Periodic Reporting for period 1 - Inhibicov (Hit-to-Lead Development of potent broad-spectrum coronavirus fusion inhibitors)
Periodo di rendicontazione: 2022-11-01 al 2024-04-30
The COVID-19 pandemic has highlighted the urgent need for effective therapeutic strategies to combat viral infections. Antiviral drugs are crucial in this context as they can help reduce hospitalizations, prevent chronic late effects, shorten the infection period, and reduce mortality. Furthermore, broad-spectrum antivirals are particularly valuable as they can be effective against new variants or other coronavirus clades. Our project aimed to address this need by developing small molecule inhibitors that target the viral fusion mechanism. The Overall Objectives were to utilize a high throughput screening (HTS) platform to identify small molecule inhibitors that target the S2 domain of the viral Spike protein, which is essential for viral fusion and infection. To conduct comprehensive primary, secondary, and tertiary screens to isolate inhibitors with high specificity and potency and validate the efficacy and specificity of these inhibitors. To prepare for commercialization by filing patents, conducting market research, and developing business models to facilitate the transition from research to market. The project advanced our understanding of viral fusion mechanisms and the development of fusion inhibitors. The innovative methodologies and technologies developed during this project, such as the high throughput screening platform, can be applied to other viral pathogens, enhancing our preparedness for future pandemics. In summary, this project significantly contributed to public health by providing effective therapeutic options against current and future viral outbreaks.
Activities Performed and Main Achievements
High-Throughput Screening Platform Development
Objective: Establish a robust high-throughput screening (HTS) platform to identify small molecule inhibitors targeting the S2 domain of the coronavirus (CoV) Spike (S) protein.
Methodology: Developed a pseudotyped vesicular stomatitis virus (VSV) model that enabled robust quantification of CoV S glycoprotein-mediated infection using fluorescent reporters. This system allowed direct visualization and quantification of single infection events, facilitating high-throughput screening.
Outcome: Successfully produced pseudoviruses featuring the S glycoprotein of SARS-CoV-2, MERS-CoV, and several SARS-CoV-2 variants, achieving high titer production essential for the screening process.
Screening Process
Primary Screen: Screened approximately 200,000 compounds against pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Identified 733 compounds capable of inhibiting infection by at least 35%.
Secondary Screen: Counter-screened the 733 hits against pseudoviruses with the VSV glycoprotein (VSV-G) to identify S-specific inhibitors. This yielded 65 compounds.
Tertiary Screen: Further screened the 65 S-specific inhibitors against pseudoviruses bearing the MERS-CoV S glycoprotein to isolate inhibitors acting downstream of receptor binding. Identified 22 compounds, of which 11 were unreported previously.
Validation and Characterization
Compound Validation: Validated the activity of the 11 most promising compounds and Nafamostat (a known TMPRSS2 protease inhibitor) against pseudoviruses bearing the S glycoprotein of SARS-CoV-2 variants (Alpha, Delta, Omicron) and MERS-CoV.
Dose Response and Cytotoxicity: Evaluated the cytotoxicity (CC50) and inhibitory concentration (IC50) of each compound before and after high-performance liquid chromatography (HPLC) purification. Identified compounds showing selective activity against CoV S glycoproteins with minimal cytotoxicity.
Lead Compound Identification
Lead Compounds: Identified PCM-0163855 and its sulfoxide derivative PCM-0282478 as promising inhibitors. PCM-0282478 demonstrated broader selectivity and higher potency against SARS-CoV-2 Delta and Omicron variants.
Enantiomer Analysis: Separated enantiomers of PCM-0282478, identifying PCM-0296174 as the most potent and selective inhibitor against SARS-CoV-2 Delta and MERS-CoV.
Outcomes
1. Publication: The findings were summarized and published (https://doi.org/10.1016/j.isci.2024.110019(si apre in una nuova finestra))
2. Scientific Impact: The project successfully identified and validated several broad-spectrum CoV inhibitors, highlighting the S2 domain as a viable target for antiviral development. This work contributes to the understanding of CoV entry mechanisms and provides a foundation for future antiviral development.
3. Technological Advancement: Developed a robust HTS platform that can be adapted for screening inhibitors against other viral pathogens, enhancing preparedness for future pandemics.
