Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.
Nitroarylmethyl quaternary (NMQ) ammonium salts have potential as prodrugs for enzymatic or radiolytic reduction to release amine effectors under hypoxia. Earlier studies demonstrated one-electron release of the cytotoxic amine mechlorethamine (HN2) from 4-nitroimidazolyl and 2-nitropyrrolyl NMQ prodrugs (but not from nitrobenzyl analogs) through intramolecular electron transfer. In this study we determined whether this is a general feature of heterocyclic NMQ prodrugs of HN2 and examined the reductive pathways in detail using pulse and steady-state radiolysis. The kinetics of radical fragmentation varied by more than four orders of magnitude, independently of the one-electron reduction potential, within the series of eight nitroheterocycles examined. In addition to the compounds identified previously, new 2-nitropyrrole and 3-nitrothiophene NMQ prodrugs were found to provide efficient HN2 release (G > 0.5 micromol/J in anoxic formate buffer). However, the nitrothiophene was sensitive to nucleophilic displacement of HN2, making it less promising. Product analysis by HPLC/mass spectrometry identified symmetrical dimers arising from benzyl-type radical intermediates but also demonstrated that these dimers are not reliable markers for the intramolecular fragmentation of the initial nitro radical anion. This study elucidated multiple competing pathways for reductive fragmentation of NMQ prodrugs and identified the preferred electron acceptors for use in the development of analogs that release more potent cytotoxins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.