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Non-competitive inhibition

About: Non-competitive inhibition is a(n) research topic. Over the lifetime, 4121 publication(s) have been published within this topic receiving 147000 citation(s).
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Journal ArticleDOI
Yung-Chi Cheng1, William H. Prusoff1Institutions (1)
TL;DR: The analysis described shows K I does not equal I 50 when competitive inhibition kinetics apply; however, K I is equal to I 50 under conditions of either noncompetitive or uncompetitive kinetics.
Abstract: A theoretical analysis has been made of the relationship between the inhibition constant ( K I ) of a substance and the ( I 50 ) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between K I and I 50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I 50 values in agreement with the admonitions previously described in the literature. The analysis described shows K I does not equal I 50 when competitive inhibition kinetics apply; however, K I is equal to I 50 under conditions of either noncompetitive or uncompetitive kinetics.

12,173 citations

Journal ArticleDOI
TL;DR: It is suggested that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.
Abstract: Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.

1,086 citations

Journal ArticleDOI
TL;DR: The effects of cholinergic agonists and antagonists on the enzyme were examined and found none and the possibility that the inhibition of this enzyme by lithium ion is related to the pharmacological actions of lithium is discussed.
Abstract: myo-Inositol-1-phosphatase has been partially purified from bovine brain. The enzyme has a molecular weight of about 58,000. Both L-myo-inositol 1-phosphate and D-myo-inositol 1-phosphate are hydrolyzed by the enzyme as well as (-)-chiro-inositol 3-phosphate and 2'-AMP. Triphosphoinositide is not a substrate. The phosphatase is completely dependent on Mg2+, which has a Km of 1 mM. Calcium and manganese ions are competitive inhibitors of Mg2+ binding with Ki values of 18 microM and 2 microM, respectively. Lithium chloride inhibits the hydrolysis of both L- and D-myo-inositol 1-phosphate to the extent of 50% at a concentration of 0.8 mM. The phosphatase from testis is similarly inhibited by lithium. Lithium ion is a noncompetitive inhibitor of Mg2+ binding and an uncompetitive inhibitor of myo-inositol 1-phosphate binding. Because lithium chloride administration elicits both an increase in the levels of myo-inositol 1-phosphate and a decrease in the levels of myo-inositol in rat brain (Allison, 1978), and because these actions are blocked by anticholinergic agents, we examined the effects of cholinergic agonists and antagonists on the enzyme and found none. The possibility that the inhibition of this enzyme by lithium ion is related to the pharmacological actions of lithium is discussed.

