Development of a cell-based target engagement assay for pyruvate dehydrogenase kinase

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2025-03-21 DOI:10.1016/j.slasd.2025.100227

Mya D. Gough , Matthew B. Robers , Cesear R. Corona , Ranjit K. Mehta , Mukesh K. Nyati , Peter L. Toogood

{"title":"Development of a cell-based target engagement assay for pyruvate dehydrogenase kinase","authors":"Mya D. Gough , Matthew B. Robers , Cesear R. Corona , Ranjit K. Mehta , Mukesh K. Nyati , Peter L. Toogood","doi":"10.1016/j.slasd.2025.100227","DOIUrl":null,"url":null,"abstract":"<div><div>Pyruvate dehydrogenase kinases (PDHKs) are non-canonical serine/threonine kinases that regulate the pyruvate dehydrogenase complex. Given their central role in metabolism, dysregulation of PDHKs has been linked with a broad variety of pathological conditions, such as cardiovascular disease, diabetes, lactic acidosis, and cancer. While there are many small molecule PDHK inhibitors, including several that have advanced into clinical development, no PDHK inhibitor has been approved for therapeutic use for any indication. Currently the field lacks well-characterized tool compounds that can probe PDHK biology and differentiate between PDHK isoforms. Moreover, disconnects between biochemical and cell-based assays have complicated efforts to understand the biological effect of inhibiting PDHK catalytic activity. To better understand how PDHK inhibitors function in cells, we have developed a cell-based assay using NanoBRET Target Engagement technology. Here, we describe the use of NanoBRET to evaluate binding at the PDHK ATP and lipoamide sites. Using these assays, we have profiled previously described PDHK inhibitors and demonstrated the ability of NanoBRET to distinguish between PDHK inhibitors with different mechanisms of action and to elucidate isoform selectivity.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100227"},"PeriodicalIF":2.7000,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"SLAS Discovery","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2472555225000206","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

Pyruvate dehydrogenase kinases (PDHKs) are non-canonical serine/threonine kinases that regulate the pyruvate dehydrogenase complex. Given their central role in metabolism, dysregulation of PDHKs has been linked with a broad variety of pathological conditions, such as cardiovascular disease, diabetes, lactic acidosis, and cancer. While there are many small molecule PDHK inhibitors, including several that have advanced into clinical development, no PDHK inhibitor has been approved for therapeutic use for any indication. Currently the field lacks well-characterized tool compounds that can probe PDHK biology and differentiate between PDHK isoforms. Moreover, disconnects between biochemical and cell-based assays have complicated efforts to understand the biological effect of inhibiting PDHK catalytic activity. To better understand how PDHK inhibitors function in cells, we have developed a cell-based assay using NanoBRET Target Engagement technology. Here, we describe the use of NanoBRET to evaluate binding at the PDHK ATP and lipoamide sites. Using these assays, we have profiled previously described PDHK inhibitors and demonstrated the ability of NanoBRET to distinguish between PDHK inhibitors with different mechanisms of action and to elucidate isoform selectivity.

Abstract Image

查看原文本刊更多论文

求助全文

约1分钟内获得全文求助全文

来源期刊

SLAS Discovery Chemistry-Analytical Chemistry

CiteScore

7.00

自引率

3.20%

发文量

审稿时长

39 days

期刊介绍： Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).