Emergence of Dengue as one of the deadliest viral diseases prompts the need for development of effective therapeutic agents. Dengue virus (DV) exists in four different serotypes and infection caused by one serotype predisposes its host to another DV serotype heterotypic re-infection. We undertook virtual ligand screening (VLS) to filter compounds against DV that may inhibit inclusively all of its serotypes. Conserved non-structural DV protein targets such as NS1, NS3/NS2B and NS5, which play crucial role in viral replication, infection cycle and host interaction, were selected for screening of vital antiviral drug leads. A dataset of plant based natural antiviral derivatives was developed. Molecular docking was performed to estimate the spatial affinity of target compounds for the active sites of DV’s NS1, NS3/NS2B and NS5 proteins. The drug likeliness of the screened compounds was followed by ADMET analysis whereas the binding behaviors were further elucidated through molecular dynamics (MD) simulation experiments. VLS screened three potential compounds including Canthin-6-one 9-O-beta-glucopyranoside, Kushenol W and Kushenol K which exhibited optimal binding with all the three conserved DV proteins. This study brings forth novel scaffolds against DV serotypes to serve as lead molecules for further optimization and drug development against all DV serotypes with equal effect against multiple disease causing DV proteins. We therefore anticipate that the insights given in the current study could be regarded valuable towards exploration and development of a broad-spectrum natural anti-dengue therapy.

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5' guanosine monophosphate (5'-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αβ possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αβ structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αβ structural stability to the experimentally observed disease phenotypes.

The need of circumventing life-threatening cardiovascular disorders (CVDs) and pulmonary hypertension (PHT) worldwide prompts researchers to develop effective therapeutic agents. Crucial role of cyclic nucleotide phosphodiesterase-5 (PDE5A) and cyclic nucleotide phosphodiesterase-3 (PDE3A) in cardiovascular signaling makes them potential drug targets for the treatment of CVDs and PHT. In this study, one-drug-multiple-target strategy has been employed to screen inhibitors exhibiting dual specificity through Phase-generated and statistically validated e-pharmacophore models of PDE5A and PDE3A. An extensive CoCoCo database of 7 million compounds with similar to 150,000,000 conformations was virtually screened by sequential e-pharmacophore mapping followed by Lipinski Rule of Five (RO5) evaluation and hierarchical docking simulations. Finally, docked hits were subjected to rigorous MMGBSA analysis to estimate the relative spatial affinity of the drug-like compounds. The hits (354 and 366 ligands against PDE5A and PDE3A, respectively) were further optimized through 2D clustering followed by a comprehensive 2D and 3D interaction analysis. Five structurally diverse hits mapped equally well with the e-pharmacophore models and showed promising inhibitory interactions with conserved four catalytic features of PDE5A and PDE3A, thus exhibiting dual specificity. Proposed lead compounds exhibited the lowest MMGBSA binding energies and were found to be in agreement with Lipinski Rule of Five (RO5) and ADME/Tox criteria as compared to sildenafil. The proposed dual inhibitors could thus provide promising outcomes for the discovery of dual as well as multipotent drug like compounds after lead optimization and primary therapeutic interventions.

Soluble guanylate cyclase (sGC) is a key enzyme implicated in various physiological processes such as vasodilation, thrombosis and platelet aggregation. The enzyme’s Heme-Nitric oxide/Oxygen (H-NOX) binding domain is the only sensor of nitric oxide (NO) in humans, which on binding with NO activates sGC to produce the second messenger cGMP. H-NOX is thus a hot target for drug design programs. BAY60-2770 and BAY58-2667 are two widely studied activators of sGC. Here we present comparative molecular dynamics studies to understand the molecular details characterizing the binding of BAY60-2770 and BAY58-2667 with the human H-NOX (hH-NOX) and bacterial H-NOX (bH-NOX) domains. HartreeFock method was used for parametrization of both the activators. A 50 ns molecular dynamics (MD) simulation was run to identify the functionally critical regions of the H-NOX domains. The CPPTRAJ module was used for analysis. BAY60-2770 on binding with bH-NOX, triggered rotational movement in signaling helix F and significant dynamicity in loops α and β, but in hH-NOX domain the compound showed relatively lesser aforementioned structural fluctuations. Conversely, hH-NOX ligated BAY58-2667 experienced highest transitions in its helix F due to electrostatic interactions with D84, T85 and R88 residues which are not conserved in bH-NOX. These conformational transformations might be essential to communicate with downstream PAS, CC and cyclase domains of sGC. Comparative MD studies revealed that BAY bound bHNOX dynamics varied from that of hH-NOX, plausibly due to some key residues such as R40, F74 and Y112 which are not conserved in bacteria. These findings will help to the design of novel drug leads to cure diseases associated to human sGC.

Protein kinase Iα (PKGIα) is a pivotal cyclic guanosine monophosphate (cGMP) signalling protein. Major steps related to the structural plasticity of PKGIα have been inferred but the structural aspects of the auto-inhibition and multidomain tertiary organization of human PKGIα in active and inactive form are not clear. Here we combine computational comparative modelling, protein–protein docking and molecular dynamics (MD) simulations to investigate structural details of the repressed state of the catalytic domain of PKGIα. Exploration of the potential inhibitory conformation of the auto-inhibitory domain (AI) within the catalytic cleft reveals that the pseudo-substrate motif binds with residues of the glycine rich loop and substrate-binding lobe. Dynamic changes as a result of coupling of the catalytic and AI domains are also investigated. The three-dimensional homodimeric models of PKGIα in the active and inactive state indicate that PKGIα in its inactive-state attains a compact globular structure where cyclic nucleotide binding (CNB-A/B) domains are buried, whereas the catalytic domains are inaccessible with their substrate-binding pockets facing the N-terminal of CNB-A. Contrary to this, the active-state model of PKGIα shows an extended conformation where CNB-A/B domains are slightly rearranged and the catalytic domains of homodimer flanking the C-terminal with their substrate binding lobes free to entrap downstream proteins. These findings are consistent with previously reported static images of the multidomain organization of PKGIα. Structural insights pertaining to the conformational heterogeneity and auto-inhibition of PKGIα provided in this study may help to understand the dynamics-driven effective regulation of PKGIα.

Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low density lipoprotein receptor (LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly reduced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combination of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events.