Determination of inhibitory effects of coumarin derivatives on Bacteroides fragilis D-LDH enzyme

Bacteroides fragilis is an anaerobic bacteria naturally hosted in the human colon flora. B. fragilis D-lactate dehydrogenase (BfD-LDH) is an important enzyme which catalyzes the conversion of D-lactate to pyruvate and regulates anaerobic glycolysis. Recent studies show that B. fragilis has been associated with colon cancer. Antibiotic resistance has been reported worldwide against infections caused by enterotoxigenic strains of B. fragilis. Therefore, it is necessary to find new approaches to treating infections caused by resistant bacteria. In this thesis study, BfD-LDH is selected as a target for suppressing of infections caused by B. fragilis in the colon flora. BfD-LDH, that has been cloned and expressed by our laboratory group, is purified with affinity chromatography using Ni-NTA agarose column. SDS-PAGE analysis results show that high amounts of protein was obtained with purity of %95. Inhibitory activities of 29 previously synthetized coumarin derivatives, bearing hydroxyl, acetoxyl and methoxyl substitutions, on BfD-LDH were investigated. In the screening it was found those 15 coumarin derivatives were potently inhibited BfD-LDH with IC50 values lower than 10µM. For the coumarin derivatives with the highest inhibitory activity (K5, K21 and K23) inhibition mechanism and kinetic parameters of the enzymatic reaction were studied in detail. Enzyme inhibition mechanism of these compounds was determined as noncompetitive inhibition (Ki = 0.4532, 0.5108 and 0.3603 µM, respectively). For in silico analysis, chemicals were sketched and confermers were identified by ChemDraw v10 and MarvinSketch v6.2 programs. Docking of the coumarins was carried out using Molegro Virtual Docker program and interaction between the coumarin derivatives and active side residues of the enzyme were demonstrated. This study is a pioneer in the inhibition of BfD-LDH. High inhibitory activity of coumarin derivatives and the absence of the equivalent enzyme in human, give hope for future in vivo studies.