A Nonradioactive High-Throughput Screening-Compatible Cell-Based Assay to Identify Inhibitors of the Monocarboxylate Transporter Protein 1.

Solute carrier proteins (SLCs) are a superfamily of transmembrane transporters that control essential physiological functions such as nutrient uptake, ion transport, and cellular waste elimination. Although many SLCs are associated with various disease states and are considered "druggable," they remain underexplored as a drug target class. One subfamily of SLCs that has gained attention for its therapeutic potential is the monocarboxylate solute transporter family. The monocarboxylate transporter protein 1 (MCT1) is a passive transporter of lactate and has gained significant attention for its role(s) in cancer progression; moreover, upregulation of MCT1 connotes poor patient outcome and survival. Consequently, small molecule inhibitors of MCT1 activity are being pursued as anticancer therapies. However, typical for members of this SLC subfamily, there is a paucity of potent and selective modulators of MCT1. This is in part due to methods used for their identification, typically relying on the use of radiolabeled substrate tracing. In addition to the safety concerns associated with radioactivity, this methodology is also expensive and time consuming. In this study, we describe the use of an MCT1 cytotoxic substrate as a tool to enable the development of a nonradioactive cell-based homogeneous assay that facilitates industry-scale high-throughput screening (HTS) of large compound libraries to identify novel MCT1 inhibitors to interrogate the therapeutic potential of MCT1. Our assay is robust, reproducible, HTS amenable, and establishes a conceptually novel way to identify chemical probes to investigate the therapeutic potential of SLC proteins.

Measuring urinary N-acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine (IPMA3) as a potential biomarker of isoprene exposure.

Isoprene, the 2-methyl analog of 1,3-butadiene, is identified as a possible human carcinogen by the International Agency for Research on Cancer (IARC). Isoprene is ubiquitous in the environment with numerous natural and anthropogenic sources. Tobacco smoke is the main exogenous source of isoprene exposure in indoor environments. Among smoke constituents, isoprene is thought to contribute significantly to cancer risk; however, no selective urinary biomarkers of isoprene exposure have been identified for humans. In this manuscript, we measured the minor isoprene metabolite IPMA1 (mixture of N-acetyl-S-(1-[hydroxymethyl]-2-methyl-2-propen-1-yl)-L-cysteine and N-acetyl-S-(2-hydroxy-3-methyl-3-buten-1-yl)-L-cysteine), and we identified IPMA3 (N-acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine) as a major isoprene metabolite and novel isoprene exposure biomarker for humans. Urinary isoprene metabolites were measured using ultra high performance liquid chromatography coupled with electrospray ionization triple quad tandem mass spectrometry (UPLC/ESI-MSMS). The detection rates of IPMA1 and IPMA3 are <20% and 82%, respectively. The selectivity and abundance of IPMA3 make it a useful urinary biomarker of isoprene exposure. The limit of detection of IPMA3 in urine was 0.5 ng mL-1. IPMA3 was stable under different storage temperatures and following ten freeze-thaw cycles. The average recovery of urine spiked with IPMA3 at three different levels was 99%. IPMA3 was measured in urine samples received from 75 anonymous subjects; the median (25th percentile, 75th percentile) IPMA3 level in smokers was 36.2 (18.2, 56.8) ng mL-1 and non-smokers 2.31 (2.31, 4.38) ng mL-1. Application of this method to large population studies will help to characterize isoprene exposure and assess potential health impact.