Development of Next-GenerationBroad-Spectrum Antivirals: Prepare for Future Pandemic Risks

I) Scientific/Technological Excellence. 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 likely it would not be the last. During a new CoV emergence already available and effective broad-spectrum antivirals endowed with an anti-hCoV activity are fundamental in slowing down the spread of the new infection, saving life, and gaining time for development of specific CoV-specific vaccines and antivirals. Thus, effective pan-CoV antivirals that can be rapidly deployed against future emerging CoVs need to be urgently developed in time before a new emergence. The MEDSynth group (www.medsynth.unito.it) in UniTo, led by Prof. Marco Lolli (Italian PI), is a Medicinal Chemistry research group specialized in the discovery advanced preclinical candidate, particularly focused in the design of broad-spectrum antivirals. In particular, MEDSynth was able to discover MEDS433 as best-in-class human dihydroorotate dehydrogenase inhibitor able to exhibits low nM range activity against SARS-CoV-2 and a wide panel of other viruses (Influenza A/B, Respiratory Syncytial, HSV-1/2 virus…). The Alagoz group in AU, led by Prof. Zeynep Alagoz (Turkish PI), is a medicinal chemistry research team specialized in synthesis of heterocyclic compounds

II) Methodology 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 PANDORA preparatory work to design and synthesize a library of 20 TIZ analogues. The library was screened against the hCOV-OC43, as a prototype human CoV, and the Respiratory Syncytial Virus (RSV), another important human respiratory virus, to identify two different chemotypes able to show antiviral activities with EC₅₀ values in the low 𝜇M range. Among them, D1, D2, D3 and D4, potently inhibited hCoV-OC43 replication (EC₅₀ 0.24 𝜇M, 3.63 𝜇M, 5.11 𝜇M and 1.0 𝜇M respectively) with an acceptable safety index (SI = 17 -74), superior if compared to NTZ. As the molecular targets of these new HTAs, the activity of the human protein disulphide isomerase A3 (PDIA3 or ERp57), a cellular thiol oxidoreductase involved in protein folding, can be proposed. PDIA3 is in fact an ER-resident enzyme that assists folding and maturation of viral glycoproteins, thus playing a critical role in the efficient completion of the replication cycle of many human viruses. Starting from these robust preliminary observations, PANDORA aims to complete the hit-to-lead process of D1-4, as well as others selected representative molecules within the proposed chemotypes, to develop innovative PDIA3 inhibitors acting as HTA lead candidates effective against both present and future hCoVs, already ADME-optimized, and thus ready to be moved toward PK/in vivo studies in the next future.

III) Project Management. We will follow a structured and collaborative approach to ensure success in each operational phase, following the detailed plan here included (objectives, deliverables, and timelines for each task). We’ll use project management tools like Asana or Trello to track progress, assign tasks, and facilitate effective communication among team members. Regular meetings will be held for updates, feedback, and timely resolution of any obstacles. Risk management will play a crucial role: we will identify and assess potential risks, establishing mitigation strategies to minimize impacts. Very important will be the collaborative and inclusive environment, encouraging active participation and idea sharing among team members. We will organize staff exchanges, one/year in alternate countries.

IV) Importance of International Collaboration. We strongly believe in the power of International Collaborations, which drive innovation and scientific breakthroughs that may not be achievable independently and promote cultural exchange. This proposal brings together the expertise of Italian (UniTO) and Turkish (AU) academic research partners, who have been collaborating since 2020 to design a common drug design platform, with the aim of securing EU-level funding in the future. The MEDSynth group has already hosted two Turkish researchers supported by the TUBITAK program. These researchers spent a total of 24 months conducting experimental research in Torino, enhancing their Medicinal Chemistry skills. In June 2024, Prof Marco Lolli was invited to deliver a plenary lecture at the 14^th International Symposium on Pharmaceutical Sciences (ISOPS-14) in Ankara (Turkey), where he had the opportunity to strengthen the collaboration with Prof. Zeynep Alagoz. This ongoing collaboration has facilitated the sharing of information and procedures, effectively merging the two entities into a single research group. The PANDORA project aims to further strengthen this established partnership.

V) Impact. This proposal aims to contribute to 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.