Key result
A scalable colorimetric assay using mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid successfully validated known inhibitors and identified chlorpromazine as a CPT1 inhibitor.
Why the study?
Existing methods to quantify CPT1 activity are not amenable to scalable, high-throughput screening for characterizing small molecule inhibitors.
The development of a scalable, high-throughput colorimetric assay using Expi293 cell extracts enables efficient screening and discovery of novel CPT1a modulators.
Enables scalable CPT1a inhibitor screening; leaves open clinical translation pending in vivo validation.
Carnitine palmitoyltransferase 1 (CPT1), which catalyzes the rate-limiting step of fatty acid oxidation, has been implicated in therapeutic approaches to several human diseases characterized by aberrant lipid metabolism. The isoform-specific quantification of CPT1 activity is essential in the characterization of small molecule inhibitors of CPT1, but several existing means to quantify enzymatic activity, including the use of radioisotope-labeled carnitine, are not amenable to scalable, high throughput screening. Here, we demonstrate that mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid are a reliable and robust source of catalytically active human CPT1. Moreover, with a source of catalytically active enzyme in hand, we modified a previously reported colorimetric method of coenzyme A (CoA) easily scalable to a 96-well format for the screening of CPT1a inhibitors. This assay platform was validated by two previously reported inhibitors of CPT1a: R-etomoxir and perhexiline. To further demonstrate the applicability of this method in small molecule screening, we prepared and screened a library of 87 known small molecule APIs, validating the inhibitory effect of chlorpromazine on CPT1.
No takes yet. Share an insight, caveat, or question.
Tran et al. (2025) studied this question. High throughput screening platform for CPT1a modulators was evaluated on Quantification of CPT1a enzymatic activity and screening of inhibitors. A scalable colorimetric assay using mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid successfully validated known inhibitors and identified chlorpromazine as a CPT1 inhibitor.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: