Characterization of the mechanism of action of new HIV-1 reverse transcriptase-associated ribonuclease H inhibitors

HIV-1 the Reverse Transcriptase (RT), the most renowned retroviral specific enzyme, was the first anti-HIV target to be exploited, as. HIV-1 RT combines two functions essential for viral replication: DNA polymerase, synthesis DNA either in a RNA dependent (RDDP) or DNA dependent (DDDP) manner, and Ribonuclease H (RNase H) . The RNase H activity catalyzes highly specific hydrolytic events on the RNA strand of the RNA/DNA replication intermediate, critical to the synthesis of integration-competent double-stranded proviral DNA. Because of its essential role, RNase H is a promising target for drug development. However, despite years of efforts, no RNase H inhibitor (RHI) has yet reached clinical approval. In this work we pursued the identification and characterization of new promising RHIs targeting either the RNase H active site itself (RNase H active site chelating agents) or both RNase H and RDDP activities (allosteric dual inhibitors). The first approach faced the challenging nature of the RNase H active site region, the morphology of wich is, more open than that of the relatively similar,HIV-1 integrase (IN). This hampers the identification of a druggable pocket. We initially used Foamy Virus RT as a tool, to perform NMR and docking analyses on the interaction between FV RT RNase H domain and a previously identified diketo acid (DKA) derivative, inhibitor RDS1643. The amino acid residues of the FV RNase H active site region (T641, I647, Y672 and W703) were established to be important for the interaction with the inhibitor and analogous residues were successfully identified in the HIV-1 RNase H domain using structural overlays. Further docking and site directed mutagenesis studies were performed using six couples of ester/acid DKA, derived from RDS1643, showing for the first time, a broad interaction between RHIs and conserved residues in the HIV-1 RNase H active site region (R448, N474, Q475, Y501 and R557). Moreover, ester and acid derivatives exhibited a different binding orientation, that reflected a different specificity for RNase H versus IN. Among the synthesised derivatives one, RDS1759, showed to be an RNase H selective active site inhibitor characterized also, for the first time,in cell-based assays. The second approach focused on the determination of the mechanism of action of a new isatine-derived RNase H/RDDP dual inhibitor, RMNC6. Docking analysis and site directed mutagenesis results suported the hypothesis of a two-sites mode of action, with an independent role for two pockets,to be further characterized for a rational optimization of the scaffold

Ribonuclease H, an unexploited target for antiviral intervention against HIV and hepatitis B virus

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-hydroxyixoquinolinediones 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 activity, broad specificity and efforts to decrease their toxicity

Insights into Ebola Virus VP35 and VP24 Interferon inhibitory functions and their initial exploitation as drug targets

Upon viral infection, the interferon (IFN) system triggers potent antiviral mechanisms limiting viral growth and spread. Hence, to sustain their infection, viruses evolved efficient counteracting strategies to evade IFN control. Ebola virus (EBOV), member of the family Filoviridae, is one of the most virulent and deadly pathogen ever faced by humans. Etiological agent of the Ebola virus disease (EVD), EBOV can be undoubtedly considered the perfect example of a powerful inhibitor of the host organism immune response activation. Particularly, the efficacious suppression of the IFN cascade contributes to disease progression and severity. Among the EBOV-encoded proteins, the viral proteins 35 (VP35) and 24 (VP24) are responsible for the EBOV extreme virulence, representing the core of such inhibitory function through which EBOV determines its very effective shield to the cellular immune defenses. VP35 inhibits the activation of the cascade leading to IFN production, while VP24 inhibits the activation of the IFN-stimulated genes. A number of studies demonstrated that both VP35 and VP24 are validated target for drug development. Insights of the structural characteristics of VP35 and VP24 domains revealed crucial pockets exploitable for drug development. Considered the lack of therapy for EVD, restoring the immune activation is a promising approach for drug development. In the present review we summarize the importance of VP35 and VP24 proteins in counteracting the host IFN cellular response and discuss their potential as druggable viral targets as a promising approach toward attenuation of EBOV virulence

Natural and Nature-Derived Products Targeting Human Coronaviruses

The ongoing pandemic of severe acute respiratory syndrome (SARS), caused by the SARS-CoV-2 human coronavirus (HCoV), has brought the international scientific community before a state of emergency that needs to be addressed with intensive research for the discovery of pharmacological agents with antiviral activity. Potential antiviral natural products (NPs) have been discovered from plants of the global biodiversity, including extracts, compounds and categories of compounds with activity against several viruses of the respiratory tract such as HCoVs. However, the scarcity of natural products (NPs) and small-molecules (SMs) used as antiviral agents, especially for HCoVs, is notable. This is a review of 203 publications, which were selected using PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar, evaluates the available literature since the discovery of the first human coronavirus in the 1960s; it summarizes important aspects of structure, function, and therapeutic targeting of HCoVs as well as NPs (19 total plant extracts and 204 isolated or semi-synthesized pure compounds) with anti-HCoV activity targeting viral and non-viral proteins, while focusing on the advances on the discovery of NPs with anti-SARS-CoV-2 activity, and providing a critical perspective

Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

We analyzed a multi-drug resistant (MR) HIV-1 re- verse transcriptase (RT), subcloned from a patient- derived subtype CRF02 AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azi- dothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to an- tagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most signif- icant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimi- nation, indicating that the two mechanisms are mu- tually exclusive and that the Q151M pathway is ob- viously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, ad- ditionally harboring the TAM K70R and the rever- sions M151Q as well as R65K since K65R antago- nizes excision. MR-R65K-K70R-M151Q was compe- tent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still pro- moted discrimination. To tackle the multi-drug resis- tance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors be- longing to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs

Garcinol from Garcinia indica inhibits HIV-1 reverse transcriptase-associated ribonuclease H

The bioactive components of Garcinia indica, garcinol (camboginol), and isogarcinol (cambogin), are suitable drug candidates for the treatment of various human diseases. HIV-1-RNase H assay was used to study the RNase H inhibition by garcinol and isogarcinol. Docking of garcinol into the active site of the enzyme was carried out to rationalize the difference in activities between the two compounds. Garcinol showed higher HIV-1-RNase H inhibition than the known inhibitor RDS1759 and retained full potency against the RNase H of a drug-resistant HIV-1 reverse transcriptase form. Isogarcinol was distinctly less active than garcinol, indicating the importance of the enolizable β-diketone moiety of garcinol for anti-RNase H activity. Docking calculations confirmed these findings and suggested this moiety to be involved in the chelation of metal ions of the active site. On the basis of its HIV-1 reverse transcriptase-associated RNase H inhibitory activity, garcinol is worth being further explored concerning its potential as a cost-effective treatment for HIV patients

The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBO

Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action

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

Suramin inhibits SARS-CoV-2 nucleocapsid phosphoprotein genome packaging function

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines - a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals