BIOCHEMICAL DRUG ASSAY METHODS
CONTENTS 1 WHY ENZYMES AS DRUG TARGETS? ENZYMES ARE ESSENTIAL FOR LIFE ENZYME STRUCTURE AND CATALYSIS PERMUTATIONS OF ENZYME STRUCTURE DURING CATALYSIS OTHER REASONS FOR STUDYING ENZYMES SUMMARY REFERENCES 2 ENZYME REACTION MECHANISMS INITIAL BINDING OF SUBSTRATE NONCOVALENT FORCES IN REVERSIBLE LIGAND BINDING TO ENZYMES ELECTROSTATIC FORCES HYDROGEN BONDS HYDROPHOBIC FORCES VAN DER WAALS FORCES TRANSFORMATIONS OF THE BOUND SUBSTRATE STRATEGIES FOR TRANSITION STATE STABILIZATION ENZYME ACTIVE SITES ARE MOST COMPLEMENTARY TO THE TRANSITION STATE STRUCTURE STEADY STATE ANALYSIS OF ENZYME KINETICS FACTORS AFFECTING THE STEADY STATE KINETIC CONSTANTS GRAPHICAL DETERMINATION OF KCAT AND KM REACTIONS INVOLVING MULTIPLE SUBSTRATES BISUBSTRATE REACTION MECHANISMS 3 REVERSIBLE MODES OF INHIBITOR INTERACTIONS WITH ENZYMES ENZYME–INHIBITOR BINDING EQUILIBRIA COMPETITIVE INHIBITION NONCOMPETITITIVE INHIBITION MUTUAL EXCLUSIVITY STUDIES UNCOMPETITIVE INHIBITION INHIBITION MODALITY IN BISUBSTRATE REACTIONS VALUE OF KNOWING INHIBITOR MODALITY QUANTITATIVE COMPARISONS OF INHIBITOR AF?NITY RELATING KI TO BINDING ENERGY DE?NING TARGET SELECTIVITY BY KI VALUES POTENTIAL ADVANTAGES AND DISADVANTAGES OF DIFFERENT INHIBITION MODALITIES IN VIVO KNOWING INHIBITION MODALITY IS IMPORTANT FOR STRUCTURE-BASED LEAD OPTIMIZATION 4 ASSAY CONSIDERATIONS FOR COMPOUND LIBRARY SCREENING DE?NING INHIBITION, SIGNAL ROBUSTNESS, AND HIT CRITERIA MEASURING INITIAL VELOCITY END-POINT AND KINETIC READOUTS EFFECTS OF ENZYME CONCENTRATION OTHER FACTORS AFFECTING INITIAL VELOCITY BALANCED ASSAY CONDITIONS BALANCING CONDITIONS FOR MULTISUBSTRATE REACTIONS ORDER OF REAGENT ADDITION USE OF NATURAL SUBSTRATES AND ENZYMES COUPLED ENZYME ASSAYS HIT VALIDATION AND PROGRESSION 5 LEAD OPTIMIZATION AND STRUCTURE-ACTIVITY RELATIONSHIPS FOR REVERSIBLE INHIBITORS CONCENTRATION–RESPONSE PLOTS AND IC50 DETERMINATION THE HILL COEF?CIENT GRAPHING AND REPORTING CONCENTRATION–RESPONSE DATA TESTING FOR REVERSIBILITY DETERMINING REVERSIBLE INHIBITION MODALITY AND DISSOCIATION CONSTANT COMPARING RELATIVE AF?NITY COMPOUND SELECTIVITY ASSOCIATING CELLULAR EFFECTS WITH TARGET ENZYME INHIBITION CELLULAR PHENOTYPE SHOULD BE CONSISTENT WITH GENETIC KNOCKOUT OR KNOCKDOWN OF THE TARGET ENZYME CELLULAR ACTIVITY SHOULD REQUIRE A CERTAIN AF?NITY FOR