Modern Methods of Drug Design and Development / edited by Matthew Lloyd.

Format:

Book

Contributor:

Lloyd, Matthew, editor.

Series:

Methods in Enzymology Series

Methods in Enzymology Series ; v.Volume 690

Language:

English

Subjects (All):

Drugs--Design.

Drugs.

Physical Description:

1 online resource (600 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Academic Press, [2023]

Summary:

Modern Methods of Drug Design and Development, a volume in the Methods in Enzymology series highlights new advances in the field with this new volume presenting interesting chapters on a variety of topics, including Recombinant protein purification for structural and kinetic studies, Steady-state kinetic analysis of reversible enzyme inhibitors.

Contents:

Front Cover

Series Page

Title Page

Copyright

Contents

Contributors

Preface

References

Chapter One: Recombinant protein production for structural and kinetic studies: A case study using M. tuberculosis α-methylacyl-CoA racemase (MCR)

1 Introduction

2 Recombinant protein production

2.1 Key resources table

2.2 Materials and equipment

2.2.1 Equipment

2.2.2 Solutions and consumables

Alternatives

2.3 Step-by-step method details

2.3.1 Production of competent cells and plasmid transformation

2.3.2 Growth of E. coli starter cultures

2.3.3 Growth of E. coli cultures and production of recombinant protein

2.4 Expected outcomes, advantages and disadvantages

2.5 Optimization and troubleshooting

2.6 Safety considerations and standards

3 Recombinant protein purification

3.1 Key resources table

3.2 Materials and equipment

3.2.1 Equipment

3.2.2 Solutions and consumables

3.3 Step-by-step method details

3.3.1 Cell lysis and purification using DEAE-Sepharose

3.3.2 Purification of pooled DEAE-Sepharose fractions using RESOURCE-Q column

3.3.3 Purification of pooled RESOURCE-Q fractions using size-exclusion chromatography

3.3.4 Buffer-exchange of purified target protein

3.4 Expected outcomes, advantages and disadvantages

3.5 Optimization and troubleshooting

3.6 Safety considerations and standards

4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

4.1 Key resources table

4.2 Materials and equipment

4.2.1 Equipment

4.2.2 Solutions and consumables

4.3 Step-by-step method details

4.4 Expected outcomes, advantages and disadvantages

4.5 Optimization and troubleshooting

4.6 Safety considerations and standards

Acknowledgements

Conflict of interest

References.

Chapter Two: Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase

2 Measurements of 4-nitrophenol pKa

2.2.1 Alternatives

2.3.1 Making the series of buffers

2.3.1.1 Timing: Day 1

2.3.2 Measuring the 4-nitrophenol pKa value

2.3.2.1 Timing: Day 1 or 2

2.3.3 Data analysis

2.3.3.1 Timing: Day 1 or 2

2.4 Expected outcomes, advantages and limitations

2.5 Statistical analysis

2.6 Optimization and troubleshooting

2.7 Safety considerations and standards

3 Alkaline phosphatase assays

3.2.1 Alternatives

3.3.1 Determining the amount of enzyme required in the assay

3.3.2 Calculation of adjusted 4-nitrophenol extinction coefficient

3.3.3 Calculation of reaction rates

3.3.4 Determining the effect of additives on the enzyme activity

3.3.5 Determining kinetic parameters for alkaline phosphatase

3.3.6 Determination of Km and Vmax values using the direct linear plot

3.3.7 Determination of Km and Vmax values using the enzyme kinetics macro

3.3.8 Calculation of numerical values for kinetic parameters

3.3.9 Measurement of Z′

3.3.10 Dose-response curves and determination of pIC50 values

3.3.10.1 Data collection

3.3.11 Data processing and determination of pIC50 values

3.3.12 Rapid dilution experiment to determine inhibitor reversibility

3.3.12.1 Collection of data

3.3.13 Data processing

3.3.14 Determination of Ki values

3.3.15 Collection of data

3.3.16 Data processing

3.3.17 Determining the type of inhibition

3.3.18 Statistical analysis of the results and determination of significance.

3.4 Expected outcomes, advantages and limitations

3.5 Statistical analysis

3.6 Optimization and troubleshooting

3.7 Safety considerations and standards

Acknowledgments

Chapter Three: Non-equilibrium modalities of inhibition: Characterizing irreversible inhibition for the ErbB receptor family membersHigh-throughput experimentation and analysis of irreversible inhibition

