Development of a novel CPT1 enzymatic assay for drug screening. Synthesis and evaluation of UB006 derivatives and flavonoids’ effect on lipid

Abstract

[eng] Lipid metabolism (biosynthesis, storage, and mobilization of lipids within an organism) is a very important aspect of energy homeostasis and plays an essential. Obesity is a complex multifactorial disease characterized by sustained positive energy intake relative to expenditure leading to excessive accumulation of fat in adipose tissue, which poses significant health risks. Cancer cells also present alterations in lipid anabolism consequence of a metabolic reprogramming. This enables the cells to meet their high energy demands and building blocks necessary for rapid proliferation. CPT1 and FAS are key enzymes in lipid metabolism. The former is the rate-limiting step in fatty acid oxidation and the latter is essential for de novo fatty acid biosynthesis. Therefore, they have been object of study and pharmacological intervention with small molecules. In this work, we have (1) developed a novel CPT1 enzymatic assay, aiming to provide a screening tool for potential new CPT1 modulators. (2) We have also synthesized and tested new derivatives of (±)-UB006, a known FAS inhibitor without CPT1 effect. (3) Finally, we evaluated the effect of several flavonoids on lipid accumulation and browning of white adipocytes. Our results in the first chapter validated a CPT1 enzymatic activity assay suitable for LCMS quantification, using 1/4th of reagents per sample and up to 6 times more the number of samples per experiment compared to the radiometric assay currently used. This method was responsive to known CPT1 inhibitors and was applied to a variety of mouse tissues’ mitochondria-enriched fractions. The assay was also successfully applied to cell culture and ex vivo muscular fibres, allowing evaluation of CPT1 activity in different metabolic states and the study of known inhibitors. In the second chapter we synthesized six (±)-UB006 derivatives by modifying the lactone’s β-side chain. (±)-UB031 and (±)-UB035 improved the cytotoxicity profile against the OVCAR-3 cell line compared to (±)-UB006. (±)-UB035 and (±)-UB036 displayed at least 4-times higher solubility and comparable FAS inhibition profiles to (±)-UB006, despite (±)-UB035 showing higher cytotoxicity in OVCAR-3 cells. In the third chapter, we found that chrysin did not induce an mRNA expression profile of genes related to beiging such as Ucp1, Pparγ, or Pgc-1α in 3T3-L1 cells. Of the flavonoids tested, compound C increased overall lipid accumulation while baicalein inhibited differentiation to adipocytes of 3T3-L1 cells. In C. elegans, baicalein reduced lipid accumulation during larval development. In conclusion, we have a flexible CPT1 enzymatic assay that can be applied to mitochondria-enriched fractions, cell culture, and ex vivo muscular fibres. We have synthesized a more potent and soluble (±)-UB006 derivative, (±)-UB035. Finally, flavonoids have failed to induce beiging or affect lipid accumulation directly except baicalein. However, the effects observed in living organisms suggests that other mechanisms might be in play.