Synthesis, inhibitory activity and oral dosing formulation of AV5124, the structural analogue of influenza virus endonuclease inhibitor baloxavir.

BACKGROUND: The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. OBJECTIVES: To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. METHODS: The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. RESULTS: Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. CONCLUSIONS: Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

Non-rigid Diarylmethyl Analogs of Baloxavir as Cap-Dependent Endonuclease Inhibitors of Influenza Viruses.

4-Substituted 2,4-dioxobutanoic acids inhibit influenza virus cap-dependent endonuclease (CEN) activity. Baloxavir marboxil, 4, is approved for treating influenza virus infections. We describe here the synthesis and biological evaluation of active compounds, 5a-5g, and their precursors (6a, 6b, 6d, and 6e) with flexible bulky hydrophobic groups instead of the rigid polyheterocyclic moieties. In silico docking confirmed the ability of 5a-5g to bind to the active site of influenza A CEN (PDB code: 6FS6) like baloxavir acid, 3. These novel compounds inhibited polymerase complex activity, inhibited virus replication in cells, prevented death in a lethal influenza A virus mouse challenge model, and dramatically lowered viral lung titers. 5a and 5e potently inhibited different influenza genera in vitro. Precursors 6a and 6d demonstrated impressive mouse oral bioavailability with 6a, providing effective in vivo protection. Thus, these novel compounds are potent CEN inhibitors with in vitro and in vivo activity comparable to baloxavir.

The Comparative Analysis of Antiviral Activity of Native and Modified Fucoidans from Brown Algae Fucus evanescens In Vitro and In Vivo.

The enzymatic depolymerization of fucoidans from brown algae allowed the production of their standardized derivatives with different biological activities. This work aimed to compare the antiviral activities of native (FeF) and modified with enzyme (FeHMP) fucoidans from F. evanescens. The cytotoxicity and antiviral activities of the FeF and FeHMP against herpes viruses (HSV-1, HSV-2), enterovirus (ECHO-1), and human immunodeficiency virus (HIV-1) in Vero and human MT-4 cell lines were examined by methylthiazolyltetrazolium bromide (MTT) and cytopathic effect (CPE) reduction assays, respectively. The efficacy of fucoidans in vivo was evaluated in the outbred mice model of vaginitis caused by HSV-2. We have shown that both FeF and FeHMP significantly inhibited virus-induced CPE in vitro and were more effective against HSV. FeF exhibited antiviral activity against HSV-2 with a selective index (SI) > 40, and FeHMP with SI ˃ 20, when they were added before virus infection or at the early stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that after intraperitoneal administration (10 mg/kg), both FeF and FeHMP protected mice from lethal intravaginal HSV-2 infection to approximately the same degree (44-56%). Thus, FeF and FeHMP have comparable potency against several DNA and RNA viruses, allowing us to consider the studied fucoidans as promising broad-spectrum antivirals.

Off-target effects of an insect cell-expressed influenza HA-pseudotyped Gag-VLP preparation in limiting postinfluenza Staphylococcus aureus infections.

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.

The Potential of Influenza HA-Specific Immunity in Mitigating Lethality of Postinfluenza Pneumococcal Infections.

: Influenza virus infections pre-dispose an individual to secondary pneumococcal infections, which represent a serious public health concern. Matching influenza vaccination was demonstrated helpful in preventing postinfluenza bacterial infections and associated illnesses in humans. Yet, the impact of influenza hemagglutinin (HA)-specific immunity alone in this dual-infection scenario remains elusive. In the present study, we assessed the protective effect of neutralizing and non-neutralizing anti-hemagglutinin immunity in a BALB/c influenza-pneumococcus superinfection model. Our immunogens were insect cell-expressed hemagglutinin-Gag virus-like particles that had been differentially-treated for the inactivation of bioprocess-related baculovirus impurities. We evaluated the potential of several formulations to restrain the primary infection with vaccine-matched or -mismatched influenza strains and secondary bacterial replication. In addition, we investigated the effect of anti-HA immunity on the interferon status in mouse lungs prior to bacterial challenge. In our experimental setup, neutralizing anti-HA immunity provided significant but incomplete protection from postinfluenza bacterial superinfection, despite effective control of viral replication. In view of this, it was surprising to observe a survival advantage with non-neutralizing adaptive immunity when using a heterologous viral challenge strain. Our findings suggest that both neutralizing and non-neutralizing anti-HA immunity can reduce disease and mortality caused by postinfluenza pneumococcal infections.

Umifenovir susceptibility monitoring and characterization of influenza viruses isolated during ARBITR clinical study.

Antiviral drugs can play a significant role in the control of influenza. Umifenovir (Arbidol) is licensed and widely used in Russia for the prophylaxis and/or treatment of influenza. We evaluated the susceptibility to umifenovir of reference influenza A and B viruses and influenza A viruses isolated from patients in the ARBITR clinical trial in 2012-2014 seasons. Using an MDCK cell-based enzyme-linked immunosorbent assay (ELISA), we showed that the replication of antigenically dominant human influenza A and B viruses was efficiently inhibited by umifenovir. The wild-type А/Perth/265/2009 (H1N1)pdm09, A/Fukui/45/2004 (H3N2), and B/Perth/211/2001 viruses and their oseltamivir-resistant counterparts were susceptible to umifenovir among in vitro laboratory assays. All 18 clinical isolates of influenza A viruses obtained before and during therapy were susceptible to umifenovir with 50% effective concentration (EC 50 ) ranging from 8.4 ± 1.1 to 17.4 ± 5.4 µM. No molecular markers of umifenovir resistance were identified in influenza viruses isolate d from patients at 3, 5, and 7 days after initiation of therapy. None of the viruses isolated before and during umifenovir therapy displayed reduced susceptibility to neuraminidase (NA) inhibitors. Thus, umifenovir is effective against influenza viruses circulating in 2012-2014 seasons, and therapy did not lead to the emergence of drug-resistant variants.