SCREENING OF COMPETITIVE INHIBITOR OF HEPARAN SULFATE IN JAPANESE ENCEPHALITIS

ABSTRACTObjective: Japanese encephalitis virus (JEV) causes central nervous system inflammatory disease Japanese encephalitis (JE), which is mainly causedin children below 15 years of age. On an estimate, there are around 3 billion people at the risk and the disease is continuously spreading globally. TheJEV belongs to Flavivirdiae family and has RNA genome. JEV envelope protein domain III (D-III) binds to the Heparan sulfate present on the cell surfaceand initiates the infection which causes the disease in children.Methods: The drug discovery and development process has become more quantitative and much more computational in recent years. In this study,comparative molecular docking studies of 200 zinc database compounds and Heparan sulfate were done with D-III of JEV using Autodock 4.2 and theresults were analyzed on the basis of binding energy, inhibition constant, and number of hydrogen bonds. The results were also analyzed by studyingthe absorption, distribution, metabolism, and excretion (ADME-T) properties of the compounds using admetSAR server.Results: Best three lead molecules zinc_8964844 zinc_8964845, zinc_12660861 were chosen based on the binding energy, inhibition constant andADME properties among a set of 200 ligands that can act as the competitive inhibitor of the Heparan sulfate, which presents on the surface of the hostcell and mediates the attachment and binding of the virus to the host cell.Conclusion: These compounds can act as the competitive inhibitor of the Heparan sulfate and they can be validated further as a drug for the treatment of JE.Keywords: Japanese encephalitis, Domain-III, Heparan sulfate, Autodock, Autodock, Absorption, Distribution, Metabolism, Excretion

IDENTIFICATION OF INHIBITORS OF DENGUE VIRUS (DENV1, DENV2 AND DENV3) NS2B/ NS3 SERINE PROTEASE: A MOLICULAR DOCKING AND SIMULATION APPROACH

 Dengue is one of the fatal diseases, which are becoming a global health burden from few decades. Dengue fever, dengue hemorrhagic fever anddengue shock syndrome, caused by dengue virus (DENV), which completes its life cycle in mosquito i.e. Aedes aegyti, and human (DENV), and infectabout various individuals every year. The objective of this study is to find a potent inhibitor of DENV (DENV1, DENV2 and DENV3). In the presentstudy, NS2b/NS3 serine protease complex in targeted for the screening of the suitable inhibitors for DENV (DENV1, DENV2 and DENV 3). Therefore,the NS2b/NS3 serine protease complex structures were retrieved from the RCSB Protein Databank. The unliganded protein structures were docked,and best three selected and analyzed. A molecular dynamic simulation is also performed to investigate the conformational and positional changesof ligand that provide insights into the binding stability. It was observed that three of screened compounds have the maximum potential against theprotein. The analysis was performed on the basis of scoring and binding ability and one of them indicated minimum energy score with high numberof interactions with active site residues and the simulation study revealed that this selected ligand could efficiently bind to the NS2b/NS3 protease.These findings conclude that this selected ligand could be a promising inhibitor of all three serotypes of DENV as drug targets.Keywords: Dengue virus, Aedes aegyti, Flaviviridae, Serine protease, Docking

In silico studies on Zika NS3 helicase: bedrock for antiviral drug design.

