pyrimidinones and formic-acid

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are genotoxic DNA lesions and mainly generated on thymine-thymine (T-T) dinucleotides upon UV irradiation. Regarding the sensitivity, specificity, and accuracy of analytical methods, it is of first choice to develop a reliable assay for simultaneous detection of these DNA lesions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the dilemma is the low detection sensitivity of the phosphate-containing dimeric photoproducts even using most favorable negative-ion mode for LC-MS/MS analysis. Unexpectedly, we observed that the detection sensitivity of T-T CPD and 6-4PP could be significantly improved using formic acid/acetic acid (∼ppm) as an additive of the mobile phase for reversed-phase LC-MS/MS analysis. This is the first demonstration of the enhancement of LC-MS/MS signals by formic acid/acetic acid in negative-ion mode. Of note, these acidic agents are often used for positive-ion mode in LC-MS assays. Benefited from the developed method, we could quantify both T-T CPD and 6-4PP in mouse embryonic stem cells upon UVC irradiation at low dosage. This sensitive method is applicable to the screening and identification of genes involved in formation, signaling, and repair of UV lesion.

Topics: Animals; Cells, Cultured; Chromatography, Liquid; DNA; DNA Damage; Formates; Mice; Molecular Structure; Photochemical Processes; Pyrimidine Dimers; Pyrimidinones; Tandem Mass Spectrometry; Ultraviolet Rays

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.

Topics: Biological Assay; Excitatory Amino Acid Transporter 2; Formates; Glutamate Plasma Membrane Transport Proteins; Heating; HEK293 Cells; Humans; Lactic Acid; Ligands; Monocarboxylic Acid Transporters; Protein Binding; Protein Stability; Pyrimidinones; Thiophenes; Uracil