4. Potential Therapeutics: Identified PCM-0296174 as a potent broad-spectrum CoV inhibitor, demonstrating the potential for developing new therapeutic agents against current and emerging CoV strains.
5. Commercialization: Patent filed
These outcomes underscore the project's success in addressing the urgent need for broad-spectrum antiviral therapies, offering promising candidates for further development and potential commercialization.
High-Throughput Screening Platform Development
Objective: Establish a robust high-throughput screening (HTS) platform to identify small molecule inhibitors targeting the S2 domain of the coronavirus (CoV) Spike (S) protein.
Methodology: Developed a pseudotyped vesicular stomatitis virus (VSV) model that enabled robust quantification of CoV S glycoprotein-mediated infection using fluorescent reporters. This system allowed direct visualization and quantification of single infection events, facilitating high-throughput screening.
Outcome: Successfully produced pseudoviruses featuring the S glycoprotein of SARS-CoV-2, MERS-CoV, and several SARS-CoV-2 variants, achieving high titer production essential for the screening process.
Screening Process
Primary Screen: Screened approximately 200,000 compounds against pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Identified 733 compounds capable of inhibiting infection by at least 35%.
Secondary Screen: Counter-screened the 733 hits against pseudoviruses with the VSV glycoprotein (VSV-G) to identify S-specific inhibitors. This yielded 65 compounds.
Tertiary Screen: Further screened the 65 S-specific inhibitors against pseudoviruses bearing the MERS-CoV S glycoprotein to isolate inhibitors acting downstream of receptor binding. Identified 22 compounds, of which 11 were unreported previously.
Validation and Characterization
Compound Validation: Validated the activity of the 11 most promising compounds and Nafamostat (a known TMPRSS2 protease inhibitor) against pseudoviruses bearing the S glycoprotein of SARS-CoV-2 variants (Alpha, Delta, Omicron) and MERS-CoV.
Dose Response and Cytotoxicity: Evaluated the cytotoxicity (CC50) and inhibitory concentration (IC50) of each compound before and after high-performance liquid chromatography (HPLC) purification. Identified compounds showing selective activity against CoV S glycoproteins with minimal cytotoxicity.
Lead Compound Identification
Lead Compounds: Identified PCM-0163855 and its sulfoxide derivative PCM-0282478 as promising inhibitors. PCM-0282478 demonstrated broader selectivity and higher potency against SARS-CoV-2 Delta and Omicron variants.
Enantiomer Analysis: Separated enantiomers of PCM-0282478, identifying PCM-0296174 as the most potent and selective inhibitor against SARS-CoV-2 Delta and MERS-CoV.
Outcomes
1. Publication: The findings were summarized and published (https://doi.org/10.1016/j.isci.2024.110019(si apre in una nuova finestra))
2. Scientific Impact: The project successfully identified and validated several broad-spectrum CoV inhibitors, highlighting the S2 domain as a viable target for antiviral development. This work contributes to the understanding of CoV entry mechanisms and provides a foundation for future antiviral development.
3. Technological Advancement: Developed a robust HTS platform that can be adapted for screening inhibitors against other viral pathogens, enhancing preparedness for future pandemics.
4. Potential Therapeutics: Identified PCM-0296174 as a potent broad-spectrum CoV inhibitor, demonstrating the potential for developing new therapeutic agents against current and emerging CoV strains.
5. Commercialization: Patent filed
These outcomes underscore the project's success in addressing the urgent need for broad-spectrum antiviral therapies, offering promising candidates for further development and potential commercialization.
The identified inhibitors have the potential to serve as broad-spectrum antiviral drugs, effective against multiple CoV strains, including emerging variants. This can significantly reduce hospitalizations and mortality rates associated with CoV infections. These inhibitors can be part of a strategic reserve, providing a rapid response tool against future CoV outbreaks. The project has advanced the understanding of CoV entry mechanisms, providing a foundation for future antiviral research. The HTS platform developed can be adapted for other viral pathogens, enhancing global pandemic preparedness. Continued optimization of lead compounds to enhance their drug-like properties and efficacy and rigorous preclinical and clinical testing to ensure safety and efficacy in humans are still needed. The first step will be to conduct in vivo studies to demonstrate the effectiveness of the inhibitors in animal models. Securing funding for continued research, development, and commercialization efforts through potential licensing agreements and partnerships with biotech firms is our logical next step.