Balan, M. | Search bioRxiv

bioRxiv2026

Comparative profiling of carnitine palmitoyltransferase 1 isoforms reveals vincamine as a selective carnitine palmitoyltransferase 1b inhibitor

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the rate-limiting step of fatty acid oxidation and has emerged as a therapeutic target for metabolic diseases and cancer. CPT1 exists in three isoforms, CPT1a, CPT1b, and CPT1c, with distinct tissue distributions and enzymatic properties; however, limitations of previous platforms enabling parallel isoform comparison have undermined efforts to identify selective inhibitors that could minimize off-target effects. Here, we describe a DTNB-based enzyme activity assay adapted for high-throughput screening of CPT1b, the predominant isoform in cardiac and skeletal muscle. Mitochondrial extracts from Expi293F cells transfected with CPT1a or CPT1b expression plasmids served as sources of catalytically active enzymes. The assay was validated using three previously confirmed CPT1b inhibitors: (R)-(+)-etomoxir, perhexiline, and malonyl-CoA. We then generated side-by-side inhibitory profiles for both isoforms, identifying vincamine as a lead selective inhibitor of CPT1b. Furthermore, chlorpromazine, previously characterized only as a broad CPT1 inhibitor and subsequently shown to inhibit CPT1a, is demonstrated here to also inhibit CPT1b, expanding its known isoform profile. Together, these results establish a robust platform for comparative isoform profiling and demonstrate that selective modulation of CPT1b is achievable, with implications for targeted therapeutics in metabolic and oncological disease.

cell biology↗

bioRxiv2022

Chemical and genetic screens identify new regulators of tetracycline-inducible gene expression system in mammalian cells

The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line which allows for inducible expression of an EGFP-fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated doxycyclinedependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase or TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. Together, these results suggest that none of the medically approved drugs significantly mitigates TDP-43 toxicity, identify new chemical and genetic regulators of the tetR system, and raise awareness on the limitations of this approach to conduct chemical or genetic screenings in mammalian cells.

molecular biology↗