Direct expression of CPT1a enables a high throughput platform for the discovery of CPT1a modulators

Jason Chen; Tuyen Tran; Anthony Wong; Luofei Wang; Pranavi Annaluru; Vibha Sreekanth; Samika Murthy; Laasya Munjeti; Tanya Park; Utkarsh Bhat; Glynnis Leong; Y Li; Simeng Chen; Natalie Kong; Rushika Raval; Yining Xie; Shreya Somani; Aditi Manohar Bhambhani; Zoey Zhu; Landen Chu; Kimai Dosch; Edward Njoo; Zhan Chen

doi:10.3390/applbiosci4020025

BiochemistryApplied BiosciencesMay 12, 2025

Peer-reviewed journal article

Direct expression of CPT1a enables a high throughput platform for the discovery of CPT1a modulators

Jason Chen, Tuyen Tran iD, Anthony Wong iD, Luofei Wang iD, Pranavi Annaluru iD, Vibha Sreekanth iD, Samika Murthy iD, Laasya Munjeti iD, Tanya Park iD, Utkarsh Bhat iD, Glynnis Leong iD, Y Li iD, Simeng Chen iD, Natalie Kong iD, Rushika Raval iD, Yining Xie iD, Shreya Somani iD, Aditi Manohar Bhambhani iD, Zoey Zhu iD, Landen Chu iD, Kimai Dosch iD, Edward Njoo iD, Zhan Chen iD

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Abstract

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. 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/628575v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f69f0corg.highwire.dtl.DTLVardef@43eb95org.highwire.dtl.DTLVardef@11fa21corg.highwire.dtl.DTLVardef@1cbe405_HPS_FORMAT_FIGEXP M_FIG C_FIG