1,023 citations

28 Mar 2005-
TL;DR: This work has shown that knowing Inhibitor Modality is important for Structure-Based Lead Organization and Associating Cellular Effects with Target Enzyme Inhibition should Require a Certain Affinity for the target Enzyme.
Abstract: Foreword. Preface. Acknowledgments. 1. Why Enzymes as Drug Targets? 1.1 Enzymes Are Essentials for Life. 1.2 Enzyme Structure and Catalysis. 1.3 Permutations of Enzyme Structure During Catalysis. 1.4 Other Reasons for Studying Enzymes. 1.5 Summary. References. 2. Enzyme Reaction Mechanisms. 2.1 Initial Binding of Substrate. 2.2 Noncovalent Forces in Reversible Ligand Binding to Enzymes. 2.2.1 Electrostatic Forces. 2.2.2 Hydrogen Bonds. 2.2.3 Hydrophobic Forces. 2.2.4 van der Waals Forces. 2.3 Transformations of the Bond Substrate. 2.3.1 Strategies for Transition State Stabilization. 2.3.2 Enzyme Active Sites Are Most Complementary to the Transition State Structure. 2.4 Steady State Analysis of Enzyme Kinetics. 2.4.1 Factors Affecting the Steady State Kinetic Constants. 2.5 Graphical Determination of k cat and K M 2.6 Reactions Involving Multiple Substates. 2.6.1 Bisubstrate Reaction Mechanisms. 2.7 Summary. References. 3. Reversible Modes of Inhibitor Interactions with Enzymes. 3.1 Enzyme-Inhibitor Binding Equilibria. 3.2 Competitive Inhibition. 3.3 Noncompetitive Inhibition. 3.3.1 Mutual Exclusively Studies. 3.4 Uncompetitive Inhibition. 3.5 Inhibition Modality in Bisubstrate Reactions. 3.6 Value of Knowing Inhibitor Modality. 3.6.1 Quantitative Comparisons of Inhibitor Affinity. 3.6.2 Relating K i to Binding Energy. 3.6.3 Defining Target Selectivity by K i Values. 3.6.4 Potential Advantages and Disadvantages of Different Inhibition Modalities In Vivo. 3.6.5 Knowing Inhibition Modality Is Important for Structure-Based Lead Organization. 3.7 Summary. References. 4. Assay Considerations for Compound Library Screening. 4.1 Defining Inhibition Signal Robustness, and Hit Criteria. 4.2 Measuring Initial Velocity. 4.2.1 End-Point and Kinetic Readouts. 4.2.2 Effects of Enzyme Concentration. 4.3 Balanced Assay Conditions. 4.3.1 Balancing Conditions for Multisubstrate Reactions. 4.4 Order of Reagent Addition. 4.5 Use of Natural Substrates and Enzymes. 4.6 Coupled Enzyme Assays. 4.7 Hit Validation and Progression. 4.8 Summary. References. 5. Lead Optimization and Structure-Activity Relationships for Reversible Inhibitors. 5.1 Concentration-Response Plots and IC 50 Determination. 5.1.1 The Hill Coefficient. 5.1.2 Graphing and Reporting Concentration-Response Data. 5.2 Testing for Reversibility. 5.3 Determining Reversible Inhibition Modality and Dissociation Constant. 5.4 Comparing Relative Affinity. 5.4.1 Compound Selectivity. 5.5 Associating Cellular Effects with Target Enzyme Inhibition. 5.5.1 Cellular Phenotype Should Be Consistent with Genetic Knockout or Knockdown of the Target Enzyme. 5.5.2 Cellular Activity Should Require a Certain Affinity for the target Enzyme. 5.5.3 Buildup of Substrate and/or Diminution of Product for the Target Enzyme Should Be Observed in Cells. 5.5.4 Cellular Phenotype Should Be Reversed by Cell-Permeable Product or Downstream Metabolites of the Target Enzyme Activity. 5.5.5 Mutation of the Target Enzyme Should Lead to Resistance or Hypersensitivity to Inhibitors. 5.6 Summary. References. 6. Slow Binding Inhibitors. 6.1 Determining k obs : The Rate Constant for Onset of Inhibition. 6.2 Mechanisms of Slow Binding Inhibition. 6.3 Determination of Mechanism and Assessment of True Affinity. 6.3.1 Potential Clinical Advantages of Slow Off-rate Inhibitors. 6.4 Determining Inhibition Modality for Slow Binding Inhibitors. 6.5 SAR for Slow Binding Inhibitors. 6.6 Some Examples of Pharmacologically Interesting Slow Binding Inhibitors. 6.6.1 Examples of Scheme B: Inhibitors of Zinc Peptidases and Proteases. 6.6.2 Example of Scheme C: Inhibition of Dihydrofolate Reductase by Methotresate. 6.6.3 Example of Scheme C: Inhibition of Calcineurin by FKBP-Inhibitor Complexes. 6.6.4 Example of Scheme C When K i << K i : Aspartyl Protease Inhibitors. 6.6.5 Example of Scheme C When k 6 Is Very Small: Selective COX2 Inhibitors. 6.7 Summary. References. 7. Tight Binding Inhibitors. 7.1 Effects of Tight Binding Inhibition Concentration-Response Data. 7.2 The IC 50 Value Depends on K i app and [E] T . 7.3 Morrison's Quadratic Equation for Fiting Concentration-Response Data for Tight Binding Inhibitors. 7.3.1 Optimizing Conditions for K i app Determination Using Morrison's Equation. 7.3.2 Limits on K i app Determinations. 7.3.3 Use of a Cubic Equation When Both Substrate and Inhibitor Are Tight Binding. 7.4 Determining Modality for Tight Binding Enzyme Inhibitors. 7.5 Tight Binding Inhibitors Often Display Slow Binding Behavior. 7.6 Practical Approaches to Overcoming the Tight Binding Limit in Determine K i . 7.7 Enzyme-Reaction Intermediate Analogues as Example of Tight Binding Inhibitors. 7.7.1 Bisubstrate Analogues. 7.7.2 Testing for Transition State Mimicry. 7.8 Potential Clinical Advantages of Tight Binding Inhibitors. 7.9 Determination of [E] T Using Tight Binding Inhibitors. 7.10 Summary. References. 8. Irreversible Enzyme Inactivators. 8.1 Kinetic Evaluation of Irreversible Enzyme Inactivators. 8.2 Affinity Labels. 8.2.1 Quiescent Affinity Labels. 8.2.2 Potential Liabilities of Affinity Labels as Drugs. 8.3 Mechanism-Based Inactivators. 8.3.1 Distinguishing Features of Mechanism-Based Inactivation. 8.3.2 Determination of the Partition Ratio. 8.3.3 Potential Clinical Advantages of Mechanism-Based Inactivators. 8.3.4 Examples of Mechanism-Based Inactivators as Drugs. 8.4 Use of Affinity Labels as Mechanistic Tools. 8.5 Summary. References. Appendix 1. Kinetic of Biochemical Reactions. A1.1 The Law of Mass Action and Reaction Order. A1.2 First-Order Reaction Kinetics. A1.3 Second-Order Reaction Kinetics. A1.4 Pseudo-First-Order Reaction Conditions. A1.5 Approach to Equilibrium: An Example of the Kinetics of Reversible Reactions. References. Appendix 2. Derivation of the Enzyme-Ligand Binding Isotherm Equation. References. Appendix 3. Serial Dilution Schemes. Index.

840 citations

Journal ArticleDOI
John O. Miners1, Donald J. Birkett1Institutions (1)
TL;DR: Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates.
Abstract: Accumulating evidence indicates that CYP2C9 ranks amongst the most important drug metabolizing enzymes in humans. Substrates for CYP2C9 include fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and numerous NSAIDs. CYP2C9 activity in vivo is inducible by rifampicin. Evidence suggests that CYP2C9 substrates may also be induced variably by carbamazepine, ethanol and phenobarbitone. Apart from the mutual competitive inhibition which may occur between alternate substrates, numerous other drugs have been shown to inhibit CYP2C9 activity in vivo and/or in vitro. Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, sulphinpyrazone, sulphaphenazole and certain other sulphonamides. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino acid residues 144 (Arg144Cys) and 359 (Ile359Leu) of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, although the frequency of this allele is relatively low. Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. Individualisation of dose is essential for those CYP2C9 substrates with a narrow therapeutic index.

808 citations

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