Ashutosh Kumar

Synthesis and molecular docking studies of 1-phenyl-4-glycosyl-dihydropyridines as potent antileishmanial agents

A series of 1-phenyl-4-glycosyl-dihydropyridines (4-17 and 19-21) were prepared by the one pot multicomponent reaction of glcosyl aldehyde, beta-keto compounds and aniline or substituted aniline in the presence of TBAHS as catalyst. The compounds were screened in vitro and in vivo for their antileishmanial activities. Most of the compounds exhibited moderate to good activity against amastigotes and promastigotes of Leishmania donovani. The compounds 4, 11, 12, 13, and 17 exhibited potent in vivo activity with selectivity index (SI) values 7.43-18.93. Molecular docking studies with these compounds revealed L. donovani PTR1 as the possible target to show antileishmanial activities.

CoMFA based de novo design of Pyrrolidine Carboxamides as Inhibitors of Enoyl Acyl Carrier Protein Reductase from Mycobacterium tuberculosis

InhA, the enoyl acyl carrier protein reductase (EACP reductase) from Mycobacterium tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective target for the development of anti-microbial agents. We report here, comparative molecular field analysis (CoMFA) studies and subsequent de novo ligand design using the LeapFrog program on pyrrolidine carboxamides, which have been reported as selective inhibitors of EACP reductase from Mycobacterium tuberculosis. The CoMFA model, constructed from the inhibitors used in this study has been successfully used to rationalize the structure-activity relationship of pyrrolidine carboxamides. The CoMFA model produced statistically significant results with cross-validated and conventional correlation coefficients of 0.626 and 0.953 respectively. Further, the predictive ability of CoMFA model was determined using a test set which gave predictive correlation coefficient r2pred of 0.880, indicating good predictive power. Finally, Leapfrog was used to propose 13 new pyrrolidine carboxamide analogues, based on the information derived from the CoMFA contour maps. The designed molecules showed better predicted activity using the CoMFA model with respect to the already reported systems; hence suggesting that newly proposed molecules in this series of compounds may be more potent and selective toward EACP reductase inhibition.

Molecular cloning and characterization of Plasmodium falciparum transketolase

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.

Leishmania donovani pteridine reductase 1: Biochemical properties and structure-modeling studies

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors.

Virtual screening against Mycobacterium tuberculosis dihydrofolate reductase: Suggested workflow for compound prioritization using structure interaction fingerprints

In this study, we suggest a new workflow for the identification and prioritization of potential compounds targeted against Mycobacterium tuberculosis dihydrofolate reductase, an important folate cycle enzyme and a validated target for the development of anti-tubercular agents. First, we have performed an integrated pharmacophore and structure-based virtual screening using Maybridge small molecule database, subsequently interaction patterns from known actives to the receptor were applied for scoring and ranking the virtual screening hits using structure interaction fingerprint (SIFt)-based similarity approach. In addition, agglomerative hierarchical clustering of the structure interaction fingerprints permits the easy separation of active from inactive binding modes. Using this approach we screened 59275 Maybridge compounds and 20 compounds were prioritized as promising virtual screening hits. Though using a receptor interaction scoring approach, the results were not biased toward the chemical classes of the known actives and the proposed compounds were structurally diverse with low molecular weights and structural complexities. Our results suggest that structure-based virtual screening coupled with the SIFt should be a valuable tool for prioritization of virtual screening hits.

