Prabu Manoharan

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables (X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

Discovery of Safe and Orally Effective 4-Aminoquinaldine Analogues as Apoptotic Inducers with Activity against Experimental Visceral Leishmaniasis

ABSTRACT Novel antileishmanials are urgently required to overcome emergence of drug resistance, cytotoxic effects, and difficulties in oral delivery. Toward this, we investigated a series of novel 4-aminoquinaldine derivatives, a new class of molecules, as potential antileishmanials. 4-Aminoquinaldine derivatives presented inhibitory effects on L. donovani promastigotes and amastigotes (50% inhibitory concentration range, 0.94 to 127 μM). Of these, PP-9 and PP-10 were the most effective in vitro and demonstrated strong efficacies in vivo through the intraperitoneal route. They were also found to be effective against both sodium antimony gluconate-sensitive and -resistant Leishmania donovani strains in BALB/c mice when treated orally, resulting in more than 95% protection. Investigation of their mode of action revealed that killing by PP-10 involved moderate inhibition of dihydrofolate reductase and elicitation of the apoptotic cascade. Our studies implicate that PP-10 augments reactive oxygen species generation, evidenced from decreased glutathione levels and increased lipid peroxidation. Subsequent disruption of Leishmania promastigote mitochondrial membrane potential and activation of cytosolic proteases initiated the apoptotic pathway, resulting in DNA fragmentation and parasite death. Our results demonstrate that PP-9 and PP-10 are promising lead compounds with the potential for treating visceral leishmaniasis (VL) through the oral route.

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property

More than 100 years of research on Alzheimer’s disease didn’t yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein–ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model’s ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.

Rationalizing lead optimization by consensus 2D- CoMFA CoMSIA GRIND (3D) QSAR guided fragment hopping in search of γ -secretase inhibitors

Abstractγ-Secretase (Gamma Secretase) is a potential drug target in Alzheimer’s disease therapeutics. A sequel lead design study was undertaken on a series of bicyclononanes with an aim of identifying potent isofunctional chemotypes. Fragment-based bioisosteric replacement, which considers shape, chemistry, and electrostatics was carried out to mine over four million medicinally relevant fragments of Brood database. The resulting subset, thus, obtained was further mined using consensus QSAR developed from 2D and CoMFA, CoMSIA, GRIND (3D) QSAR predicted endpoints with superior statistical results. The employed consensus prediction and the predicted endpoint values were found to be in good agreement with the experimental values. The predictive ability of the generated model was validated using different statistical metrics, and similarity-based coverage estimation was carried out to define applicability boundaries. Few analogs designed, using the concept of bioisosterism, were found to be promising and could be considered for synthesis and subsequent screening.

Protein-bound fragment based virtual screening (PFVS) approach to identify potential lead fragments as BACE1 inhibitors

P1-g17 The effects of electrical stimulation of dorsal raphe nucleus on neuronal response properties of layer IV of barrel cortex following long-term sensory deprivation Hamid Sheikhkanloui-Milan 1,2 , Vahid Sheibani 1, Saeed Esmaeili-Mahani 3, Ali Shamsizadeh 4, Golamreza Sepehri 1, Mohammadreza Afarinesh 1 1 Kerman Neuroscience Research Center (KNRC), Kerman, Iran 2 Department of Physiology, School of Medicine, Ardebil University of Medical Sciences, Ardebil, Iran 3 Department of Biology, Faculty of Sciences, Shahid Bahonar University, Kerman, Iran 4 Department of Physiology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran

Stigmatellin Y – An anti-biofilm compound from Bacillus subtilis BR4 possibly interferes in PQS–PqsR mediated quorum sensing system in Pseudomonas aeruginosa

Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett-Burman analysis and central composite rotatable design (CCRD). It was evident that at 35mlL-1 glycerol and 3.8gL-1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC-MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC-MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS-PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy.