Masters Degree. University of KwaZulu-Natal, Durban.Zika virus is a re-emerging infectious disease, which was declared to be a public health emergency of international concern due to its various reported complications ranging from microcephaly in newborn to Guillain-Barré Syndrome (GBS). Because less attention has been paid to this virus over time, literature has been lacking regarding the structural and conformational features of its proteins particularly the NS3 helicase protein. This dissertation has addressed two major aspects of Zika NS3 helicase protein: (i) the binding interactions and (ii) structural dynamics and conformational changes of the protein. Investigations were carried out on the various Zika NS3 helicase ligand binding landscapes using 10 ligands via molecular docking, of which the best 3 were subjected to molecular dynamic simulations and several post dynamics analyses. Ivermectin, HMC-HO1α and lapachol emerged as the best 3 ligands. The result of the analysis showed that the binding of Ivermectin to ssRNA site and Lapachol and HMC-HO1α to the ATPase site induces a more compact protein structure, thus stabilizing residue fluctuations. The pharmacophoric characteristics found in Lapachol, HMC-HO1α and Ivermectin may be utilized in the design of a potent hybrid drug that can show efficient inhibition of a multitude of diseases including the detrimental co-infection of ZIKV, Dengue and Chikungunya. Also in this study, a detailed structural dynamic analysis was carried out on the structural flexibility of the NS3 helicase protein after NITD008 binding via molecular dynamics simulation and other posts dynamic analysis including the Principal Component Analysis (PCA) and the Dynamic Cross Correlation (DCC) analysis. Result revealed a prominent shift in the P-Loop found at the ATP site of the helicase. This loop and helical flexible regions give new insight into the dynamic structural features of ZIKV NS3 Helicase. The PCA and DCC analysis result revealed a significant structural flexibility of the NITD008-NS3 Helicase system compared to the rigid unbound form of the protein. Furthermore, the NITD008-NS3 Helicase complex stability was also ensured via a 130ns molecular dynamic simulation, this has proven NITD008 as an effective potential inhibitor of the NS3 helicase protein. This research is of immense importance to the discovery of a potent Zika inhibitor and in medicine as it has proven potential inhibitors with a good binding affinity towards Zika NS3 helicase. Also, this study hopes to fill in the gap of information that has been missing regarding molecular studies on Zika virus to some extent

Mechanistic Elucidation of Protease–Substrate and Protein–Protein Interactions for Targeting Viral Infections

Viral infections represent an old threat to global health, with multiple epidemics and pandemics in the history of mankind. Despite several advances in the development of antiviral substances and vaccines, many viral species are still not targeted. Additionally, new viral species emerge, posing a menace without precedent to humans and animals and causing fatalities, disabilities, environmental harm, and economic losses. In this thesis, we present rational modeling approaches for targeting specific protease-substrate and protein-protein interactions pivotal for the viral replication cycle. Over the course of this work, antiviral research is supported beginning with the development of small molecular antiviral substances, going through the modeling of a potential immunogenic epitope for vaccine development, towards the establishment of descriptors for susceptibility of animals to a viral infection. Notably, all the research was done under scarce data availability, highlighting the predictive power of computational methods and complementarity between in-silico and in-vitro or in-vivo methods

Inhibitory Potential of Chromene Derivatives on Structural and Non-Structural Proteins of Dengue Virus

Dengue fever is a mosquito-borne viral disease that has become a serious health issue across the globe. It is caused by a virus of the Flaviviridae family, and it comprises five different serotypes (DENV-1 to DENV-5). As there is no specific medicine or effective vaccine for controlling dengue fever, there is an urgent need to develop potential inhibitors against it. Traditionally, various natural products have been used to manage dengue fever and its co-morbid conditions. A detailed analysis of these plants revealed the presence of various chromene derivatives as the major phytochemicals. Inspired by these observations, authors have critically analyzed the anti-dengue virus potential of various 4H chromene derivatives. Further, in silico, in vitro, and in vivo reports of these scaffolds against the dengue virus are detailed in the present manuscript. These analogues exerted their activity by interfering with various stages of viral entry, assembly, and replications. Moreover, these analogues mainly target envelope protein, NS2B-NS3 protease, and NS5 RNA-dependent RNA polymerase, etc. Overall, chromene-containing analogues exerted a potent activity against the dengue virus and the present review will be helpful for the further exploration of these scaffolds for the development of novel antiviral drug candidates

The in silico investigation of pharmacological targets of the zika virus : insights into the structural characteristics of the NS5 and NS3 proteins from atomistic molecular simulations.

