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.

中文翻译：

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通过细胞热位移分析检测溶质载体蛋白的化学参与。

溶质载体（SLC）是跨膜蛋白，可跨细胞膜转运各种营养物质，代谢产物和药物。尽管SLC与细胞稳态，代谢和疾病状态相关，但对于大多数SLC，我们缺乏有关同源配体性质的实验证据，无论是内生菌还是异源菌。而且，即使对于已表征抑制剂的大约20种SLC，细胞中的结合分析也仅限于放射性标记或荧光探针的可及性。已引入细胞热转移测定（CETSA）作为一种强大的方法，通过监测配体诱导的细胞蛋白热稳定性变化来评估靶标结合。我们讨论了是否可以将CETSA修改为常规适用于SLC等膜转运蛋白的问题。我们以SLC16A1（MCT1）和SLC1A2（EAAT2）为目标，建立了可检测SLC的化学结合的稳定条件。使用免疫印迹，我们证明了用SLC16A1抑制剂AZD3965和AR-C155858处理可稳定HEK293细胞裂解液和完整细胞中的内源性SLC16A1。另外，SLC16A1的高亲和力配体l-乳酸酯和低亲和力的配体甲酸酯分别导致SLC16A1的强和弱稳定化。此外，我们观察到用选择性抑制剂WAY-213613治疗后SLC1A2的稳定性。我们建议，此处介绍的实验方法应普遍且容易适用于监测SLC在细胞环境中对化学配体的参与，从而帮助其脱孤。