Throughout the project, we developed and validated a novel class of peptide-porphyrin conjugates (PPCs) with antiviral activity and the ability to cross the blood-brain and blood-placenta barriers. A pool of PPCs was synthesized and tested in vitro for barrier penetration and antiviral efficacy against ZIKV, DENV, HIV, and SARS-CoV-2. Two of these lead compounds showed promising results, with P-H1 and P-H3 advancing to in vivo testing. These conjugates significantly reduced ZIKV viral load in neonatal mouse brains and demonstrated good tolerability across multiple animal models. ADME-tox analyses highlighted P-H3 for its favorable pharmacokinetic profile and strong brain penetration, identifying it as a lead candidate for further development.
During the final reporting period (months 61–72), UPF conducted stability studies on P-H1 and P-H3 under dry storage at 5°C, room temperature, and 40°C for up to five months. LC-MS/MS analyses identified key synthetic impurities, and HPLC purification ensured the conjugates were essentially endotoxin-free. Long-term storage is recommended at -20°C or lower. GIMM continued in vivo efficacy studies using neonatal mouse models of ZIKV infection. PPCs were evaluated for their ability to cross the BBB and reduce brain viral load, brain damage, and inflammation, as assessed by histology and immunolabelling. In Brazil, Andrea Da Poian’s team tested the therapeutic effect of PPCs in adult mice treated 1- and 6-days post-infection. Kátia da Conceição’s team assessed the toxicity of P-H1 and P-H3 in Galleria mellonella larvae and Danio rerio (zebrafish). In larvae, both conjugates were well tolerated up to 140 mg/kg, with survival rates above 90%. In zebrafish embryos, mild developmental effects such as delayed hatching were observed, within acceptable limits for toxicity assessment. Synovo conducted a preliminary safety study in pregnant mice treated with 35 mg/kg of PPCs. A slight increase in alkaline phosphatase was noted, but no significant changes were detected in other liver function markers or in animal behavior. The No-Observed-Effect Level (NOEL) is likely above 35 mg/kg/day, although further monitoring is warranted.
The project also advanced regulatory and translational pathways through interactions with the EMA, clinical sponsors, and industry. Dissemination activities included 5 peer-reviewed publications, 2 patent applications, and 41 scientific presentations. Public engagement reached broad audiences through 38 media appearances and educational outreach. NOVIRUSES2BRAIN exceeded its dissemination targets and established a strong foundation for clinical translation of PPC-based antivirals.
