Structure based de novo design of IspD inhibitors as anti-tubercular agents

Tuberculosis is one of the leading contagious diseases, caused by Mycobacterium tuberculosis. Despite improvements in anti-tubercular agents, it remains one of the most prevalent infectious diseases worldwide, responsible for a total of 1.6 million deaths annually. The emergence of multidrug resistant strains highlighted the need of discovering novel drug targets for the development of anti-tubercular agents. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) is an enzyme involved in MEP pathway for isoprenoid biosynthesis, which is considered an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. In the present study, we have employed structure based drug design approach to develop novel and potent inhibitors for IspD receptor. To explore binding affinity and hydrogen bond interaction between the ligand and active site of IspD receptor, docking studies were performed. ADMET and synthetic accessibility filters were used to screen designed molecules. Finally, ten compounds were selected and subsequently submitted for the synthesis and in vitro studies as IspD inhibitors

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation

A hydroquinone-specific screening system for directed P450 evolution

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). © 2018, The Author(s)

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

1.5Full digital generat a partir de la base topogràfica 1:5 000. Els fulls d'aquesta sèrie corresponen a la divisió 4 x 4 de la malla de distribució del Mapa topográfico nacional de España 1:50 000. Cada full inclou 2 finestres (Mapa índex de la sèrie; Mapa guia). - Projecció Universal Transversa Mercator (UTM), fus 31, sobre el·lipsoide internacional i datum europeu. Equidistància de les corbes de nivell: 5 m.Imatge digital de 90 x 67 cm1:5 000300 PP

3D-QSAR and molecular docking analysis of (4-piperidinyl)-piperazines as acetyl-CoA carboxylases inhibitors

Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme, which plays a vital role in fatty acid metabolism and obesity induced type 2 diabetes. Herein, we have performed 3D-QSAR and molecular docking analysis on a novel series of (4-piperidinyl)-piperazines to design potent ACC inhibitors. This study correlates the ACC inhibitory activities of 68 (4-piperidinyl)-piperazine derivatives with several stereo-chemical parameters representing steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The CoMFA and CoMSIA models exhibited excellent rncv2 values of 0.974 and 0.985, and rcv2 values of 0.671 and 0.693, respectively. CoMFA predicted rpred2 of 0.910 and CoMSIA predicted rpred2 of 0.963 showed that the predicted values were in good agreement with experimental values. Glide5.5 program was used to explore the binding mode of inhibitors inside the active site of ACC. We have accordingly designed novel ACC inhibitors by utilising the LeapFrog and predicted with excellent inhibitory activity in the developed models

The calculated enthalpies of the nine pyrazole anions, cations, and radicals: a comparison with experiment

Enthalpies of 12 pyrazole species including neutral, anions, cations, and radicals have been calculated at the G3B3 level. The main conclusions are: (i) there are ten equilibria between species of which six have been measured experimentally and the agreement is excellent; (ii) two structures, cyclic and chain, have been found for the pyrazolium-radical 8 that are able to explain the electrochemistry of pyrazolium salts; (iii) the aromaticity, calculated as the NICS indexes, is related to the unexpected stability of the pyrazole anion 3. © 2006 Elsevier Ltd. All rights reserved.Peer Reviewe

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation