Evaluation of FASN inhibitors by a versatile toolkit reveals differences in pharmacology between human and rodent FASN preparations and in antiproliferative efficacy in vitro vs. in situ in human cancer cells.

Autor: Singha PK; School of Medicine Institute of Biomedicine Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Mäklin K; School of Medicine Institute of Biomedicine Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Vihavainen T; School of Medicine Institute of Biomedicine Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Laitinen T; School of Pharmacy Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Nevalainen TJ; School of Pharmacy Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Patil MR; School of Pharmacy Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Tonduru AK; School of Pharmacy Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Poso A; School of Pharmacy Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland; Department of Internal Medicine VIII, University Hospital Tuebingen, 72076, Tuebingen, Germany., Laitinen JT; School of Medicine Institute of Biomedicine Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland., Savinainen JR; School of Medicine Institute of Biomedicine Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland. Electronic address: juha.savinainen@uef.fi.
Jazyk: angličtina
Zdroj: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences [Eur J Pharm Sci] 2020 Apr 07; Vol. 149, pp. 105321. Date of Electronic Publication: 2020 Apr 07.
DOI: 10.1016/j.ejps.2020.105321
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest None.
(Copyright © 2020 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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