amoxicillin	Penicillin binding proteins	Bacterial infection
Captopril, enalapril	Angiotensin converting enzyme	Hypertension
Carbidopa	Dopa decarboxylase	Parkinson’s disease
Celebrex, Vioxx	Cyclooxygenase-2	Inflammation
CI-1040, PD0325901	MAP kinase kinase	Cancer
Clavulanate	β-Lactamase	Bacterial resistance
Digoxin	Sodium, potassium ATPase	Heart disease
Efavirenz, nevirapine	HIV reverse transcriptase	AIDS
Epristeride, finasteride, dutasteride	Steroid 5α-reductase	Benign prostate hyperplasia, male pattern baldness
Fluorouracil	Thymidylate synthase	Cancer
Leflunomide	Dihydroorotate	Inflammation
	Dehydrogenase
Lovastatin and other statins	HMG-CoA reductase	Cholesterol lowering
Methotrexate	Dihydrofolate reductase	Cancer, immunosuppression
Nitecapone	Catechol-O-methyltransferase	Parkinson’s disease
Norfloxacin	DNA gyrase	Urinary tract infections
Omeprazole	H^+, K^+ ATPase	Peptic ulcers
PALA	Aspartate	Cancer
	Transcarbamoylase
Sorbinol	Aldose reductase	Diabetic retinopathy
Trimethoprim	Bacterial dihydrofolate reductase	Bacterial infections
Viagra, Levitra	Phosphodiesterase	Erectile dysfunction

Although enzymes are essential for life, dysregulated enzyme activity can also lead to disease states. In some cases mutations in genes encoding enzymes can lead to abnormally high concentrations of the enzyme within a cell (overexpression). Alternatively, point mutations can lead to an enhancement of the specific activity (i.e., catalytic efficiency) of the enzyme because of structural changes in the catalytically critical amino acid residues. By either of these mechanisms, aberrant levels of the reaction product’s formation can result, leading to specific pathologies. Hence human enzymes are also commonly targeted for pharmacological intervention in many diseases.

Enzymes, then, are attractive targets for drug therapy because of their essential roles in life processes and in pathophysiology. Indeed, a survey reported in 2000 found that close to 30% of all drugs in clinical use derive their therapeutic efficacy through enzyme inhibition (Drews, 2000). More recently Hopkins and Groom (2002) updated this survey to include newly launched drugs and found that nearly half (47%) of all marketed small molecule drugs inhibit enzymes as their molecular target (Figure 1.1). Worldwide sales of small molecule drugs that function as enzyme inhibitors exceeded 65 billion dollars in 2001, and this market was expected to grow to more than 95 billion dollars by 2006 (see Figure 1.2). Some contraction of the worldwide market has occurred due to withdrawal of several products since 2005. Revised forecasts suggest that the worldwide market will now grow at a rate of about 6.7% as of 2005 (Business Communications Company, Inc., 2006, “Enzyme Inhibitors with Broad Therapeutic Application”).

Figure 1.1 Distribution of marketed drugs by biochemical target class. GPCRs = G-Protein coupled receptors.

Source: Redrawn from Hopkins and Groom (2002).

Figure 1.2 Worldwide market for small molecule drugs that function as enzyme inhibitors in 2001 and projected for 2006. AAGR = average annual growth rate.

Source: Business Communications Company, Inc. Report RC-202R: New Developments in Therapeutic Enzyme Inhibitors and Receptor Blockers, www.bccresearch.com.

The attractiveness of enzymes as drug targets results not only from the essentiality of their catalytic activity but also from the fact that enzymes, by their very nature, are highly amenable to inhibition by small molecular weight, drug-like molecules. Because of this susceptibility to inhibition by small molecule drugs, enzymes are commonly the target of new drug discovery and design efforts at major pharmaceutical and biotechnology companies today; my own informal survey suggests that between 50 and 75% of all new drug-seeking efforts at several major pharmaceutical companies in the United States are focused on enzymes as primary targets.

While the initial excitement generated by the completion of the Human Genome Project was in part due to the promise of a bounty of new targets for drug therapy, it is now apparent that only a portion of the some 30,000 proteins encoded for by the human genome are likely to be amenable to small molecule drug intervention. A recent study suggested that the size of the human “druggable genome” (e.g., human genes encoding proteins that are expected to contain functionally necessary binding pockets with appropriate structures for interactions with drug-like molecules) is more on the order of 3000 target proteins (i.e., about 10% of the genome), a significant portion of these being enzymes (Hopkins and Groom, 2002). As pointed out by Hopkins and Groom, just because a protein contains a druggable binding pocket does not necessarily make it a good target for drug discovery; there must be some expectation that the protein plays some pathogenic role in disease so that inhibition of the protein will lead to a disease modification. Furthermore the same study estimates that of the nearly 30,000 proteins encoded by the human genome, only about 10% (3000) can be classified as “disease-modifying genes” (e.g., genes that, when knocked out in mice, effect a disease-related phenotype). The intersection of the druggable genome and the disease-modifying genome thus defines the number of bona fide drug targets of greatest interest to pharmaceutical scientists. This intersection, according to Hopkins and Groom (2002), contains only between 600 and 1500 genes, again with a large proportion of these genes encoding for enzyme targets.

The “druggability” of enzymes as targets reflects the evolution of enzyme structure to efficiently perform catalysis of chemical reactions, as discussed in the following section.

1.2 Enzyme Structure and Catalysis

From more than a thousand years of folk remedies and more recent systematic pharmacology, it is well known...

Table of contents

• Cover
• Title page
• Copyright page
• Dedication
• Foreword to Second Edition
• Preface to Second Edition
• Foreword to First Edition
• Preface to First Edition
• Acknowledgments (From First Edition)
• Epigraph
• CHAPTER 1: Why Enzymes as Drug Targets?
• CHAPTER 2: Enzyme Reaction Mechanisms
• CHAPTER 3: Reversible Modes of Inhibitor Interactions with Enzymes
• CHAPTER 4: Assay Considerations for Compound Library Screening
• CHAPTER 5: Lead Optimization and Structure–Activity Relationships for Reversible Inhibitors
• CHAPTER 6: Slow Binding Inhibitors
• CHAPTER 7: Tight Binding Inhibition
• CHAPTER 8: Drug–Target Residence Time
• CHAPTER 9: Irreversible Enzyme Inactivators
• CHAPTER 10: Quantitative Biochemistry in the Pharmacological Evaluation of Drugs
• APPENDIX 1: Kinetics of Biochemical Reactions
• APPENDIX 2: Derivation of the Enzyme–Ligand Binding Isotherm Equation
• APPENDIX 3: Serial Dilution Schemes
• APPENDIX 4: Relationship between [I]/IC50 and Percentage Inhibition of Enzyme Activity When h = 1
• APPENDIX 5: Propagation of Uncertainties in Experimental Measurements
• APPENDIX 6: Useful Physical Constants at Different Temperatures
• APPENDIX 7: Common Radioactive Isotopes Used in Studies of Enzymes
• APPENDIX 8: Common Prefixes for Units in Biochemistry
• APPENDIX 9: Some Aromatic Ring Systems Commonly Found in Drugs
• APPENDIX 10: Residual Plots
• Index