Through the SARS-CoV-2 pandemic, it has become clear that the development of antivirals is essential for the health and wellbeing of the population. In this study, novel active site protease inhibitors against SARS-CoV-2 were tested for their inhibitory activity against the viral 3-Chymotrypsin like protease through the means of FRET based enzymatic assays. Additionally, Compound 104 targeting the NS2B-NS3 protease was tested against Zika virus through yield reduction assays as a means to assess whether these assays are suitable for the assessment of peptide hybrid compounds in Zika virus.Novel compounds against SARS-CoV-2 were screened and five of the selected six active compounds were found to inhibit the viral protease at a half-maximal inhibitory concentration (IC50) of below 0.075 µM.In Zika virus, the yield reduction assay was assessed and it was found that under the conditions tested, this assay is not suitable for the assessment of peptide hybrid compounds in Zika virus.The active novel compounds against SARS-CoV-2 should be taken for further assessment in cell based assays as the next step of development. Compound 104 should be assessed under different experimental conditions to identify whether different conditions can make this assay suitable for the intended use.

Viral proteases play crucial roles in the life cycle and maturation of many viruses by processing the viral polyprotein after translation and in some cases cleaving host proteins associated with the immune response. The essential role of viral proteases makes them attractive therapeutic targets. In this thesis, I provide an introductory summary of viral proteases, their structure, mechanism, and inhibition, while the breadth of this thesis focuses on the Hepatitis C virus (HCV) NS3/4A and Zika virus (ZIKV) NS2B/NS3 viral proteases. HCV NS3/4A protease inhibitors (PIs) have become a mainstay in combination therapies. However, drug resistance remains a major problem against these PIs. In this thesis, I applied insights from the HCV substrate envelope (SE) model to develop strategies for designing PIs that are less susceptible to resistance. Also, I used the HCV NS3/4A protease as a model system to decipher the molecular mechanism and role of fluorination in HCV PIs potency and drug resistance. The drug design strategies described in this thesis have broad applications in drug design. The ZIKV is an emerging global threat, and currently, with no treatment available. In this thesis, I described the discovery, biochemical and antiviral evaluation of novel noncompetitive quinoxaline-based inhibitors of the ZIKV NS2B/NS3 protease. The inhibitors are proposed to interfere with NS2 binding to NS3, thereby preventing the protease from adopting the closed and active conformation. The inhibitors from this work will serve as lead compounds for further inhibitor development toward the goal of developing antivirals.