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Coronary artery disease (CAD) is one of the major causes of mortality worldwide. Recent genome-wide association studies have started to unravel the genetic architecture of the disease. Such efforts have identified Calcitonin receptor-like (CALCRL), an important mediator of the endothelial fluid shear stress response, associated with CAD risk variants. In this study we functionally characterized the non-coding regulatory elements carrying CAD risks SNPs and studied their role in the regulation ofCALCRLexpression in endothelial cells. We demonstrate that rs880890-harboring regulatory element exhibits high enhancer activity and significant allelic bias with A allele showing 40% more activity than G allele. We also observed that the A allele of rs880890 is favored over the G allele under shear stress. CRISPR deletion of rs880890-enhancer resulted in downregulation ofCALCRLexpression. EMSA further showed that heat shock factors are binding to the enhancer with a preference for A allele over the G allele. In line with this, HSF1 knockdown resulted in a significant decrease inCALCRLexpression.CALCRLknockdown as well as variant perturbation experiments confirmed the role of CALCRL in the regulation of eNOS, apelin, angiopoietin, prostaglandins and endothelin-1 signaling pathways while demonstrating a significant decrease in cell proliferation and tube formation. Overall, our results demonstrate the existence of an endothelial-specific heat shock factor regulated transcriptional enhancer carrying a CAD risk SNP rs880890 that regulatesCALCRLexpression. Better understanding ofCALCRLgene regulation and the role of SNPs in modulation ofCALCRLexpression could provide important steps towards understanding genetic regulation of shear stress signaling responses.

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.