Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal exchange reporters (IERs), which directly monitor changes to exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical exchange correction, thereby improving reproducibility.
ID 16894 Poster Board 460Benzalkonium chlorides (BACs) are widely used antimicrobials in a variety of consumer products and settings, including disinfectants, medical products, and food-processing industry. Though initially recognized as safe, the FDA has called for additional safety data on BAC use in consumer products. Their toxicity becomes especially important during the COVID-19 pandemic due to the increased use of BAC-containing disinfectant products. BAC exposure in rats led to their accumulation primarily in the kidney (42fold higher than blood); in contrast, accumulation did not occur in the liver, likely due to efficient metabolism of BACs by hepatic cytochromes P450 (CYPs), mainly CYP4F isoforms and CYP2D6. Thus, we hypothesized that BAC-metabolizing capacity determines the extent of the buildup of BACs in the kidney and subsequent kidney injury. Human kidney and liver microsomes were incubated with 1 mM C12-BAC over 8 time points between 0 and 64 minutes, and LC-MS analysis showed C12-BAC was consumed much slower in the kidney microsomes than the liver microsomes, with the half-lives being 50 and 2.5 min, respectively. We then used cell viability assays to investigate the nephrotoxicity of C10-, C12-, C14-and C16-BAC and their v-OH metabolites ranging from 0-40 mM over 48 hours in 2D-cultured human proximal tubule epithelial cells (PTECs). In general, the parent compounds displayed much larger cytotoxicity (at least 7-17-fold) than their v-OH metabolites, suggesting BAC metabolism by CYPs detoxifies BACs. EC 50 for C12-BAC is approximately 3 mM while the EC50 for the C14-and C16-BACs is around 1 mM. The EC 50 value for C10-BAC was not captured in the concentration range examined. BAC-metabolizing CYP4F11 and CYP4F12 are expressed in the kidney, but administration of a CYP4F inhibitor, HET0016, to inhibit the metabolism of BACs did not significantly increase the cytotoxicity of parent BACs, suggesting that BAC metabolism by the kidney is not sufficient to detoxify them. We then examined the nephrotoxicity of BACs using a kidney-on-a-chip microphysiological system (MPS), in which the microenvironment of the chip mimics the functional characteristics of human proximal tubules. We found that 1 mM of C14-BAC exerts potent toxicity to human PTECs in the kidney MPS system, compared to the untreated channel. However, kidney injury molecule 1 (KIM-1) displayed no difference between the C14-BAC and untreated channels, suggesting an alternative cell death mechanism by BACs. To summarize, our data suggest that the kidney has smaller BAC-metabolizing capacities than the liver and that BACs can potentially cause nephrotoxicity in humans.
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