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ABSTRACT: Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70-80 Ca(2+) ions. However, the nature of Ca(2+)-binding sites has not yet been fully characterized. In this study, we employed in silico approaches to identify the Ca(2+) binding sites and to understand the molecular basis of CASQ1-Ca(2+) recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: ) and adding the missing C-terminal residues (aa350-364). Using this model we performed extensive 30 ns molecular dynamics simulations over a wide range of Ca(2+) concentrations ([Ca(2+)]). Our results show that the Ca(2+)-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca(2+)-binding sites share a similar geometry and interestingly, the majority of them were found to be induced by increased [Ca(2+)]. We also found that the system shows maximal Ca(2+)-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data show that the CASQ1 back-to-back stacking is progressively stabilized by the emergence of an increasing number of hydrophobic interactions with increasing [Ca(2+)]. Further, this study shows that the CAS domain assumes a compact structure with an increase in Ca(2+) binding, which suggests that the CAS domain might function as a Ca(2+)-sensor that may be a novel structural motif to sense metal. We propose the term "Dn-motif" for the CAS domain.
Molecular BioSystems 04/2013; · 3.53 Impact Factor
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ABSTRACT: Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70-80 Ca2+ ions. However, the nature of Ca2+-binding sites has not yet been fully characterized. In this study, we employed in-silico approaches to identify the Ca2+ binding sites and to understand the molecular basis of CASQ1-Ca2+ recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: 3UOM) and adding the missing C-terminal residues (aa350-364). Using this model we performed extensive 30 ns molecular dynamics simulations exposed to wide range of Ca2+ concentrations ([Ca2+]). Our results show that the Ca2+-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca2+-binding sites share a similar geometry and interestingly, majority of them were found to be induced by increased [Ca2+]. We also found that the system undergoes maximal Ca2+-binding to the CAS (consecutive asparta
Molecular BioSystems 01/2013; · 3.53 Impact Factor
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ABSTRACT: The structure-based approaches were implemented to design and rationally select the molecules for synthesis and anti-HCV activity evaluation. The systematic structure-activity relationships of previously discovered molecules (types I, II, III) were analyzed to design new molecules (type IV) by bioisosteric replacement of the amino group. The ligand conformation, binding mode studies and drug like properties were major determinant for selection of molecules for final synthesis. The replacement of amino group with methyl restored the interactions with RNA-template (Tem 799) through bifurcated weak H-bond (C-H…O). This is an interesting finding observed from molecular modeling studies. It was found that 6c-e has anti-HCV activity (EC(50) in 37-46μM) while 6a, 6b and 6g were inactive. The compound 6f (EC(50) 28μM) was the most active among the series however it also showed some cytotoxicity (CC(50) 52.8μM). Except 6f, none of the compounds were found to be cytotoxic (CC(50)>100μM). The present study discloses structure-based approach for novel anti-HCV lead discovery and opens a future scope of lead optimization.
Bioorganic & medicinal chemistry letters 10/2012; · 2.65 Impact Factor
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ABSTRACT: Among approximately 65 kinases of the malarial genome, RIO2 (right open reading frame) kinase belonging to the atypical class of kinase is unique because along with a kinase domain, it has a highly conserved N-terminal winged helix (wHTH) domain. The wHTH domain resembles the wing like domain found in DNA binding proteins and is situated near to the kinase domain. Ligand binding to this domain may reposition the kinase domain leading to inhibition of enzyme function and could be utilized as a novel allosteric site to design inhibitor. In the present study, we have generated a model of RIO2 kinase from Plasmodium falciparum utilizing multiple modeling, simulation approach. A novel putative DNA-binding site is identified for the first time in PfRIO2 kinase to understand the DNA binding events involving wHTH domain and flexible loop. Induced fit DNA docking followed by minimization, molecular dynamics simulation, energetic scoring and binding mode studies are used to reveal the structural basis of PfRIO2-ATP-DNA complex. Ser105 as a potential site of phosphorylation is revealed through the structural studies of ATP binding in PfRIO2. Overall the present study discloses the structural facets of unknown PfRIO2 complex and opens an avenue toward exploration of novel drug target.
Journal of Molecular Modeling 09/2012; · 1.80 Impact Factor
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Structural Chemistry 01/2012; · 1.85 Impact Factor
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ABSTRACT: An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4. The Simmons-Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermediate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog 8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC(50) values of 0.273 and 0.368 μM in genotypes 1A and 1B, respectively.
Bioorganic & medicinal chemistry letters 07/2011; 21(13):3982-5. · 2.65 Impact Factor
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ABSTRACT: CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.
Biochemical Journal 01/2011; 435(2):391-9. · 4.90 Impact Factor
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ABSTRACT: Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.
