ABSTRACT
Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC50 of 3.5 µM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.
Subject(s)
DNA Gyrase/metabolism , Topoisomerase II Inhibitors/pharmacology , Biocatalysis , Dose-Response Relationship, Drug , Escherichia coli/enzymology , Molecular Structure , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemical synthesis , Topoisomerase II Inhibitors/chemistryABSTRACT
BACKGROUND: The aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an endogenous neurotoxin implicated in Parkinson's Disease. Elucidating protein targets of DOPAL is essential in understanding it's pathology. The enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of DOPAL. METHODS: GAPDH activity was measured via reduction of NAD+ cofactor (340 nm). Protein aggregation was assessed with SDS-PAGE methods and specific modification via chemical probes. RESULTS: Low micromolar levels of DOPAL caused extensive GAPDH aggregation and irreversibly inhibited enzyme activity. The inactivation of GAPDH was dependent on both the catechol and aldehyde moieties of DOPAL. It is suggested that Cys are modified and oxidized by DOPAL. CONCLUSIONS: The mechanism by which DOPAL modifies GAPDH can serve as a mechanistic explanation to the pathological events in Parkinson's Disease.