Doctor of Philosophy in Pharmacological Science. University of KwaZulu-Natal, Durban 2017.The re-emerging Zika virus has evolved into a catastrophic epidemic during the past year, with an estimated 1.5 million reported cases of Zika infections worldwide, since the 2015 outbreak in Brazil. The virus has received considerable attention during 2016 with a flood of new discoveries, from evolving modes of viral transmission to viral-linked neurological disorders, unique specificity to host cells and increasing mutation rates. However, prior to the devastating 2015 outbreak in Brazil, the virus was classified as a neglected pathogen similar to Dengue and the West Nile virus. Despite the wide-scale research initiative, there is still no cure for the virus. There are currently vaccine clinical trials that are on-going but there has not been a breakthrough with regard to small molecule inhibitors. A lot of experimental resources have been allocated to repuposing FDA-approved drugs as possible inhibitors, however, even some of the most potent flavivirus inhibitors have adverse toxic effects. The first crystal structure of the zika virus was released in May 2016 and since then, six viral protein structures have been made available. Due to this lack in structural information, there is little known regarding the structural dynamics, active binding sites and the mechanism of inhibition of ZIKV enzymes. This study delves into the structural characteristics of three of the most crucial enzymatic targets of the zika virus, the NS5 RNA-dependent RNA polymerase and Methyltransferase as well as the NS3 Helicase. With emerging diseases, such as ZIKV, computational techniques including molecular modeling and docking, virtual screening and molecular dynamic simulations have allowed chemists to screen millions of compounds and thus funnel out possible lead drugs. These in silico approaches have warranted Computer-Aided Drug Design as a cost-effective strategy to fast track the drug discovery process. The The above techniques, amongst numerous other computational tools were employed in this study to provide insights into conformational changes that elucidate potential inhibitory mechanisms, active site identification and characterization and pharmacophoric features leading to promising small molecule inhibitor cadidates. The first study (Chapter 4), provided a comprehensive review on potential host/viral targets as well as provided a concise route map depicting the steps taken toward identifying potential inhibitors of drug targets when no crystal structure is available. A homology model case study, of the NS5 viral protein, was also demonstrated. The second study (Chapter 5) used the validated NS5 homology model to investigate the active sites at both the RNA-dependent RNA polymerase and Methyltransferase domains and subsequently employ a generated pharmacophore model to screen for potential inhibitors. Chapter 6 reports the third study, which investigates the structural dynamics and in turn, the possible mechanism of inhibition of the ZIKV NS3 Helicase enzyme when bound to ATP-competitive inhibitor, NITD008. The study also provides insight on the binding mode at the ATPase active site, thus assisting in the design of effective inhibitors against this detrimental viral target. Chapter 7 maps out the binding landscape of the ATPase and ssRNA site by demonstrating the chemical characteristics of potent flavivirus lead compounds, Lapachol, HMC-HO1α and Ivermectin at the respective NS3 Helicase binding sites. This study offers a comprehensive in silico perspective to fill the gap in drug design research against the Zika virus, thus giving insights toward the structural characteristics of pivotal targets and describing promising drug candidates. To this end, the work presented in this study is considered to be a fundamental platform in the advancements of research toward targeted drug design/delivery against ZIKV

The methyltransferase and helicase enzymes as therapeutic targets of Zika virus : a bio- computational analysis of interactions with potential inhibitors.