Bioorganic & medicinal chemistry 05/2010; 18(10):3403-12. · 2.82 Impact Factor
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ABSTRACT: Calsequestrin undergoes dynamic polymerization with increasing calcium concentration by front-to-front dimerization and back-to-back
packing forming wire-shaped structures. Recent findings that a point mutation R33Q leads to lethal Catecholaminergic Polymorphic
Ventricular Tachycardia (CPVT) implied a crucial role for the N-terminus. In this study we demonstrate that this mutation
resides in a highly conserved alternately charged residue cluster #1 (DGKDR) in the N-terminal end of calsequestrin. We further
show that the cluster configures itself as a ring system and the dipolar arrangement within the cluster brings about a critical
conformational flip of residues K31-D32 essential for dimer stabilization by formation of hydrogen bond network. We additionally
show that Ca2+-induced calsequestrin aggregation is non-linear, reversible and can regain native conformation by Ca2+-chelation
with EGTA. These studies suggest that the cluster #1 works as a molecular switch and governs the bidirectional transition
between CASQ2 monomer and dimer. We further demonstrate that mutations disrupting the alternating charge pattern of the cluster,
including the R33Q impair Ca2+-CASQ2 interaction leading to altered polymerization-depolymerization dynamics. These studies
provide new mechanistic insight on the functional effects of the R33Q mutation and its potential role in CPVT.
Journal of Biological Chemistry 03/2010; · 4.77 Impact Factor
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ABSTRACT: Calsequestrin undergoes dynamic polymerization with increasing calcium concentration by front-to-front dimerization and back-to-back packing, forming wire-shaped structures. A recent finding that point mutation R33Q leads to lethal catecholaminergic polymorphic ventricular tachycardia (CPVT) implies a crucial role for the N terminus. In this study, we demonstrate that this mutation resides in a highly conserved alternately charged residue cluster (DGKDR; cluster 1) in the N-terminal end of calsequestrin. We further show that this cluster configures itself as a ring system and that the dipolar arrangement within the cluster brings about a critical conformational flip of Lys(31)-Asp(32) essential for dimer stabilization by formation of a H-bond network. We additionally show that Ca(2+)-induced calsequestrin aggregation is nonlinear and reversible and can regain the native conformation by Ca(2+) chelation with EGTA. This study suggests that cluster 1 works as a molecular switch and governs the bidirectional transition between the CASQ2 monomer and dimer. We further demonstrate that mutations disrupting the alternating charge pattern of the cluster, including R33Q, impair Ca(2+)-CASQ2 interaction, leading to altered polymerization-depolymerization dynamics. This study provides new mechanistic insight into the functional effects of the R33Q mutation and its potential role in CPVT.
Journal of Biological Chemistry 03/2010; 285(22):17188-96. · 4.77 Impact Factor
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ABSTRACT: Enantiomerically pure cyclopentyl cytosine [(-)-carbodine 1] was synthesized from d-ribose and evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine, (+/-)-carbodine and ribavirin. (-)-Carbodine 1 exhibited potent antiviral activity against various strains of influenza A and B viruses.
Bioorganic & medicinal chemistry letters 02/2010; 20(8):2601-4. · 2.65 Impact Factor
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ABSTRACT: 3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO) inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity index in the magnitude of 10(3)-10(5). The docking studies were carried out to gain further structural insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the theoretical (K(i)) values obtained by molecular docking studies were in congruence with their experimental (K(i)) values.
Bioorganic & medicinal chemistry 01/2010; 18(5):1875-81. · 2.82 Impact Factor
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ABSTRACT: GSK-3beta, one of the vital enzymes responsible for various phosphorylation catalysis. Induced fit mechanism and the presence of conserved water molecule(s) in the active site poses complexity during the process of virtual screening. The present investigation reveals the practical strategy to handle the induced fit mechanism of GSK-3beta though flexible docking protocol. This protocol provides an enrichment of 70% in top 1% of the dataset with a rank correlation of >0.9 and found better in comparison to earlier reported protocols.
Bioorganic & medicinal chemistry letters 09/2009; 19(19):5582-5. · 2.65 Impact Factor
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Hyo-Joong Kim, Ashoke Sharon,
Chandralata Bal,
Jianing Wang,
Madhan Allu,
Zhuhui Huang,
Michael G Murray,
Leda Bassit,
Raymond F Schinazi,
Brent Korba,
Chung K Chu
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ABSTRACT: A series of 7-deazaneplanocin A (7-DNPA, 2) analogues were synthesized and evaluated for in vitro antiviral activity against HBV and HCV. The syntheses of target carbocyclic nucleosides were accomplished via a convergent procedure. 7-Substitutions were introduced by using 7-substituted-7-deaza heterocyclic base precursors (F, Cl, Br, and I) or via substitution reactions after the synthesis of the carbocyclic nucleosides. Among the synthesized compounds, 2, 13-15, 24, and 27 exhibited significant anti-HCV activity (EC(50) ranged from 1.8 to 20.1 microM) and compounds 2, 15, 22, and 24 demonstrated moderate to potent anti-HBV activity (EC(50) = 0.3-3.3 microM). In addition, compound 24 also showed activity against lamivudine- and adefovir-associated HBV mutants.
