Identification of dengue viral RNA-dependent RNA polymerase inhibitor using computational fragment-based approaches and molecular dynamics study

Dengue is a major public health concern in tropical and subtropical countries of the world. There are no specific drugs available to treat dengue. Even though several candidates targeted both viral and host proteins to overcome dengue infection, they have not yet entered into the later stages of clinical trials. In order to design a drug for dengue fever, newly emerged fragment-based drug designing technique was applied. RNA-dependent RNA polymerase, which is essential for dengue viral replication is chosen as a drug target for dengue drug discovery. A cascade of methods, fragment screening, fragment growing, and fragment linking revealed the compound [2-(4-carbamoylpiperidin-1-yl)-2-oxoethyl]8-(1,3-benzothiazol-2-yl)naphthalene-1-carboxylate as a potent dengue viral polymerase inhibitor. Both strain energy and binding free energy calculations predicted that this could be a better inhibitor than the existing ones. Molecular dynamics simulation studies showed that the dengue polymerase–lead complex is stable and their interactions are consistent throughout the simulation. The hydrogen-bonded interactions formed by the residues Arg792, Thr794, Ser796, and Asn405 are the primary contributors for the stability and the rigidity of the polymerase–lead complex. This might keep the polymerase in closed conformation and thus inhibits viral replication. Hence, this might be a promising lead molecule for dengue drug designing. Further optimization of this lead molecule would result in a potent drug for dengue.     

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.     

Computational selection of flavonoid compounds as inhibitors against SARS-CoV-2 main protease,RNA-dependent RNA polymerase and spike proteins: A molecular docking study

An outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been recognized as a global health concern. Since, no specific antiviral drug is proven effective for treatment against COVID-19, identification of new therapeutics is an urgent need. In this study, flavonoid compounds were analyzed for its inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. Virtual docking was performed for screening of flavonoid compounds retrieved from PubChem against the main protease of SARS-CoV-2 using COVID-19 docking server. The cut off of dock score was set to >?9 kcal/mol and screened compounds were individually docked against main protease, RNA-dependent RNA polymerase, and spike proteins using AutoDock 4.1 software. Finally, lead flavonoid compounds were subjected to ADMET analysis. A total of 458 flavonoid compounds were virtually screened against main protease target and 36 compounds were selected based on the interaction energy value >?9 kcal/mol. Furthermore, these compounds were individually docked against protein targets and top 10 lead compounds were identified. Among the lead compounds, agathisflavone showed highest binding energy value of ?8.4 kcal/mol against main protease, Albireodelphin showed highest dock score of ?9.8 kcal/mol and ?11.2 kcal/mol against RdRp, and spike proteins, respectively. Based on the high dock score and ADMET properties, top 5 lead molecules such as Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate were identified as potent inhibitors against main protease, RdRp, and spike protein targets of SARS-CoV-2. These all compounds are having non-carcinogenic and non-mutagenic properties. This study finding suggests that the screened compounds include Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate could be the potent inhibitors of SARS-CoV-2 targets.     

Substrate/inhibition studies of bacteriophage T7 RNA polymerase with the 5'-triphosphate derivative of a ring-expanded ('fat') nucleoside possessing potent antiviral and anticancer activities

As part of an effort to explore the mechanism of potent, broad spectrum antiviral and anticancer activities of a number of ring-expanded ('fat') nucleosides that we recently reported, a representative 'fat' nucleoside 4,6-diamino-8-imino-8H-1-beta-D-ribofuranosylimidazo[4,5-e][1,3]di azepine (1) was converted to its 5'-triphosphate derivative (2), and biochemically screened for possible inhibition of nucleic acid polymerase activity, employing synthetic DNA templates and the bacteriophage T7 RNA polymerase as a representative polymerase. Our results suggest that 2 is a moderate inhibitor of T7 RNA polymerase, and that the 5'-triphosphate moiety of 2 appears to be essential for inhibition as nucleoside 1 alone failed to inhibit the polymerase reaction.     

Coronaviral RNA-methyltransferases: function,structure and inhibition

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.     

Backbone 1H, 13C and 15N resonance assignments of dengue virus NS2B–NS3p in complex with aprotinin

Dengue virus, belongs to Flaviviridae, is an arthropod transmitted virus that threatens millions of people’s lives. As with other flaviviruses, a positive single-stranded 11-kilobases RNA in the dengue virus genome encodes three structural proteins (capsid protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The two component protease NS2B–NS3p is essential for viral replication and is believed to be a potential antiviral drug target. Aprotinin, a native inhibitor, is proved to retard the activity of NS2B–NS3p. The backbone assignments of NS2B–NS3p will be essential for determining the high resolution solution structure of NS2B–NS3p and screening new antiviral drugs. Herein, we report the backbone ¹H, ¹⁵N, ¹³C resonance assignments of the N terminal fragment of NS2B (4.8 kDa) and NS3p (18.5 kDa) in complex with aprotinin (6.5 kDa) by high resolution NMR.     

A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan

ABSTRACT: Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.     

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.     

Identification of the phospholipid binding regions of the envelope E protein of flaviviruses by molecular dynamics

Abstract

The Flavivirus genus comprise several important human pathogens, including dengue, West Nile, Yellow fever, Japanese encephalitis, Zika, and tick-borne encephalitis viruses. These enveloped viruses affect more than 2 billion people in the world, mainly in less developed countries. Although some vaccines exist for some flaviviruses, these vaccines are not universally available due to many factors and since their infections are a world-wide public health issue, the development of antiviral molecules is fundamental. Flavivirus membranes, through the help of the envelope E glycoprotein, fuse with endosomal compartments in a pH-dependent way to release their genome into the cytoplasm and require specific lipids, such as bis(monoacylglycero)phosphate (BMP), for efficient fusion. The fundamental role the envelope E protein has on viral entry and membrane fusion suggest that it is an essential antiviral target. In this work, we have used atomistic molecular dynamics simulations to study the binding of the head-group of BMP to the tip of the envelope E proteins of ZIKV, DENV, TBEV and JEV viruses whose three-dimensional structures are known. Our results indicate that, apart from the fusion loop, there are different amino acid residues in different regions of the envelope E proteins of flaviviruses capable of binding the head-group of BMP. These regions should work together to accomplish the binding and fusion of the envelope and endosomal membranes and represent a new target to develop and design potent and effective antiviral agents capable of blocking flavivirus-endosome membrane fusion.      

Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

Structure of hepatitis C virus polymerase in complex with primer-template RNA

The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.     

In Vivo Therapeutic Protection against Influenza A (H1N1) Oseltamivir-Sensitive and Resistant Viruses by the Iminosugar UV-4

Our lead iminosugar analog called UV-4 or N-(9-methoxynonyl)-1-deoxynojirimycin inhibits activity of endoplasmic reticulum (ER) α-glucosidases I and II and is a potent, host-targeted antiviral candidate. The mechanism of action for the antiviral activity of iminosugars is proposed to be inhibition of ER α-glucosidases leading to misfolding of critical viral glycoproteins. These misfolded glycoproteins would then be incorporated into defective virus particles or targeted for degradation resulting in a reduction of infectious progeny virions. UV-4, and its hydrochloride salt known as UV-4B, is highly potent against dengue virus in vitro and promotes complete survival in a lethal dengue virus mouse model. In the current studies, UV-4 was shown to be highly efficacious via oral gavage against both oseltamivir-sensitive and -resistant influenza A (H1N1) infections in mice even if treatment was initiated as late as 48-72 hours after infection. The minimal effective dose was found to be 80-100 mg/kg when administered orally thrice daily. UV-4 treatment did not affect the development of protective antibody responses after either influenza infection or vaccination. Therefore, UV-4 is a promising candidate for further development as a therapeutic intervention against influenza.     

Asunaprevir,a Potent Hepatitis C Virus Protease Inhibitor,Blocks SARS-CoV-2 Propagation

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a global health concern. Various SARS-CoV-2 vaccines have been developed and are being used for vaccination worldwide. However, no therapeutic agents against coronavirus disease 2019 (COVID-19) have been developed so far; therefore, new therapeutic agents are urgently needed. In the present study, we evaluated several hepatitis C virus direct-acting antivirals as potential candidates for drug repurposing against COVID-19. Theses include asunaprevir (a protease inhibitor), daclatasvir (an NS5A inhibitor), and sofosbuvir (an RNA polymerase inhibitor). We found that asunaprevir, but not sofosbuvir and daclatasvir, markedly inhibited SARS-CoV-2-induced cytopathic effects in Vero E6 cells. Both RNA and protein levels of SARS-CoV-2 were significantly decreased by treatment with asunaprevir. Moreover, asunaprevir profoundly decreased virion release from SARS-CoV-2-infected cells. A pseudoparticle entry assay revealed that asunaprevir blocked SARS-CoV-2 infection at the binding step of the viral life cycle. Furthermore, asunaprevir inhibited SARS-CoV-2 propagation in human lung Calu-3 cells. Collectively, we found that asunaprevir displays broad-spectrum antiviral activity and therefore might be worth developing as a new drug repurposing candidate for COVID-19.     

HCV NS5B polymerase-bound conformation of a soluble sulfonamide inhibitor by 2D transferred NOESY

HCV NS5B RNA-dependent RNA polymerase (NS5B) is essential for viral replication and is therefore considered a target for antiviral drug development. From our ongoing screening effort in the search for new anti-HCV agents, a novel inhibitor 1 with low microM activity against the HCV NS5B polymerase was identified. SAR analysis indicated the optimal substitution pattern required for activity, for example, carboxylic acid group at 2-position of thiophene ring. We describe the steps taken to identify and solve the bioactive conformation of derivative 6 through the use of the transferred NOE method (trNOE).     

Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease

Background

Due to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug.

Results

In this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties.

Conclusion

We hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures.

     

Inhibition of hepatitis B virus replication by cIAP2 involves accelerating the ubiquitin-proteasome-mediated destruction of polymerase

Cellular inhibitor of apoptosis protein 2 (cIAP2) is a potent suppressor of apoptotic cell death. We have shown previously that cIAP2 is involved in the tumor necrosis factor alpha (TNF-α)-induced anti-hepatitis B virus (HBV) response; however, the mechanism for this antiviral effect remains unclear. In the present study, we demonstrate that cIAP2 can significantly reduce the levels of HBV DNA replication intermediates but not the total viral RNA or core protein levels. Domain-mapping analysis revealed that the carboxy-terminal domains of cIAP2 were indispensable for this anti-HBV ability and that an E3 ligase-deficient mutant of cIAP2 (termed cIAP2*) completely lost its antiviral activity. We further identified HBV polymerase as the target of cIAP2. Overexpression of cIAP2 but not cIAP2* reduced polymerase protein levels, while cIAP2 knockdown increased polymerase expression. In addition, we observed that cIAP2 promoted the degradation of the viral polymerase through a proteasome-dependent pathway. Further experiments demonstrated that cIAP2 can bind to polymerase and promote its polyubiquitylation. Finally, we found that cIAP2 downregulated the encapsidation of HBV pregenomic RNA. Taken together, these data reveal a novel mechanism for the inhibition of HBV replication by cIAP2 via acceleration of the ubiquitin-proteasome-mediated decay of polymerase and reduction of the encapsidation of HBV pregenomic RNA, making this mechanism a novel strategy for HBV therapy.