Doctoral of Philosophy in Pharmaceutical Sciences. University of KwaZulu-Natal, Westville, 2019.The rampant Zika virus has received worldwide attention after becoming a global crisis following the Brazilian epidemic in 2015. From an obscure and neglected pathogen, Zika virus is now a notorious virus associated with neurological disorders in infants and adults. Since 2016, the rapid research response from the global scientific community have led to the discovery of numerous potential small molecule inhibitors and vaccines against the Zika virus. Although, in spite of this massive research initiative, there is still no effective antiviral nor vaccine that has made it out of clinical trials. The design and development of new chemical entities demands excessive cost, time and resources. Therefore, this study applies computer-aided drug design techniques, which accelerates the rational drug design process. Computational approaches including molecular docking, virtual screening, molecular modeling and molecular dynamics facilitate the filtration of large databases of compounds to sift out potential lead compounds. Furthermore, research has dedicated several resources toward FDA-approved drug repurposing. Generally, drugs have similar effects on viruses of the same family; hence drugs that have previously been effective in treating other flaviviruses, such as Dengue virus and West Nile virus, are being tested for its potential inhibition of Zika virus. However, the ability of these drugs to pass the bloodbrain barrier to treat infected neurons poses a challenge to anti-Zika virus drug discovery. This study proposes innovative strategies to design drugs that are capable of passing the blood-brain barrier, and to be able to use drugs that are impermeable via drug delivery mechanisms. This study also assesses the bioavailability and blood-brain barrier permeability of screened drugs to scrutinize the list of potential Zika virus inhibitors. Apart from identifying potential inhibitors, understanding the structural dynamics of viral targets and molecular mechanisms underlying potential inhibition of the virus is imperative. This study explores the structural and molecular dynamics of key targets of the Zika virus, the NS3 helicase and the NS5 methyltransferase enzymes, using computational approaches mentioned above and several others elaborated in this thesis. These computational methods also allowed the identification of precise interactions, amino acid residues, inhibitory mechanisms and pharmacophoric features involved in binding of lead compounds to these enzymes. IX Chapter 4 represents the first study of this thesis, which presents a concise literature background of Zika virus and identifies blood-brain barrier permeability as a core challenge in anti-Zika virus drug development. This study also provides approaches that may enable researchers to create effective anti-Zika virus drugs. Chapter 5 is the subsequent study of this thesis, which applies molecular dynamics to comparatively investigate the mechanism of inhibition and binding mode of two potential inhibitors, sinefungin and compound 5, to the NS5 methyltransferase. The specific pharmacophoric moieties of the most stable inhibitor are also identified in this study. Chapter 6 is the final study of this thesis, which examines the structural dynamics of the Zika virus NS3 helicase enzyme upon binding of ATPase inhibitor and flavivirus lead compound, resveratrol, and reports the key interactions and amino acid residues of the NS3 helicase that contribute highly to binding of resveratrol. This thesis presents an all-inclusive in silico assessment to advance research in drug design and development of Zika virus inhibitors, thus providing a greater understanding of the structural dynamics that occur in unbound and inhibitor-bound Zika virus target enzymes. Therefore, the constituents of this thesis are considered an essential platform in the progression of research toward anti-ZIKV drug design, discovery and delivery against Zika virus

In silico and in vitro antiviral activity evaluation of prodigiosin from Serratia marcescens against enterovirus 71

Prodigiosin, a red linear tripyrrole pigment found in Serratia marcescens, is one such naturally occurring compound that has gained wide attention owing to its numerous biological activities, including antibacterial, antifungal, antimalarial, anticancer, and immunosuppressive properties. This study was conducted to evaluate the possible antiviral activity of prodigiosin against Enterovirus 71, a causative agent of hand, foot, and mouth disease (HFMD). Preliminary studies were done in silico by analyzing the interaction of prodigiosin with amino acid residues of five EV71-target proteins. Interaction refinement analysis with FireDock revealed that 2C helicase (-48.01 kcal/moL) has the most negative global energy, followed by capsid (-36.52 kcal/moL), 3C protease (-34.16 kcal/moL), 3D RNA polymerase (-30.93 kcal/moL) and 2A protease (-20.61 kcal/moL). These values are indicative of the interaction strength. Prodigiosin was shown to form chemical bonds with specific amino acid residues in capsid (Gln-30, Asn-223), 2A protease (Trp-33, Trp-142), 2C helicase (Tyr-150, His-151, Gln-169, Ser-212), 3C protease (Glu-50), and 3D RNA polymerase (Ala-239, Tyr-237). To investigate further, prodigiosin was extracted from S. marcescens using a methanolic extraction method. In vitro studies revealed that prodigiosin, with an IC50 value of 0.5112 μg/mL, reduced virus titers by 0.17 log (32.39%) in 30 min and 0.19 log (35.43%) in 60 min. The findings suggest that prodigiosin has antiviral activity with an intermediate inhibitory effect against EV71. As a result of this research, new biological activities of prodigiosin have been identified