1.1 Life as a kinetic state of matter

1.2 Brief theory of slow binding and irreversible inhibition

2 Protocol

2.1 Key resource table

2.2 Materials and equipment employed in this study

2.3.1 Making the buffers, substrate and enzyme

2.3.2 Step by step guide on how to run the assay

2.3.4 Considerations when interpreting assay results

2.4 Optimization and troubleshooting

Declaration of conflicting interests

Data and software availability

Funding

Chapter Four: Analysis of continuous enzyme kinetic data using ICEKAT

2 Program implementation

3 User's guide to ICEKAT

3.1 Required materials

3.2 Continuous enzyme kinetic assay data analysis in ICEKAT

3.2.1 Preparing and uploading data to ICEKAT

3.2.2 Semi-automated ICEKAT data analysis

3.3 Special considerations for specific ICEKAT analysis models

3.3.1 pEC50/pIC50 model

3.3.2 HTS model

3.4 Special considerations for specific ICEKAT analysis modes

3.4.1 Schnell-Mendoza mode

3.4.2 Logarithmic Fit mode

4 Data processing

5 Expected outcomes, advantages, and limitations

6 Conclusions

Chapter Five: You get what you screen for: Standards for experimental design and data fitting in drug discoveryData fitting in drug discovery

1 An introduction to mechanism-based computer simulation and data fitting

2.1 Modeling steady-state kinetic data

2.2 Modeling competition

2.3 Kinetics of substrate binding: Confidence contour analysis

3 Modeling and fitting equilibrium binding data

3.1 Special syntax for entering a titration

3.2 Fitting dose-response curves

3.3 Time to reach equilibrium

3.4 Fitting dose/response curves using the Hill equation

3.5 Fitting cooperative binding

3.6 Asymmetry in titration curves

4 Signals for measuring equilibrium binding and kinetics

4.1 Fluorescence methods combining kinetic and equilibrium measurements

4.2 Alternative labeling methods

4.3 Modeling coupled reactions

5 Global data fitting

6 Summary

Funding support/Acknowledgments

Chapter Six: Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR)

2 Purification of recombinant human AMACR 1A

2.3.1 Transformation of plasmid and growth of recombinant E. coli cells

2.3.2 Purification of recombinant human AMACR using metal-chelate chromatography

3 Synthesis and quantification of acyl-CoA substrates

3.3.1 General synthetic procedures

3.3.1.1 General Method A: Acid coupling to evans auxiliary

3.3.1.2 General Method B: Methylation.

3.3.1.3 General Method C: Evan's auxiliary cleavage

3.3.1.4 General Method D: Synthesis of acyl-CoA esters

3.3.2 Synthesis of S-2-[13C]-2-[2H]-2-methyldecanoyl-CoA 10

3.3.2.1 Synthesis of ethyl [2-13C]-2-ethoxycarbonyldecanoate 12

3.3.2.2 Synthesis of 2-[13C]-2-[2H2]-decanoic acid 13

3.3.3 Synthesis of Anti-(2R,3R)-3-fluoro-2-methyldecanoyl-CoA (17)

3.3.3.1 Synthesis of (R)-4-benzyl-3-propanoyloxazolidin-2-one 19

3.3.3.2 Synthesis of (R)-4-benzyl-3-[(2R,3S)-3-hydroxy-2-methyldecanoyl]oxazolidin-2-one 20

3.3.3.3 Synthesis of (R)-4-benzyl-3-[(2S,3R)-3-fluoro-2-methyldecanoyl]oxazolidin-2-one 21

3.3.3.4 Synthesis of (2S,3R)-3-fluoro-2-methyldecanoic acid 22

3.3.3.5 Synthesis of (2R,3R)-3-fluoro-2-methyldecanoyl-CoA 17

3.3.4 Synthesis of colorimetric substrate 23

3.3.4.1 Synthesis of 2R,S-3-(2,4-dinitrophenoxy)-2-methylpropan-1-ol 26

3.3.4.2 Synthesis of 2R,S-3-(2,4-dinitrophenoxy)-2-methylpropanoic acid 27

3.3.4.3 Synthesis of 2R,S-3-(2,4-dinitrophenoxy)-2-methylpropanoyl-CoA 23

3.4 Expected outcomes, advantages and limitations

3.4.1 Synthesis of precursor acids

3.4.2 Synthesis of acyl-CoA esters (General Method D)

4 NMR assays of recombinant human AMACR 1A

4.3.1 2H-exchange assay for acyl-CoA ester substrates

4.3.2 Analysis of the stereochemical outcome of the reaction of 2-methylacyl-CoA esters with AMACR

4.4 Expected outcomes, advantages and limitations

4.5 Quantification and data analysis methods

4.5.1 Data collection and referencing

4.5.2 Data processing

4.5.3 NMR assays with double labeled substrates

4.5.4 NMR assays with eliminating substrates

4.6 Optimization and troubleshooting.

4.7 Safety considerations and standards.

Notes:

Description based on print version record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-443-15871-1

0-443-15872-X

OCLC:

1405367035