Repurposing SARS-CoV-2 Main Protease Inhibitors for HIV-1 Protease Inhibition

Severe acute respiratory syndrome (SARS-CoV-2) led to the COVID-19 global pandemic, with over 178 million people infected since the start of the pandemic. SARS-CoV-2 is a retrovirus that utilizes a main protease (Mpro ). Mpro is a catalytic cys/his protease. Several treatments were proposed to stop the pandemic including repurposing drugs to inhibit the Mpro . Another retrovirus that uses a protease is human immunodeficiency virus (HIV-1); which has been a global epidemic for 40 years and is a devastating disease that attacks the immune system. HIV-1 has infected 77.5 million people since the start of the epidemic in 1981 and is still prevalent today. HIV-1 retrovirus utilizes the host cell to transcribe viral RNA and translate gag-pol protein. HIV-1 protease is a dual aspartyl protease used to cleave this gag-pol protein, thereby activating the protein, allowing for viral replication and infection of other cells. The focus of this research is to investigate whether these two proteases have a similar enough mechanism that SARS-CoV-2 Mpro inhibitors will also inhibit HIV-1 protease. Four repurposed SARS-CoV-2 inhibitors: carmofur, leupeptin, rosuvastatin, and withanone were chosen to be repurposed for HIV-1 protease due to their affordability and affinity for SARS-CoV-2 Mpro . Initially, computational analysis was utilized to acquire binding information of the repurposed SARS-CoV-2 inhibitors on HIV-1 protease. POCASA (Pocket Cavity Search Algorithm) was used to identify the main binding pockets of HIV-1 protease. Swissdock identified the center of the HIV-1 protease homodimer being the most prevalent binding pocket. Analysis of this pocket showed thermodynamically favorable binding to leupeptin, carmofur, rosuvastatin, and withanone. Since computational analysis showed promising prevalence for the inhibitors, the next step was to test these experimentally; therefore HIV-1 protease was purified from E. Coli cells. The pure HIV-1 protease was used in a fluorescent binding assay to derive binding affinity of the inhibitors. Preliminary experimental binding results show carmofur binding (Kd = 1 ± 0.63 mM), and rosuvastatin binding (Kd = 0.025 ± 0.007 mM); leupeptin did not show binding. The next steps are to evaluate binding for withanone as well as activity inhibition of HIV-1 protease using these repurposed SARS-CoV-2 Mpro inhibitors to further investigate whether these two proteases have a similar enough mechanism that SARS-CoV-2 Mpro inhibitors will also inhibit HIV-1 protease. © FASEB.