Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro, including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits Cellular Entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7, and B.1.351 Variants.

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 µM, in contrast to an IC50 of 28.3 µM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 µM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 µM). Notably, (-)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.

Design, synthesis and screening of a drug discovery library based on an Eremophila-derived serrulatane scaffold.

Chemical studies of the aerial parts of the Australian desert plant Eremophila microtheca afforded the targeted and known diterpenoid scaffolds, 3,7,8-trihydroxyserrulat-14-en-19-oic acid and 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid. The most abundant serrulatane scaffold was converted to the poly-methyl derivatives, 3-hydroxy-7,8-dimethoxyserrulat-14-en-19-oic acid methyl ester and 3,7,8-trimethoxyserrulat-14-en-19-oic acid methyl ester using simple and rapid methylation conditions consisting of DMSO, NaOH and MeI at room temperature. Subsequently a 12-membered amide library was synthesised by reacting the methylated scaffolds with a diverse series of commercial primary amines. The chemical structures of the 12 undescribed semi-synthetic analogues were fully characterised following 1D/2D NMR, MS, UV, ECD and [α]D data analyses. All compounds were evaluated for their anthelmintic, anti-microbial and anti-viral activities. While none of the compounds significantly inhibited motility or development of the exsheathed third-stage larvae (xL3s) of a pathogenic ruminant parasite, Haemonchus contortus, the tri-methylated analogue induced a skinny phenotype in fourth-stage larvae (L4s) after seven days of treatment (IC50 = 14 µM). Anti-bacterial and anti-fungal activities were not observed at concentrations up to 20 µM. Activity against HIV latency reversal was tested in inducible, chronically-infected cells, with the tri-methylated analogue being the most active (EC50 = 38 µM).

Cross-Reactive Dengue Virus Antibodies Augment Zika Virus Infection of Human Placental Macrophages.

Zika virus (ZIKV), which emerged in regions endemic to dengue virus (DENV), is vertically transmitted and results in adverse pregnancy outcomes. Antibodies to DENV can cross-react with ZIKV, but whether these antibodies influence ZIKV vertical transmission remains unclear. Here, we find that DENV antibodies increase ZIKV infection of placental macrophages (Hofbauer cells [HCs]) from 10% to over 80% and enhance infection of human placental explants. ZIKV-anti-DENV antibody complexes increase viral binding and entry into HCs but also result in blunted type I interferon, pro-inflammatory cytokine, and antiviral responses. Additionally, ZIKV infection of HCs and human placental explants is enhanced in an immunoglobulin G subclass-dependent manner, and targeting FcRn reduces ZIKV replication in human placental explants. Collectively, these findings support a role for pre-existing DENV antibodies in enhancement of ZIKV infection of select placental cell types and indicate that pre-existing immunity to DENV should be considered when addressing ZIKV vertical transmission.

Antiviral Efficacy of Verdinexor In Vivo in Two Animal Models of Influenza A Virus Infection.

Influenza A virus (IAV) causes seasonal epidemics of respiratory illness that can cause mild to severe illness and potentially death. Antiviral drugs are an important countermeasure against IAV; however, drug resistance has developed, thus new therapeutic approaches are being sought. Previously, we demonstrated the antiviral activity of a novel nuclear export inhibitor drug, verdinexor, to reduce influenza replication in vitro and pulmonary virus burden in mice. In this study, in vivo efficacy of verdinexor was further evaluated in two animal models or influenza virus infection, mice and ferrets. In mice, verdinexor was efficacious to limit virus shedding, reduce pulmonary pro-inflammatory cytokine expression, and moderate leukocyte infiltration into the bronchoalveolar space. Similarly, verdinexor-treated ferrets had reduced lung pathology, virus burden, and inflammatory cytokine expression in the nasal wash exudate. These findings support the anti-viral efficacy of verdinexor, and warrant its development as a novel antiviral therapeutic for influenza infection.

Comparison of Microchip Transponder and Noncontact Infrared Thermometry with Rectal Thermometry in Domestic Swine (Sus scrofa domestica).

During disease outbreaks, core temperature is a useful health metric in swine, due to the presence of pyrexia especially during the acute phase of infection. Despite technologic advances in other facets of swine production and health management, rectal thermometry continues to be the 'gold standard' for measuring core body temperature. However, for various reasons, collecting rectal temperatures can be difficult and unsafe depending on the housing modality. In addition, the delay between insertion of the rectal thermometer and obtaining a reading can affect measurement accuracy, especially when the pig requires physical restraint. Clearly safer, faster, and more accurate and precise temperature acquisition methods that necessitate minimal or no handling of swine are needed. We therefore compared rectal thermometers, subcutaneous microchips, and an inexpensive handheld infrared thermometer by measuring the core body temperature of 24 male castrated piglets at random intervals over a 5-wk period. The core body temperature (mean ± 1 SD) was 39.3±0.5 °C by rectal thermometry, 39.0±0.7 °C by microchip transponder, and 34.3±1.0 °C by infrared thermometry; these 3 values differed significantly. Although the readings obtain by using infrared thermometry were numerically lower than those from the other methods, it is arguably the safest method for assessing the core temperature of swine and showed strong relative correlation with rectal temperature.