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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
In vitro Mutagenesis01:16

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.

Adam M Thomas1, Marta Serafini1, Emma K Grant2

  • 1Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom.

ACS Chemical Biology
|October 24, 2023
PubMed
Summary

Drug discovery faces challenges with target validation. This study introduces a novel mutate and conjugate approach using site-directed mutagenesis and electrophilic fragments to rapidly identify selective small molecule inhibitors for mutant proteins like BRD4.

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Drug Discovery

Background:

  • Target validation is a critical bottleneck in drug discovery, leading to high attrition rates, especially in Phase II clinical trials.
  • Developing new methods to enhance target validation is crucial for improving the efficiency of the drug discovery pipeline.
  • Bromodomain-containing protein 4 (BRD4) is an important target in various diseases, but selective inhibition remains challenging.

Purpose of the Study:

  • To develop and validate a novel 'mutate and conjugate' approach for rapid identification of selective small molecule inhibitors.
  • To demonstrate the efficacy of this approach using the bromodomain-4 protein (BRD4) as a model system.
  • To identify selective inhibitors for a mutated BRD4 protein (BRD4(1)L94C) and assess their cellular activity.

Main Methods:

  • Structure-based design was used to introduce a site-directed mutation (L94C) in BRD4(1) with minimal functional impact.
  • Screening of fragment libraries against wild-type and mutant BRD4(1) to identify selective binders.
  • Elaboration of identified fragments with click chemistry tags for cellular target engagement studies.
  • Assessment of compound selectivity in HEK293T cells expressing wild-type or mutant BRD4(1).

Main Results:

  • A specific mutation (L94C) was identified in BRD4(1) that minimally affected protein function.
  • A series of fragments selectively inhibiting the mutant BRD4(1)L94C over wild-type BRD4(1) were identified.
  • One elaborated compound demonstrated high selectivity for BRD4(1)L94C in cellular assays.
  • The 'mutate and conjugate' approach successfully enabled rapid identification of a selective inhibitor.

Conclusions:

  • The combination of site-directed mutagenesis and electrophilic fragments provides a powerful strategy for rapid target validation and inhibitor discovery.
  • This 'mutate and conjugate' approach facilitates the identification of selective small molecule inhibitors for specific protein targets.
  • The identified selective BRD4(1)L94C inhibitor serves as a starting point for further drug development and cellular phenotype assessment.