Periodic Reporting for period 5 - NOVIRUSES2BRAIN (''One size fits all'' unique drug to eradicate multiple viral species simultaneously from the central nervous system of co-infected individuals)
Reporting period: 2024-09-01 to 2025-08-31
Viral infections of the brain and central nervous system (CNS) represent a critical and growing public health concern, with viruses such as Zika (ZIKV), Dengue (DENV), HIV, and SARS-CoV-2 linked to severe neurological complications. The ZIKV outbreak in Latin America demonstrated the profound risks associated with neurotropic viruses, particularly during pregnancy, where placental transmission can result in devastating congenital conditions like microcephaly. Current antiviral strategies fail to account for the complexity of co-infections and the necessity of crossing biological barriers such as the blood-brain barrier (BBB) and the blood-placenta barrier (BPB), leaving a gap in effective therapeutic solutions for these high-risk infections.
The NOVIRUSES2BRAIN project was launched to address this challenge by developing a new class of peptide-based antivirals—peptide-porphyrin conjugates (PPCs)—designed to cross the BBB and BPB and effectively inhibit viral replication within the CNS. The overall objective was to combine rational molecular design, advanced synthesis, and biological testing to identify candidates with strong antiviral efficacy and brain-targeting properties. The final period of the project successfully confirmed the ability of lead PPC candidates to cross the BBB and significantly reduce viral load in relevant preclinical models, establishing in vivo proof-of-concept.
In conclusion, NOVIRUSES2BRAIN laid the scientific and technological foundation for PPC-based brain-targeting antivirals. The consortium achieved key milestones, including the design and synthesis of novel PPCs, demonstration of their antiviral activity in vitro and in vivo, and validation of their ability to reach the brain. These findings open promising avenues for future translational development and justify further investment towards the clinical advancement of PPCs.
The NOVIRUSES2BRAIN project was launched to address this challenge by developing a new class of peptide-based antivirals—peptide-porphyrin conjugates (PPCs)—designed to cross the BBB and BPB and effectively inhibit viral replication within the CNS. The overall objective was to combine rational molecular design, advanced synthesis, and biological testing to identify candidates with strong antiviral efficacy and brain-targeting properties. The final period of the project successfully confirmed the ability of lead PPC candidates to cross the BBB and significantly reduce viral load in relevant preclinical models, establishing in vivo proof-of-concept.
In conclusion, NOVIRUSES2BRAIN laid the scientific and technological foundation for PPC-based brain-targeting antivirals. The consortium achieved key milestones, including the design and synthesis of novel PPCs, demonstration of their antiviral activity in vitro and in vivo, and validation of their ability to reach the brain. These findings open promising avenues for future translational development and justify further investment towards the clinical advancement of PPCs.
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.
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.
NOVIRUSES2BRAIN applies an interdisciplinary strategy combining medicinal chemistry, biochemistry, biophysics, and in vivo virology to address the urgent need for antiviral drugs capable of targeting infections in the brain. Currently, no approved therapies exist for Zika or other Aedes-borne viruses, and available antivirals rarely cross the blood-brain barrier. By building on prior discoveries—molecules with antiviral activity and others with barrier-crossing capacity—the project successfully created a unique class of peptide-drug conjugates: peptide-porphyrin conjugates (PPCs), combining both properties.
This advancement goes beyond scientific innovation. The consortium has actively engaged with regulators such as the European Medicines Agency (EMA) to explore future clinical translation. Dissemination efforts have included international conferences, peer-reviewed publications, patent filings, and broad public outreach through media and stakeholder events.
This advancement goes beyond scientific innovation. The consortium has actively engaged with regulators such as the European Medicines Agency (EMA) to explore future clinical translation. Dissemination efforts have included international conferences, peer-reviewed publications, patent filings, and broad public outreach through media and stakeholder events.