Computational protein design and protein-ligand interaction studies for the improvement of organophosphorus degrading potential of Deinococcus radiodurans

The organophosphorus hydrolase enzyme is involved in the catalyzing reaction that involve hydrolysis of organophosphate toxic compounds. An enzyme from Deinococcus radiodurans reported as homologous to phosphotriesterase and show activity against organophosphate. In the past activity of this enzyme is low and efforts made to improve the activity by experimental mutation study. However only very few organophosphates tested against very few catalytic site mutations. In order to improve the catalytic power of the organophosphorus hydrolase enzyme, we carried out systematic functional hotspot based protein engineering strategy. The mutants tested against 46 know organophosphate compounds using molecular docking study. Finally, we carried out an extensive molecular docking study to predict the binding of 46 organophosphate compounds to wild-type protein and mutant organophosphorus hydrolase enzyme. At the end we are able to improve the degrading potential of organophosphorus hydrolase enzyme against organophosphate toxic compounds. This preliminary study and the outcome would be useful guide for the experimental scientist involved in the bioremediation of toxic organophosphate compounds.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer’s disease therapeutics.

Computational profiling of pore properties of outer membrane proteins

The β-strand forming outer membrane proteins (OMPs) are found in the outer membrane of the Gram negative bacteria, eukaryotic organelles of prokaryotic origin, mitochondria, and chloroplasts. The OMPs present in the outer membrane plays a vital role in the maintenance of cell homeostasis. Two classes of OMPs were recognized within the outer membrane, one is peripheral and another is integral membrane proteins with unique β-barrel fold architecture. In Gram negative bacteria, OMPs are classified into six families based on the function (Koebnik, Locher, & Van Gelder, 2000). There are general porins (OMPC, OMPF, and PhoE), passive transporters (LamB, ScrY, FadL, and TodX), active transporters (siderophores transporters FepA, FecA, Fhu A, and vitamin B12 transporters BtuB), enzymes (phospholipase OmpLA or protease OmpT), defensive proteins (OmpX), and structural proteins (OmpA). Understanding the structure and function of OMPs is of critical importance, in the antibiotic design and development. In an antibacterial therapy, one of the main steps for the antibiotic is to cross the outer membrane through OMPs like OMPF present in S. typhi (Balasubramaniam, Arockiasamy, Kumar, Sharma, & Krishnaswamy, 2012). Most of the antibiotic targets are present within the bacterial cell; the antibiotics are known to take two routes to reach the targets. The hydrophobic antibiotics pass through the lipid and the hydrophilic molecules translocate through water-filled porins. The lipid and protein compositions of the outer membrane have a strong impact on the permeability of antibiotics and drug resistance. So, studies conducted on physico-chemical properties of amino acid residues along the pores and by considering the lipid–OMP interactions may provide valuable insights into the antibiotic resistance. Liposomeswelling assay is used to qualitatively characterize antibiotic translocation through porin. Later quantitative electrophysiology measurements were used. The OMPmediated antibiotic permeability is still not clearly understood. The availability of high resolution porin structure and further investigation on this reveals that antibiotic translocates by interacting with the surface of the channel. Ampicillin-binding site in the channel of OMPF is identified. Facilitated diffusion through a binding site was observed in maltoporin (Danelon, Brando, & Winterhalter, 2003). Computational molecular dynamics (MD) studies in the past have shed light on the antibiotic translocation process (Kumar et al., 2010). Moreover, the alteration or modification of porin expression is associated with antimicrobial resistance. The emergence of porin-related antibiotic resistance is a challenge for designing better antibiotics (Beceiro, Tomas, & Bou, 2013; Nikaido, 2003). There are limited studies on relating computational modeling/dynamics with experiments. A search on pubmed results in only 150 hits in which very few are interesting. An interesting work by Kumar et al., demonstrates the structural and dynamical properties of the two major porins (OmpF and OmpC) in Escherichia coli, using MD simulations and showed the relationship between pore amino acid properties and antibiotic transport (Kumar, Hajjar, Ruggerone, & Ceccarelli, 2010). They presented the results of transport properties using accelerated MD simulations to probe the diffusion of norfloxacin (a fluoroquinolone antibiotic) through the two porins OmpF/OmpC. Porins perform various functions by depending upon channels, regulation of porin expression and its structure. Although OMPs are commonly made of β-strands, it is interesting to observe the variety of completely different functions, that is been performed such as diffusion pores, substrate-specific transporters, signal transduction, and enzymatic activity. The transport of various substances