The Impact of Assay Design on Medicinal Chemistry: Case Studies

Born, Joshua R.; Chenniappan, Vinoth Kumar; Davis, Danielle; Dahlin, Jayme L.; Marugán, Juan; Patnaik, Samarjit

doi:10.1177/24725552211026238

Cited by 3 publications

References 71 publications

(91 reference statements)

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Development of a Highly Selective NanoBRET Probe to Assess MAGL Inhibition in Live Cells

Gazzi,

Brennecke,

Olikauskas

et al. 2024

ChemBioChem

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Cell‐free enzymatic assays are highly useful tools in early compound profiling due to their robustness and scalability. However, their inadequacy to reflect the complexity of target engagement in a cellular environment may lead to a significantly divergent pharmacology that is eventually observed in cells. The discrepancy that emerges from properties like permeability and unspecific protein binding may largely mislead lead compound selection to undergo further chemical optimization. We report the development of a new intracellular NanoBRET assay to assess MAGL inhibition in live cells. Based on a reverse design approach, a highly potent, reversible preclinical inhibitor was conjugated to the cell‐permeable BODIPY590 acceptor fluorophore while retaining its overall balanced properties. An engineered MAGL‐nanoluciferase (Nluc) fusion protein provided a suitable donor counterpart for the facile interrogation of intracellular ligand activity. Validation of assay conditions using a selection of known MAGL inhibitors set the stage for the evaluation of over 1’900 MAGL drug candidates derived from our discovery program. This evaluation enabled us to select compounds for further development based not only on target engagement, but also on favorable physicochemical parameters like permeability and protein binding. This study highlights the advantages of cell‐based target engagement assays for accelerating compound profiling and progress at the early stages of drug discovery programs.

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Development of a Highly Selective NanoBRET Probe to Assess MAGL Inhibition in Live Cells

Gazzi,

Brennecke,

Olikauskas

et al. 2024

ChemBioChem

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Lead Generation

Narjes,

Pairaudeau,

Petrović

2023

The Handbook of Medicinal Chemistry

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The lead generation phase is the stage in early drug discovery that refers to the process of identifying hit molecules that interact with the desired target, followed by a limited optimisation of those hits. During this period the project aims to identify high-quality chemical tools suitable for advanced cellular and in vivo studies which will help to strengthen the validation of the target and increase confidence in the proposed therapeutic hypothesis. This chapter gives an overview of the major hit finding approaches together with their strengths and limitations. The process of hit profiling and hit selection, as well as hit expansion, are illustrated, and complications that can be encountered during this initial phase are highlighted. The advantage of using an integrated lead generation strategy is emphasised. A well-defined lead generation strategy, where two or more complementary hit finding approaches are executed in parallel, combined with a screening cascade containing relevant biological assays, will often result in the identification of structurally diverse lead series, leading ultimately to successful clinical candidates.

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