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

Glu-108 is essential for subunit assembly and dimer stability of d-phosphoglycerate dehydrogenase from Entamoeba histolytica

D-phosphoglycerate dehydrogenase catalyses the first step of phosphorylated serine biosynthesis pathway by oxidizing 3-phosphoglycerate, a glycolysis intermediate into phosphohydroxsy pyruvate. For Entamoeba histolytica this pathway is an integral part of the cysteine metabolism, which is considered to be vital for growth and survival of the parasite. Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a homodimer at pH 7. Mild acidic conditions induce significant changes in the functional and structural features of the protein as observed by enzymatic activity, spectropolarimetric measurements and fluorescence spectroscopy. Most interestingly the oligomeric status of the protein was lost and a functionally inactive monomer was stabilized at pH 5. Computational modeling and molecular dynamic simulations show that dimeric assembly of EhPGDH was stabilized with the help of several inter-subunit non-covalent interactions and subunit dissociation at pH 5 can be attributed to protonation of acidic amino acid residues present at the dimer interface. Site directed mutagenesis studies suggest that Glu-108 is essential for subunit assembly as the E108A mutant existed as monomer even at pH 7. The studies unequivocally show that the electrostatic interactions at the dimer interface play a crucial role in the stability of the protein and a complete dimer is essentially required for optimal enzymatic activity.

New molecular scaffolds for the design of Mycobacterium tuberculosis type II dehydroquinase inhibitors identified using ligand and receptor based virtual screening

Using ligand and receptor based virtual screening approaches we have identified potential virtual screening hits targeting type II dehydroquinase from Mycobacterium tuberculosis, an effective and validated anti-mycobacterial target. Initially, we applied a virtual screening workflow based on a combination of 2D structural fingerprints, 3D pharmacophore and molecular docking to identify compounds that rigidly match specific aspects of ligand bioactive conformation. Subsequently, the resulting compounds were ranked and prioritized using receptor interaction fingerprint based scoring and quantitative structure activity relationship model developed using already known actives. The virtual screening hits prioritized belong to several classes of molecular scaffolds with several available substitution positions that could allow chemical modification to enhance binding affinity. Finally, identified hits may be useful to a medicinal chemist or combinatorial chemist to pick up the new molecular starting points for medicinal chemistry optimization for the design of novel type II dehydroquinase inhibitors.

Receptor based 3D-QSAR to identify putative binders of Mycobacterium tuberculosis Enoyl acyl carrier protein reductase

In the current study, the applicability and scope of 3D-QSAR models (CoMFA and CoMSIA) to complement virtual screening using 3D pharmacophore and molecular docking is examined and applied to identify potential hits against Mycobacterium tuberculosis Enoyl acyl carrier protein reductase (MtENR). Initially CoMFA and CoMSIA models were developed using series of structurally related arylamides as MtENR inhibitors. Docking studies were employed to position the inhibitors into MtENR active site to derive receptor based 3D-QSAR models. Both CoMFA and CoMSIA yielded significant cross validated q2 values of 0.663 and 0.639 and r2 values of 0.989 and 0.963, respectively. The statistically significant models were validated by a test set of eight compounds with predictive r2 value of 0.882 and 0.875 for CoMFA and CoMSIA. The contour maps from 3D-QSAR models in combination with docked binding structures help to better interpret the structure activity relationship. Integrated with CoMFA and CoMSIA predictive models structure based (3D-pharmacophore and molecular docking) virtual screening have been employed to explore potential hits against MtENR. A representative set of 20 compounds with high predicted IC50 values were sorted out in the present study.

Characterization of dipeptidylcarboxypeptidase of Leishmania donovani: a molecular model for structure based design of antileishmanials.

Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4xa0mM and Vmax, 1.173 μmole/ml/min. Inhibition studies revealed that known ACE inhibitors (captopril and bradykinin potentiating peptide; BPP1) were weak inhibitors for LdDCP as compared to human testicular ACE (htACE) with Ki values of 35.8xa0nM and 3.9xa0μM, respectively. Three dimensional model of LdDCP was generated based on crystal structure of Escherichia coli DCP (EcDCP) by means of comparative modeling and assessed using PROSAII, PROCHECK and WHATIF. Captopril docking with htACE, LdDCP and EcDCP and analysis of molecular electrostatic potentials (MEP) suggested that the active site domain of three enzymes has several minor but potentially important structural differences. These differences could be exploited for designing selective inhibitor of LdDCP thereby antileishmanial compounds either by denovo drug design or virtual screening of small molecule databases.