Journal of Medicinal Chemistry 01/2009; 52(1):206-13. · 4.80 Impact Factor
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ABSTRACT: A series of (-)-beta-D-(2R,4R)-dioxolane-thymine-5'-O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1(LAI) in human peripheral blood mononuclear (PBM) cells as well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was observed for the compound 2-4 (5'-O-aliphatic acid esters) without increase in cytotoxicity in comparison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0 and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the half-lives from 12.3 to 48.0 min.
Bioorganic & medicinal chemistry 12/2008; 17(3):1404-9. · 2.82 Impact Factor
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Journal of Fluorine Chemistry 10/2008; 129(9):743-766. · 2.03 Impact Factor
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ABSTRACT: Despite the significant successes in the area of anti-HBV agents, resistance and cross-resistance against available therapeutics are the major hurdles in drug discovery. The present investigation is to understand the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of five anti-HBV agents [lamivudine (3TC, 1), adefovir (ADV, 2), entecavir (ETV, 3), telbivudine (LdT, 4) and clevudine (l-FMAU, 5)]. In this regard, homology modeled structure of HBV-polymerase was used for minimization, conformational search and induced fit docking followed by binding energy calculation on wild-type as well as on mutant HBV-polymerases (L180M, M204V, M204I, L180M+M204V, L180M-M204I). Our studies suggest a significant correlation between the fold resistances and the binding affinity of anti-HBV nucleosides. The binding mode studies reveals that the domain C residue M204 is closely associated with sugar/pseudosugar ring positioning in the active site. The positioning of oxathiolane ring of 3TC (1) is plausible due the induced fit orientation of the M204 residue in wild-type, and further mutation of M204 to V204 or I204 reduces the final binding affinity which leads to the drug resistance. The domain B residue L180 is not directly close ( approximately 6A) to the nucleoside/nucleoside analogs, but indirectly associated with other active-site hydrophobic residues such as A87, F88, P177 and M204. These five hydrophobic residues can directly affect on the incoming nucleoside analogs in terms of its association and interaction that can alter the final binding affinity. There was no sugar ring shifting observed in the case of adefovir (2) and entecavir (3), and the position of sugar ring of 2 and 3 is found similar to the sugar position of natural substrate dATP and dGTP, respectively. The exocyclic double bond of entecavir (3) occupied in the backside hydrophobic pocket (made by residues A87, F88, P177, L180 and M204), which enhances the overall binding affinity. The active site binding of LdT (4) and l-FMAU (5) showed backward shifting along with upward movement without enforcing M204 residue and this significant different binding mode makes these molecules as polymerase inhibitors, without being incorporated into the growing HBV-DNA chain. Structural results conferred by these l- and d-nucleosides, explored the molecular basis of drug resistance which can be utilized for future anti-HBV drug discovery.
Antiviral research 09/2008; 80(3):339-53. · 3.61 Impact Factor
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ABSTRACT: Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza- p-cyano-6-benzylthioinosine (IC 50 = 5.3 microM) and 7-deaza- p-methoxy-6-benzylthioinosine (IC 50 = 4.6 microM), were evaluated in cell culture to delineate their selective toxicity.
Journal of Medicinal Chemistry 08/2008; 51(13):3934-45. · 4.80 Impact Factor
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ABSTRACT: Prodrugs of (-)-beta-D-(2R,4R)-1,3-dioxolane-2,6-diamino purine (DAPD), organic salts of DAPD, 5'-L-valyl DAPD and N-1 substituted (-)-beta-D-(2R,4R)-1,3-dioxolane guanosine (DXG) have been synthesized with the objective of finding molecules which might be superior to DAPD and DXG in solubility as well as pharmacologic profiles. Synthesized prodrugs were evaluated for anti-HIV activity against HIV-1(LAI) in primary human lymphocytes (PBM cells) as well as their cytotoxicity in PBM, CEM and Vero cells. DAPD prodrugs, modified at the C6 position of the purine ring, demonstrated several folds of enhanced anti-HIV activity in comparison to the parent compound DAPD without increasing the toxicity. The presence of alkyl amino groups at the C6 position of the purine ring increased the antiviral potency several folds, and the most potent compound (-)-beta-D-(2R,4R)-1,3-dioxolane-2-amino-6-aminoethyl purine (8) was 17 times more potent than that of DAPD. 5'-L-Valyl DAPD 20 and organic acid salts 21-24 also exhibited enhanced anti-HIV activity in comparison to DAPD, while DXG prodrugs 16 and 17 exhibited lower potency than that of DXG or DAPD.
Antiviral Research 10/2007; 75(3):198-209. · 4.30 Impact Factor
