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Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

ABSTRACT: BACKGROUND: Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. RESULTS: Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. CONCLUSIONS: We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.

SUBMITTER: Grover A

PROVIDER: S-EPMC3278839 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

Grover Abhinav A Agrawal Vibhuti V Shandilya Ashutosh A Bisaria Virendra S VS Sundar Durai D

BMC bioinformatics 20111130

<h4>Background</h4>Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occ ...[more]

PMID: 22373101

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Project description:BackgroundDevelopment of a cancerous cell takes place when it ceases to respond to growth-inhibiting signals and multiplies uncontrollably and can detach and move to other parts of the body; the process called as metastasis. A particular set of cysteine proteases are very active during cancer metastasis, Cathepsins being one of them. They are involved in tumor growth and malignancy and have also been reported to be overexpressed in tumor cell lines. In the present study, a combinatorial approach comprising three-dimensional quantitative structure-activity relationship (3D QSAR), ligand-based pharmacophore modelling and search followed by cathepsin L structure-based high throughput screening was carried out using an initial set of 28 congeneric thiosemicarbazone derivatives as cathepsin L inhibitors. A 3D QSAR was derived using the alignment of a common thiosemicarbazone substructure. Essential structural features responsible for biological activity were taken into account for development of a pharmacophore model based on 29 congeneric thiosemicarbazone derivatives. This model was used to carry out an exhaustive search on a large dataset of natural compounds. A further cathepsin L structure-based screen identified two top scoring compounds as potent anti-cancer leads.ResultsThe generated 3D QSAR model showed statistically significant results with an r(2) value of 0.8267, cross-validated correlation coefficient q(2) of 0.7232, and a pred_r(2) (r(2) value for test set) of 0.7460. Apart from these, a high F test value of 30.2078 suggested low probability of the model's failure. The pharmacophoric hypothesis chosen for searching the natural compound libraries was identified as DDHRR, where two Ds denote 2 hydrogen donors, H represents a hydrophobic group and two Rs represent aromatic rings, all of which are essential for the biological activity. We report two potential drug leads ZINC08764437 (NFP) and ZINC03846634 (APQ) obtained after a combined approach of pharmacophore-based search and structure-based virtual screen. These two compounds displayed extra precision docking scores of -7.972908 and -7.575686 respectively suggesting considerable binding affinity for cathepsin L. High activity values of 5.72 and 5.75 predicted using the 3D QSAR model further substantiated the inhibitory potential of these identified leads.ConclusionThe present study attempts to correlate the structural features of thiosemicarbazone group with their biological activity by development of a robust 3D QSAR model. Being statistically valid, this model provides near accurate values of the activities predicted for the congeneric set on which it is based. These predicted activities are good for the test set compounds making it indeed a statistically sound 3D QSAR model. The identified pharmacophore model DDHRR.8 comprised of all the essential features required to interact with the catalytic triad of cathepsin L. A search for natural compounds based on this pharmacophore followed by docking studies further screened out two top scoring candidates: NFP and AFQ. The high binding affinity and presence of essential structural features in these two compounds make them ideal for consideration as natural anti-tumoral agents. Activity prediction using 3D QSAR model further validated their potential as worthy drug candidates against cathepsin L for treatment of cancer.

