For the implementation of the Chemical Weapons Convention, the Organisation for Prohibition of Chemical Weapons (OPCW) designates laboratories for the analysis of chemicals that can be used as chemical warfare agents (CWAs). In these laboratories, analytical methods for detecting CWAs have been developed for environmental and biomedical samples. Protein adducts from exposed biomedical samples are an important type of biomarker for verification of their abuse. Moreover, these adducts could also be applied in chemical impurity profiling studies of biomedical samples. Alkylating agents, as cytotoxicants, can react with Cys 34 in human serum albumin, and cysteine-proline-phenylalanine (CPF) tripeptide adducts generated from proteinase K digestion of modified albumin have been used as biomarkers for retrospective detection of exposure to sulfur mustards or nitrogen mustards. In this study, a sensitive screening method for detecting exposure to 14 alkylating agents in one analytical run was established through sample preparation and instrumental detection optimization. Ultrahigh-performance liquid chromatography–triple quadrupole mass spectrometry (UHPLC–TQ MS) operated in multiple reaction monitoring (MRM) mode and combined with an optimized chromatographic program was used for detecting the 14 corresponding CPF tripeptide adducts. The limits of detection (LODs) are in the range of 0.200–10.0 ng/mL exposure concentrations in human plasma. This method could also allow impurity profiling applicable for potential attribution of sulfur and nitrogen mustard in plasma through retrospective analysis of exposed biomedical samples with impurity compositions as low as 0.1%. This method has potential in clinical diagnosis, CWA verification and forensic identification applications. Graphical
Nicotinamide adenine dinucleotide (NAD) is essentially involved in many biological processes of cancer cells, yet chemical intervention of NAD biosynthesis failed to obtain an optimal therapeutic benefit. We herein developed a new strategy to induce catastrophic NAD depletion by concurrently impairing NAD synthesis and promoting NAD consumption. We designed a series of new compounds that conjugate an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD salvage pathway, with a DNA-alkylating agent. Among them, compound 11b exhibited potent anticancer efficacy in cancer cell lines and mouse tumor models with intrinsic resistance to the parent compound FK866 or chlorambucil. Compound 11b caused catastrophic NAD depletion via a synergistic effect between the NAD salvage pathway blockade and DNA damage-triggered NAD consumption. Our findings suggest a new intervention strategy for causing catastrophic NAD depletion in cancer cells and provide basis for the development of new inhibitors targeting NAD metabolism.
DNA alkylating agents are widely used in anticancer pharmacology. Although shown to induce cross-linking and/or methylation of DNA, how they affect the mechanical properties of DNA and activity of DNA enzymes remains to be elucidated. Here, we perform single-molecule optical tweezer experiments on DNA treated with alkylating agents, including melphalan, cisplatin, and dacarbazine. While all three drugs induce a significant increase of overstretching force and a reduction of hysteresis, suggesting stabilization of DNA against shearing forces, their effects on elasticity of DNA were quite different, with the largest change in persistence length induced by cisplatin. Furthermore, we find that these alkylating-agent-induced changes on DNA have different effects on processivity of DNA polymerase, with melphalan and cisplatin showing significantly reduced activity and dacarbazine showing little effect. Overall, our results provide new insights into the effects for these alkylating agents, which could potentially facilitate a better design of related drugs.
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