Detection of Chemical Engagement of Solute Carrier Proteins by a Cellular Thermal Shift Assay

ACS Chemical Biology
Mari HashimotoGiulio Superti-Furga

Abstract

Solute carriers (SLCs) are transmembrane proteins that transport various nutrients, metabolites, and drugs across cellular membranes. Despite the relevance of SLCs to cell homeostasis, metabolism, and disease states, for the majority of SLCs we lack experimental evidence regarding the nature of the cognate ligands, whether endobiotic or xenobiotic. Moreover, even for the roughly 20 SLCs for which inhibitors have been characterized, engagement assays in cells are limited to the accessibility of radiolabeled or fluorescent probes. The cellular thermal shift assay (CETSA) has been introduced as a powerful method to assess target engagement by monitoring ligand-induced changes in the thermal stability of cellular proteins. We addressed the question of whether CETSA could be modified to become routinely applicable to membrane transporters such as SLCs. We used SLC16A1 (MCT1) and SLC1A2 (EAAT2) as targets to establish robust conditions by which chemical engagement of SLCs can be detected. Using immunoblotting, we demonstrate that treatment with the SLC16A1 inhibitors AZD3965 and AR-C155858 stabilized endogenous SLC16A1 in HEK293 cell lysates as well as intact cells. In addition, the high-affinity ligand of SLC16A1, l-lactate, and the...Continue Reading

References

May 23, 2001·Progress in Neurobiology·N C Danbolt
Jan 15, 2002·Journal of Biomolecular Screening·M W PantolianoF R Salemme
Jul 22, 2004·The Journal of Biological Chemistry·Sandra GendreauChristoph Fahlke
Mar 13, 2008·Structure·Alexander I AlexandrovRaymond C Stevens
Mar 2, 2010·Nature Reviews. Drug Discovery·UNKNOWN International Transporter ConsortiumLei Zhang
Jul 12, 2012·Nature Biotechnology·Heesun CheongCraig B Thompson
Aug 30, 2012·Nature Methods·Caroline A SchneiderKevin W Eliceiri
Nov 10, 2012·Transplant International : Official Journal of the European Society for Organ Transplantation·Clara PåhlmanHenrik Ekberg
Mar 20, 2013·Molecular Aspects of Medicine·Matthias A HedigerElspeth A Bruford
Nov 28, 2013·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Radosław PolańskiChristopher J Morrow
Aug 8, 2014·Nature Protocols·Rozbeh JafariDaniel Martinez Molina
Oct 4, 2014·Science·Mikhail M SavitskiGerard Drewes
Dec 6, 2014·Nature Reviews. Drug Discovery·Sanjay K Nigam
Jun 27, 2015·Nature Reviews. Drug Discovery·Lawrence LinKathleen M Giacomini
Aug 2, 2015·Cell·Adrián César-RazquinGiulio Superti-Furga
Sep 20, 2015·Protein Engineering, Design & Selection : PEDS·Y AshokV-P Jaakola
Sep 22, 2015·Nature Methods·Kilian V M HuberGiulio Superti-Furga
Nov 3, 2015·Nature Methods·Friedrich B M ReinhardGerard Drewes

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Citations

Mar 7, 2020·Molecular Systems Biology·André MateusMikhail M Savitski
Mar 27, 2020·Annual Review of Biochemistry·M B RobersK V Wood
Nov 2, 2019·SLAS Discovery·Brinton Seashore-LudlowThomas Lundbäck
Apr 9, 2020·Frontiers in Cellular and Infection Microbiology·Swati GargShailja Singh
Nov 28, 2019·Journal of Medicinal Chemistry·Wesley WangChristopher G Parker
Jan 18, 2019·ACS Chemical Biology·Justin M ThomasJordan L Meier
Jul 23, 2019·ACS Chemical Biology·Aarti KawatkarM Paola Castaldi
Aug 28, 2021·Frontiers in Pharmacology·Vojtech DvorakGiulio Superti-Furga

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Methods Mentioned

BETA
thermal shift
glycosylation
chemical treatments
protein assay

Software Mentioned

GraphPad
ImageJ
GraphPad Prism

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