Exploring Ugi-Azide Four-Component Reaction Products for Broad-Spectrum Influenza Antivirals with a High Genetic Barrier to Drug Resistance

Abstract

Influenza viruses are respiratory pathogens that are responsible for seasonal influenza and sporadic influenza pandemic. The therapeutic efficacy of current influenza vaccines and small molecule antiviral drugs is limited due to the emergence of multidrug-resistant influenza viruses. In response to the urgent need for the next generation of influenza antivirals, we utilized a fast-track drug discovery platform by exploring multi-component reaction products for antiviral drug candidates. Specifically, molecular docking was applied to screen a small molecule library derived from the Ugi-azide four-component reaction methodology for inhibitors that target the influenza polymerase PAC-PB1N interactions. One hit compound 5 was confirmed to inhibit PAC-PB1N interactions in an ELISA assay and had potent antiviral activity in an antiviral plaque assay. Subsequent structure-activity relationship studies led to the discovery of compound 12a, which had broad-spectrum antiviral activity and a higher in vitro genetic barrier to drug resistance than oseltamivir. Overall, the discovery of compound 12a as a broad-spectrum influenza antiviral with a high in vitro genetic barrier to drug resistance is significant, as it offers a second line of defense to combat the next influenza epidemics and pandemics if vaccines and oseltamivir fail to confine the disease outbreak.

Potent and broad-spectrum cycloheptathiophene-3-carboxamide compounds that target the PA-PB1 interaction of influenza virus RNA polymerase and possess a high barrier to drug resistance

“…During the preparation of this manuscript, a study reporting the identification of a new PA-PB1 inhibitor of influenza virus as well as the evaluation of the barrier to drug resistance of this compound was published (Zhang et al, 2018). In keeping with our findings, Zhang and coworkers showed that OST rapidly induced the selection of resistant viral variants, while no viruses with altered susceptibility to the PA-PB1 inhibitor were isolated.…”

“…In keeping with our findings, Zhang and coworkers showed that OST rapidly induced the selection of resistant viral variants, while no viruses with altered susceptibility to the PA-PB1 inhibitor were isolated. However, they stopped the selection process at P10 (Zhang et al, 2018), while we carried out a prolonged drug-resistance selection for a total of 20/30 viral passages. Altogether, our data and the results obtained by Zhang et al suggest that drug resistance in vitro to dissociative inhibitors targeting the PA-PB1 interactions of influenza virus RdRP appears hard to develop, differently from what happens with the current anti-influenza drugs such as adamantanes and neuraminidase inhibitors and also with PA and PB2 enzymatic inhibitors approved or under clinical development (Hay et al, 1985;Molla et al, 2002;McKimm-Breschkin et al, 2012;Byrn et al, 2015;Noshi et al, 2018).…”

“…To evaluate the barrier to drug resistance of selected cHTC compounds (29, 31, and 54) and OST as a reference compound, IAV PR/8 strain was serially passaged in the presence of increasing concentrations of test compounds following a procedure previously described (Zhang et al, 2018).…”

“…To evaluate the propensity of this class of inhibitors to induce drug resistance, we tried to select in vitro influenza viruses resistant to the most promising compounds under drug selective pressure, following a procedure similar to that previously employed for other anti-influenza compounds, including PA-PB1 inhibitors (Molla et al, 2002;Shih et al, 2010;Ma et al, 2016;Hu et al, 2017;Zhang et al, 2018). The cHTC compounds that both inhibited the viral replication in PRAs with EC50 values ≤ 1 µM and disrupted specifically the PA-PB1 interaction in ELISA with IC50 values < 20 µM were selected for these studies.…”

“…Since these interactions are essential for viral replication and their binding interfaces are highly conserved among IAV and IBV strains (Pérez and Donis, 1995;Ghanem et al, 2007;Stevaert and Naesens, 2016), the disruption of such interactions represents an attractive strategy for the development of anti-influenza drugs with broad activity (Loregian et al, 2002;Loregian and Palù, 2005;Palù and Loregian, 2013;Loregian et al, 2014;Stevaert and Naesens, 2016;Massari et al, 2016). The feasibility and the effectiveness of such an approach have been demonstrated by the increasing number of PA-PB1 dissociative inhibitors developed in the last decade (Wunderlich et al, 2009;Wunderlich et al, 2011;Muratore et al, 2012a;Muratore et al, 2012b;Fukuoka et al, 2012;Massari et al, 2013;Kessler et al, 2013;Lepri et al, 2014;Pagano et al, 2014;Tintori et al, 2014;Massari et al, 2015;Trist et al, 2016;Yuan et al, 2016;Desantis et al, 2017;Watanabe et al, 2017;Massari et al, 2017;Zhang et al, 2018;D'Agostino et al, 2018).…”