Document Type

Thesis

Publication Date

4-2022

Disciplines

Amino Acids, Peptides, and Proteins | Biochemistry | Biochemistry, Biophysics, and Structural Biology | Biophysics | Chemicals and Drugs | Medicinal and Pharmaceutical Chemistry | Medicinal-Pharmaceutical Chemistry | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Pharmaceutics and Drug Design | Pharmacy and Pharmaceutical Sciences | Structural Biology

Advisor

Lisa Gentile

Abstract

Severe acute respiratory syndrome (SARS-CoV-2) led to the COVID-19 global pandemic, with over 460 million cases of infection and over 6 million deaths 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 79.5 million people and has killed an estimated 36 million people since the start of the epidemic in 1981 and is still prevalent today. HIV-1 retrovirus utilizes the host cell’s machinery to transcribe viral RNA and translate gag-pol protein. HIV-1 protease is a dual asp protease used to cleave this gag-pol protein, thereby activating the protein, allowing for viral replication and infection of other cells. HIV-1 pr can sporadically mutate into drug resistant strains which resist the common therapies used to inhibit HIV-1 pr. An approach to combat this issue is to create treatments made specifically for these drug resistant strains. In one strain, multidrug resistance is caused in the multidrug resistant hexamutant of HIV-1 pr (HIV-1 pr MDR-HM) with six amino acid mutations: (L10I/M46I/I54V/V82A/I84V/L90M). The focus of this research is to investigate whether SARS-CoV-2 Mpro and HIV-1 pr have a similar enough mechanism that SARS-CoV-2 Mpro inhibitors will also bind to both HIV-1 protease wild type (wt) and MDR-HM. Three repurposed SARS-CoV-2 inhibitors: carmofur, leupeptin, and rosuvastatin were chosen to see if they could be repurposed for HIV-1 protease due to their affordability and computational binding affinity to HIV-1 pr. 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 predict 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, and rosuvastatin. Since computational analysis showed promising prevalence for the inhibitors, the next step was to test these in vitro. A plasmid containing HIV-1 protease was transformed into E. coli BL21(DE3) cells. HIV-1 protease was purified from E. Coli cells and used in a fluorescent binding assay to derive binding affinity of rosuvastatin to HIV-1 pr wt (Kd=32.3 ± 3.5 μM) and MDR-HM (Kd=73.7 ± 15.8 μM), as well as carmofur to HIV-1 pr wt (Kd= 0.35 ± 0.01 mM) and MDR-HM (Kd=0.31 ± 0.04 mM).

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