Project description:BackgroundHSPs (Heat shock proteins) are highly conserved ubiquitous proteins among species which are involved in maintaining appropriate folding and conformation of other proteins and are thus referred to as molecular chaperones. Hsp90 (Heat-shock protein 90 kDa) is one of a group of molecular chaperones responsible for managing protein folding and quality control in cell environment. However it is also involved in the maturation and stabilization of a wide range of oncogenic client proteins which are crucial for oncogenesis and malignant progression. Hsp90 requires a series of co-chaperones to assemble into a super-chaperone complex for its function. These co-chaperones bind and leave the complex at various stages to regulate the chaperoning process. Arresting the chaperone cycle at these stages by targeting different co-chaperone/Hsp90 interactions seems to be quite a viable alternative and is likely to achieve similar consequences as that of Hsp90 direct inhibition with added favors of high specificity and reduced side effect profile. The study conducted here is an attempt to explore the potential of Withania somnifera's major constituent WA (Withaferin A) in attenuating the Hsp90/Cdc37 chaperone/co-chaperone interactions for enhanced tumor arresting activity and to elucidate the underlying mode of action using computational approaches.ResultsFormation of active Hsp90/Cdc37 complex is one of the essential steps for facilitation of chaperone client interaction, non-assembly of which can lead to prevention of the chaperone-client association resulting in apoptosis of tumor cells. From our flexible docking analysis of WA into active Hsp90/Cdc37 complex in which key interfacing residues of the complex were kept flexible, disruption of the active association complex can be discerned. While docking of WA into segregated Hsp90 leaves the interface residues untouched. Thus the molecular docking analysis of WA into Hsp90 and active Hsp90/Cdc37 complex conducted in this study provides significant evidence in support of the proposed mechanism of chaperone assembly suppression by inhibition or disruption of active Hsp90/Cdc37 complex formation being accounted by non-assembly of the catalytically active Hsp90/Cdc37 complex. Results from the molecular dynamics simulations in water show that the trajectories of the protein complexed with ligand WA are stable over a considerably long time period of 4 ns, with the energies of the complex being lowered in comparison to the un-docked association complex, suggesting the thermodynamic stability of WA complexed Hsp90/Cdc37.ConclusionsThe molecular chaperone Hsp90 has been a promising target for cancer therapy. Cancer is a disease marked by genetic instability. Thus specific inhibition of individual proteins or signalling pathways holds a great potential for subversion of this genetic plasticity of cancers. This study is a step forward in this direction. Our computational analysis provided a rationalization to the ability of naturally occurring WA to alter the chaperone signalling pathway. The large value of binding energy involved in binding of WA to the active Hsp90/Cdc37 complex consolidates the thermodynamic stability of the binding. Our docking results obtained substantiate the hypothesis that WA has the potential to inhibit the association of chaperone (Hsp90) to its co-chaperone (Cdc37) by disrupting the stability of attachment of Hsp90 to Cdc37. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent Hsp90-client modulating capability.

Project description:To determine the role of ICP22 in transcription, we performed precise nuclear run-on followed by deep sequencing (PRO-seq) and global nuclear run-on with sequencing (GRO-seq) in cells infected with a viral mutant lacking the entire ICP22-encoding α22 (US1/US1.5) gene and a virus derived from this mutant bearing a restored α22 gene. At 3 h postinfection (hpi), the lack of ICP22 reduced RNA polymerase (Pol) promoter proximal pausing (PPP) on the immediate early α4, α0, and α27 genes. Diminished PPP at these sites accompanied increased Pol processivity across the entire herpes simplex virus 1 (HSV-1) genome in GRO-seq assays, resulting in substantial increases in antisense and intergenic transcription. The diminished PPP on α gene promoters at 3 hpi was distinguishable from effects caused by treatment with a viral DNA polymerase inhibitor at this time. The ICP22 mutant had multiple defects at 6 hpi, including lower viral DNA replication, reduced Pol activity on viral genes, and increased Pol activity on cellular genes. The lack of ICP22 also increased PPP release from most cellular genes, while a minority of cellular genes exhibited decreased PPP release. Taken together, these data indicate that ICP22 acts to negatively regulate transcriptional elongation on viral genes in part to limit antisense and intergenic transcription on the highly compact viral genome. This regulatory function directly or indirectly helps to retain Pol activity on the viral genome later in infection. IMPORTANCE The longstanding observation that ICP22 reduces RNA polymerase II (Pol II) serine 2 phosphorylation, which initiates transcriptional elongation, is puzzling because this phosphorylation is essential for viral replication. The current study helps explain this apparent paradox because it demonstrates significant advantages in negatively regulating transcriptional elongation, including the reduction of antisense and intergenic transcription. Delays in elongation would be expected to facilitate the ordered assembly and functions of transcriptional initiation, elongation, and termination complexes. Such limiting functions are likely to be important in herpesvirus genomes that are otherwise highly transcriptionally active and compact, comprising mostly short, intronless genes near neighboring genes of opposite sense and containing numerous 3'-nested sets of genes that share transcriptional termination signals but differ at transcriptional start sites on the same template strand.

Dataset Information

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

Publications

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

Similar Datasets

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