Synthesis and biological evaluation of nitrogen heterocycle systems as potential antiviral agents

Viruses are obligate intracellular parasites that consist of either double- or single-stranded DNA or RNA enclosed in a protein coat called capsid. Some viruses also possess a lipid envelope that, like the capsid, may contain antigenic glycoproteins. Most of them contain or encode enzymes essential for their replicative cycle inside host cells, sometimes usurping their metabolic machinery. Traditional therapeutic approaches have mostly focused on targeting specific viral components or enzymes. This pathogen-directed strategy, while successful in numerous cases, in many others results ineffective due to the emergence of drug-resistance. A different approach, addressed to target host-factors essential for viral replication, has recently draw an increasing attention. My PhD project aimed at synthesizing new nitrogen heterocycle systems, designed especially against RNA viruses, such as those belonging to Flaviviridae, Orthomyxoviridae and Paramyxoviridae families. Among them there are, respectively, pathogens responsible for diseases with a high epidemiological impact, as BVDV in cattle and HCV in humans, influenza A and B viruses and respiratory syncytial virus (RSV). The project has been organized into the following phases: 1. Chemical synthesis of the novel compound series. During my PhD I designed and synthesized diverse chemical series of different chemotypes, in order to obtain new antiviral agents: the acridine nucleus, the dihydrotriazine scaffold, the benzimidazole ring as well as anilino and benzenesulfonamide derivatives. Previous studies performed by the research group where I develop my Ph.D. thesis identified some prototypes for the different classes endowed with intrinsic antiviral activity; thus, during my Ph.D. research work I explored various possibilities of functionalisation with the aim of increasing their potency and selectivity profiles towards the respective antiviral target. 2. Characterization of the new compounds. Each newly synthesized compound have been characterised by spectroscopic methods (such as UV, IR, 1H-NMR and 13C-NMR) and elemental analysis. 3. Evaluation of cytotoxicity, antiviral activity in vitro, enzymatic assays and computational studies have been performed in collaboration with several national and international research groups, to assess the biological activity and to identify/confirm the respective molecular targets

Re-emergence of Neglected Tropical Diseases amid the COVID-19 Pandemic : Epidemiology, Transmission, Mitigation Strategies, and Recent Advances in Chemotherapy and Vaccines

The current re-emergence of neglected tropical diseases (NTD) amid the global COVID-19 pandemic requires increased attention. These include communicable and vector-borne diseases caused by various fungi, bacteria (e.g. tuberculosis), viruses (e.g. dengue, Chikungunya fever, monkeypox, Marburg and Ebola virus disease, poliomyelitis, rabies), and parasites (e.g. filariasis, malaria, trypanosomiasis, leishmaniasis, schistosomiasis, onchocerciasis). Whilst the vast majority of such diseases remain endemic to specific regions of the world (e.g. tropical Africa), some - like those caused by the Ebola virus, the Marburg virus, and more recently the Monkeypox virus - have been reported elsewhere (e.g. Europe and America), forcing public health boards in various countries to take all necessary precautions to control such a spread. The Department for Control of Neglected Tropical Disease was created in 2005 by the World Health Organization (WHO) to tackle NTD. In 2021, the 74th World Health Assembly proposed a 9-year plan (2021-2030) intended to eradicate neglected diseases. Over the past three years, COVID-19 has had a significant impact on socio-economic activities and healthcare systems worldwide. With the WHO recently declaring the global monkeypox outbreak a Public Health Emergency of International Concern, a coordinated effort among high-income and low/middle-income countries is now more than ever recommended to address the threat posed by the worldwide re-emergence of some NTD. There is currently a lack of knowledge on understanding how such diseases are transmitted and what mitigation strategies should be put in place to control their spread. Better availability of diagnostic tests, vaccines, and drugs in affected countries is also required. In this Research Topic, we wish to address how to best tackle the re-emergence of NTD in the context of the COVID-19 pandemic. This collection welcomes a range of articles including opinion, commentary, systematic reviews, and original research articles on epidemiology, transmission, mitigation strategies, and recent advances in chemotherapy and vaccines for these NTD