Cloning, Purification, and Biophysical Characterization of FemB Protein from Methicillin-Resistant Staphylococcus aureus and Inhibitors Screening.

Methicillin-resistant Staphylococcus aureus has emerged as a leading cause of nosocomial, community acquired infections worldwide. Earlier investigations revealed that mecA-encoded FEM proteins play a role in antimicrobial resistance by developing unique peptidoglycan cross-linking which helps in the formation of protective cell membrane. In view to this, present study focused on expression, purification FEM proteins, and FemB biophysical characterization with the aid of in silico and in vitro approaches. Furthermore, we carried out biological screening assays and identified the novel potent 1,2,3-triazole conjugated 1,3,4-oxadiazole hybrid molecule which could inhibit the MRSA than the proven oxacillin.

14,15-Epoxyeicosa-5,8,11-trienoic Acid (14,15-EET) surrogates: carboxylate modifications.

The cytochrome P450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a powerful endogenous autacoid that has been ascribed an impressive array of physiologic functions including regulation of blood pressure. Because 14,15-EET is chemically and metabolically labile, structurally related surrogates containing epoxide bioisosteres were introduced and have become useful in vitro pharmacologic tools but are not suitable for in vivo applications. A new generation of EET mimics incorporating modifications to the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase (sEH). Tetrazole 19 (ED50 0.18 µM) and oxadiazole-5-thione 25 (ED50 0.36 µM) were 12- and 6-fold more potent, respectively, than 14,15-EET as vasorelaxants; on the other hand, their ability to block sEH differed substantially, i.e., 11 vs >500 nM. These data will expedite the development of potent and specific in vivo drug candidates.

14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates containing epoxide bioisosteres: influence upon vascular relaxation and soluble epoxide hydrolase inhibition.

All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED(50) 1.7 microM) to 14,15-EET as vasorelaxants but were approximately 10-35 times less potent as sEH inhibitors (IC(50) 59 and 19 microM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED(50) 3.5 microM, IC(50) 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.

Arachidonic acid inhibits basolateral K channels in the cortical collecting duct via cytochrome P-450 epoxygenase-dependent metabolic pathways.

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on basolateral 18-pS K channels in the principal cell of the cortical collecting duct (CCD) of the rat kidney. Application of AA inhibited the 18-pS K channels in a dose-dependent manner and 10 microM AA caused a maximal inhibition. The effect of AA on the 18-pS K channel was specific because application of 11,14,17-eicosatrienoic acid had no effect on channel activity. Also, the inhibitory effect of AA on the 18-pS K channels was abolished by blocking cytochrome P-450 (CYP) epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) but was not affected by inhibiting CYP omega-hydroxylase or cyclooxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase-dependent metabolites was further supported by the observation that application of 100 nM 11,12-epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18-pS K channels. In contrast, addition of either 5,6-, 8,9-, or 14,15-EET failed to inhibit the 18-pS K channels. Moreover, application of 11,12-EET was still able to inhibit the 18-pS K channels in the presence of MS-PPOH. This suggests that 11,12-EET is a mediator for the AA-induced inhibition of the 18-pS K channels. We conclude that AA inhibits basolateral 18-pS K channels by a CYP epoxygenase-dependent pathway and that 11,12-EET is a mediator for the effect of AA on basolateral K channels in the CCD.