1 research outputs found
Detection of Chemical Engagement of Solute Carrier Proteins by a Cellular Thermal Shift Assay
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 low-affinity ligand, formate,
resulted in strong and weak stabilization of SLC16A1, respectively.
Moreover, we observed stabilization of SLC1A2 upon treatment with
the selective inhibitor WAY-213613. We propose that the experimental
approach presented here should be generally and easily applicable
for monitoring the engagement of chemical ligands by SLCs in cellular
settings and thus assisting in their deorphanization