Development of innovative preclinical ready pan-coronavirus antivirals: prepare to look forward (Acronym: DIRECT)

The devastating effects of the COronaVIrus Disease (COVID-19) have taught the world weakness and general unpreparedness in tackling a new viral pandemic. SARS and MERS outbreaks indeed proved that COVID-19 is not the first zoonotic coronavirus (CoV) disease that humanity has to face, and it would likely not be the last. During a CoV pandemic surge effective antivirals are fundamental in slowing down the spread of infection, saving life, and gaining time while waiting for the specific CoV vaccine development. Thus, effective pan-CoV antivirals that can be rapidly deployed against future emerging CoVs are urgently needed. This proposal aims to contribute to both COVID-19 therapeutics and future CoVs pandemic preparedness by discovering innovative pan-CoV Host-Targeting Antivirals (HTAs) to be developed up to preclinical levels, and ready to be effectively moved from a CoV urgency to the next one (Fig. 1). The human protein disulphide isomerase A3 (PDIA3 or ERp57), a cellular thiol oxidoreductase involved in protein folding, may represent an innovative target for the design of broad-spectrum HTAs. PDIA3 is known to assists folding and maturation of viral glycoproteins, thus playing an important role in many viral infections. Previous work identified Nitazoxanide (NTZ), a FDA-approved antiparasitic drug endowed with a broad-spectrum antiviral activity, as a candidate PDIA3 inhibitor. NTZ acts as a prodrug by releasing in vivo its active metabolite Tizoxanide (TIZ), proved to potently inhibit PDIA3. Starting from the pharmacophoric features of TIZ and using hit-to-lead strategies supported by bioisosteric and in silico approaches, we have performed an extensive DIRECT preparatory work to design and synthesize a library of eighty (80) TIZ analogues. The library was screened against the hCOV-OC43, as an illustrative human CoV, and the Respiratory Syncytial Virus (RSV), another respiratory virus paragon identifing three different chemotypes able to show antiviral activities with EC50 values in the low microM range. Among them, the hydroxyindole D5, potently inhibited hCoV-OC43 replication (EC50 0.094 M) with an acceptable safety index (SI = 173), one log superior for both parameters compared to those shown by NTZ. The interaction of D5 with the recombinant PDIA3 protein was then demonstrated by both a thermal-shift assay that showed the shifting of the enzyme melting temperature by 0.9 °C in presence of D5, and in silico studies, thus suggesting that the compound may fit well into a binding pocket of PDIA3 (Fig. 1). Starting from these robust preliminary observations, DIRECT aims to complete the hit-to-lead process of D5, as well as others selected representative molecules within the proposed chemotypes, to develop innovative PDIA3 inhibitors acting as HTA lead candidates effective against both SARS-CoV-2 and hCoVs, already ADME-optimized, and thus ready to be moved toward PK/in vivo.