Synthesis of biotinylated-LPG as a chemical biology tool enabling discovery of ALCAT1 modulators

Abstract

As a mitochondrial signature phospholipid, cardiolipin (CL) is required for membrane structure, respiration, dynamics, fragmentation, and mitophagy. Alteration of CL by reactive oxygen species (ROS) can cause mitochondrial dysfunction, which is implicated in the pathogenesis of many diseases. The enzyme ALCAT1 (acyl-CoA: lysocardiolipin acyltransferase-1) facilitates the conversion of CL by incorporating polyunsaturated fatty acids into lysocardiolipin. Accumulating evidence suggests that overexpression of ALCAT1 is involved in pathological cardiolipin remodeling and mitochondrial bioenergetics. Few ALCAT1 modulators are reported in the literature, and the enzymatic activity was tested via a low-throughput TLC (thin layer chromatography) assay. To identify small molecule ALCAT1 inhibitors, a robust assay was needed to enable a full deck high throughput screen. Scintillation proximity assay (SPA) was the method of choice because it permits the rapid and sensitive measurement of a broad range of biological processes in a homogeneous system. A biotinylated ALCAT1 substrate was required as a chemical biology tool in developing SPA. Among a panel of phospholipids, lysophosphatidyl glycerol (LPG) was identified as the best substrate for ALCAT1. Herein we report the synthesis of biotinylated-LPG analogs with varied linker lengths and their activity towards ALCAT1.