THE TARGET ENZYME BUILDUP OF SUBSTRATE AND/OR DIMINUTION OF PRODUCT FOR THE TARGET ENZYME SHOULD BE OBSERVED IN CELLS CELLULAR PHENOTYPE SHOULD BE REVERSED BY CELL-PERMEABLE PRODUCT OR DOWNSTREAM METABOLITES OF THE TARGET ENZYME ACTIVITY MUTATION OF THE TARGET ENZYME SHOULD LEAD TO RESISTANCE OR HYPERSENSITIVITY TO INHIBITORS 6 SLOW BINDING INHIBITORS DETERMINING KOBS: THE RATE CONSTANT FOR ONSET OF INHIBITION MECHANISMS OF SLOW BINDING INHIBITION DETERMINATION OF MECHANISM AND ASSESSMENT OF TRUE AF?NITY POTENTIAL CLINICAL ADVANTAGES OF SLOW OFF-RATE INHIBITORS DETERMINING INHIBITION MODALITY FOR SLOW BINDING INHIBITORS SAR FOR SLOW BINDING INHIBITORS SOME EXAMPLES OF PHARMACOLOGICALLY INTERESTING SLOW BINDING INHIBITORS EXAMPLES OF SCHEME B: INHIBITORS OF ZINC PEPTIDASES AND PROTEASES EXAMPLE OF SCHEME C: INHIBITION OF DIHYDROFOLATE REDUCTASE BY METHOTREXATE EXAMPLE OF SCHEME C: INHIBITION OF CALCINEURIN BY FKBP-INHIBITOR COMPLEXES EXAMPLE OF SCHEME C WHEN KI* << KI: ASPARTYL PROTEASE INHIBITORS INHIBITORS 7 TIGHT BINDING INHIBITORS EFFECTS OF TIGHT BINDING INHIBITION CONCENTRATION–RESPONSE DATA THE IC50 VALUE DEPENDS ON KI APP AND [E]T MORRISON’S QUADRATIC EQUATION FOR FITTING CONCENTRATION–RESPONSE DATA FOR TIGHT BINDING INHIBITORS OPTIMIZING CONDITIONS FOR KI APP DETERMINATION USING MORRISON’S EQUATION LIMITS ON KI APP DETERMINATIONS USE OF A CUBIC EQUATION WHEN BOTH SUBSTRATE AND INHIBITOR ARE TIGHT BINDING DETERMINING MODALITY FOR TIGHT BINDING ENZYME INHIBITORS TIGHT BINDING INHIBITORS OFTEN DISPLAY SLOW BINDING BEHAVIOR PRACTICAL APPROACHES TO OVERCOMING THE TIGHT BINDING LIMIT IN DETERMINING KI ENZYME-REACTION INTERMEDIATE ANALOGUES AS EXAMPLES OF TIGHT BINDING INHIBITORS BISUBSTRATE ANALOGUES TESTING FOR TRANSITION STATE MIMICRY POTENTIAL CLINICAL ADVANTAGES OF TIGHT BINDING INHIBITORS DETERMINATION OF [E]T USING TIGHT BINDING INHIBITORS 7 10 SUMMARY 8 IRREVERSIBLE ENZYME INACTIVATORS KINETIC EVALUATION OF IRREVERSIBLE ENZYME INACTIVATORS AF?NITY LABELS QUIESCENT AF?NITY LABELS POTENTIAL LIABILITIES OF AF?NITY LABELS AS DRUGS MECHANISM-BASED INACTIVATORS DISTINGUISHING FEATURES OF MECHANISM-BASED INACTIVATION DETERMINATION OF THE PARTITION RATIO POTENTIAL CLINICAL ADVANTAGES OF MECHANISM-BASED INACTIVATORS EXAMPLES OF MECHANISM-BASED INACTIVATORS AS DRUGS USE OF AF?NITY LABELS AS MECHANISTIC TOOLS