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For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks., Competing Interests: Declaration of competing interest Authors AC, SD, GLD, AP, and EZ declare no conflict of interest. DS is the Medical Director of AIM ImmnunoTech. WMM is a member of Board of Directors for AIM ImmunoTech. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-β production. Blocking VP35-dsRNA interaction and IFN-β suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC 50 value of 8.5 μM. It reverts the EBOV VP35 inhibition of IFN-β production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a K D of 12 μM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC 50 value of 9.1 μM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The management of Human Immunodeficiency Virus type 1 (HIV-1) infection requires life-long treatment that is associated with chronic toxicity and possible selection of drug-resistant strains. A new opportunity for drug intervention is offered by antivirals that act as allosteric inhibitors targeting two viral functions (dual inhibitors). In this work, we investigated the effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derivatives on both HIV-1 Integrase (IN) and Reverse Transcriptase associated Ribonuclease H (RNase H) activities. Among the tested compounds, the dihydroxyindole-carboxamide 5 was able to inhibit in the low micromolar range (1-18 μM) multiple functions of IN, including functional IN-IN interactions, IN-LEDGF/p75 binding and IN catalytic activity. Docking and site-directed mutagenesis studies have suggested that compound 5 binds to a previously described HIV-1 IN allosteric pocket. These observations indicate that 5 is structurally and mechanistically distinct from the published allosteric HIV-1 IN inhibitors. Moreover, compound 5 also inhibited HIV-1 RNase H function, classifying this molecule as a dual HIV-1 IN and RNase H inhibitor able to impair the HIV-1 virus replication in cell culture. Overall, we identified a new scaffold as a suitable platform for the development of novel dual HIV-1 inhibitors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ribonucleases H (RNases H) are endonucleolytic enzymes, evolutionarily related to retroviral integrases, DNA transposases, resolvases and numerous nucleases. RNases H cleave RNA in RNA/DNA hybrids and their activity plays an important role in the replication of prokaryotic and eukaryotic genomes, as well as in the replication of reverse-transcribing viruses. During reverse transcription, the RNase H activity of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) degrades the viral genomic RNA to facilitate the synthesis of viral double-stranded DNA. HIV and HBV reverse transcriptases contain DNA polymerase and RNase H domains that act in a coordinated manner to produce double-stranded viral DNA. Although RNase H inhibitors have not been developed into licensed drugs, recent progress has led to the identification of a number of small molecules with inhibitory activity at low micromolar or even nanomolar concentrations. These compounds can be classified into metal-chelating active site inhibitors and allosteric inhibitors. Among them, α-hydroxytropolones, N-hydroxyisoquinolinediones and N-hydroxypyridinediones represent chemotypes active against both HIV and HBV RNases H. In this review we summarize recent developments in the field including the identification of novel RNase H inhibitors, compounds with dual inhibitory activity, broad specificity and efforts to decrease their toxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The 32nd International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held in Baltimore, Maryland, USA, on May 12-15, 2019. This report gives an overview of the conference on behalf of the Society. It provides a general review of the meeting and awardees, summarizing the presentations, and their main conclusions from the perspective of researchers active in many different areas of antiviral research and development. As in past years, ICAR promoted and showcased the most recent progress in antiviral research, and continued to foster collaborations and interactions in drug discovery and development. The 33rd ICAR will be held in Seattle, Washington, USA, March 30th-April 3rd, 2020., (Copyright © 2019 Elsevier B.V. All rights reserved.)

HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg 2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Double-stranded RNA (dsRNA) is synthesized during the course of infection by RNA viruses as a byproduct of replication and transcription and acts as a potent trigger of the host innate antiviral response. In the cytoplasm of the infected cell, recognition of the presence of viral dsRNA as a signature of "non-self" nucleic acid is carried out by RIG-I-like receptors (RLRs), a set of dedicated helicases whose activation leads to the production of type I interferon α/β (IFN-α/β). To overcome the innate antiviral response, RNA viruses encode suppressors of IFN-α/β induction, which block RLRs recognition of dsRNA by means of different mechanisms that can be categorized into: (i) dsRNA binding and/or shielding ("hide"), (ii) dsRNA termini processing ("mask") and (iii) direct interaction with components of the RLRs pathway ("hit"). In light of recent functional, biochemical and structural findings, we review the inhibition mechanisms of RLRs recognition of dsRNA displayed by a number of highly pathogenic RNA viruses with different disease phenotypes such as haemorrhagic fever (Ebola, Marburg, Lassa fever, Lujo, Machupo, Junin, Guanarito, Crimean-Congo, Rift Valley fever, dengue), severe respiratory disease (influenza, SARS, Hendra, Hantaan, Sin Nombre, Andes) and encephalitis (Nipah, West Nile)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) is a multifunctional enzyme which displays DNA polymerase activity, which recognizes RNA and DNA templates, and a degradative ribonuclease H (RNase H) activity. While both RT functions are required for retroviral replication, until now only the polymerase function has been widely explored as drug target. We have identified a novel diketo acid derivative, 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS 1643), which inhibits in enzyme assays the HIV-1 RT-associated polymerase-independent RNase H activity but has no effect on the HIV-1 RT-associated RNA-dependent DNA polymerase (RDDP) activity and on the RNase H activities displayed by the Avian Myeloblastosis Virus and E. coli. Time-dependence studies revealed that the compound is active independently on the order of its addition to the reaction mixture, and inhibition kinetics studies demonstrated that RDS 1643 inhibits the RNase H activity noncompetitively, with a K(I) value of 17 microM. When RDS 1643 was combined with non-nucleoside RT inhibitors (NNRTI), such as efavirenz and nevirapine, results indicated that RDS 1643 does not affect the NNRTIs anti-RDDP activity and that, vice versa, the NNRTIs do not alter the RNase H inhibition by RDS 1643. When assayed on the viral replication in cell-based assays, RDS 1643 inhibited the HIV-1(IIIB) strain with an EC(50) of 14 microM. Similar results were obtained against the Y181C and Y181C/K103N HIV-1 NNRTI resistant mutant strains. RDS 1643 may be the first HIV-1 inhibitor selectively targeted to the viral RT-associated RNase-H function.