Computational Screening Strategy for Drug Repurposing Identified Niclosamide as Inhibitor of Vascular Calcification.

Takeshi Tanaka,Masanori Aikawa,Takaharu Asano,Sasha A Singh,Takehito Okui,Shiori Kuraoka,Elena Aikawa

doi:10.3389/fcvm.2021.826529

Abstract

Vascular calcification is a cardiovascular disorder with no therapeutic options. We recently reported that o-octanoyltransferase (CROT) suppression can inhibit vascular calcification in vivo and in vitro through amelioration of mitochondrial function and fatty acid metabolism. Inhibiting calcification with a small molecule compound targeting CROT-associated mechanisms will be a promising non-invasive treatment of vascular calcification. Here we used a computational approach to search for existing drugs that can inhibit vascular calcification through the CROT pathway. For screening of the compounds that reduce CROT expression, we utilized the Connectivity Map encompassing the L1000 computational platform that contains transcription profiles of various cell lines and perturbagens including small molecules. Small molecules (n = 13) were identified and tested in human primary smooth muscle cells cultured in osteogenic media to induce calcification. Niclosamide, an FDA-improved anthelmintic drug, markedly inhibited calcification along with reduced alkaline phosphatase activity and CROT mRNA expression. To validate this compound in vivo, LDL receptor (Ldlr)-deficient mice fed a high fat diet were given oral doses of niclosamide (0 or 750 ppm admixed with diet) for 10 weeks. Niclosamide treatment decreased aortic and carotid artery calcification as determined by optical near infrared molecular imaging (OsteoSense680) and histological analysis. In addition, niclosamide improved features of fatty liver, including decreased cholesterol levels along with decreased Crot expression, while plasma total cholesterol levels did not change. Proteomic analysis of aortic samples demonstrated that niclosamide affected wingless/integrated (Wnt) signaling pathway and decreased runt-related transcription factor 2 (Runx2) expression, an essential factor for calcification. Our target discovery strategy using a genetic perturbation database with existing drugs identified niclosamide, that in turn inhibited calcification in vivo and in vitro, indicating its potential for the treatment of vascular calcification.

Highlights

Cardiovascular disease is a primary cause of death globally
The remaining 13 candidates were assessed for their ability to decrease carnitine o-octanoyltransferase (CROT) mRNA expression and suppress calcium deposition in human smooth muscle cells (hSMCs) cultured in osteogenic media (OM)
We employed a computational approach to screen small molecules that could decrease the expression of CROT, a novel target for inhibition of vascular calcification previously reported by our group [4]

Summary

Introduction

Vascular calcification, characterized as deposition of hydroxyapatite in the arterial wall, is a major contributor to cardiovascular disease [1, 2]. We recently reported carnitine o-octanoyltransferase (CROT) as a candidate target to suppress vascular calcification [4]. Small interfering RNA (siRNA)mediated suppression of CROT in hSMCs and genetic deletion of Crot in mice inhibited vascular calcification. Additional proteomics, lipidomics and network analysis studies deduced that CROT inhibition suppresses calcification potential by ameliorating mitochondrial function and fatty acid metabolism [4]. This recent study suggests CROT as a promising target, it is only the beginning of multiple steps toward confirming CROT inhibition is a viable therapy for cardiovascular calcification. Small molecule or compound inhibitor screens typically follow the target discovery stage; conventional screening assays face many challenges including escalating costs and excessive amount of time required for drug development [5]

Methods

Results

Conclusion