This work details the inhibition of BACE1, BACE2, and SARS-CoV-2 3CLpro through several novel and potent protease inhibitors. Nanomolar potency of BACE1 and BACE2 is achieved with all tested inhibitors, and the S2 subsite has been identified as a BACE2 selectivity determinant. This is supported by the observation that the novel BACE2 mutant, BACE2 L246N, displays increased potency and selectivity over BACE1 against this peptidomimetic scaffold. Nanomolar to micromolar potency of SARS-CoV-2 3CLpro is achieved with the compounds tested in this study. Kinetic data illustrates the allowed substitutions at the P1’, P1, P2, and P4 positions on two scaffolds: ML188 and GC376. Finally, this work presents the high-resolution crystal structures of four inhibitors bound to BACE1 and 12 inhibitors bound to SARS-CoV-2 3CLpro. These structural data help to explain the selectivity determinants of BACE1 and BACE2 and further enable structure-based drug design against these two enzymes for the treatments of Alzheimer's Disease and Type II Diabetes, respectively. Additionally, these structural data illustrate the flexibility of the GC376 scaffold at the P3/P4 position, providing a structural rationale for the observed differences in potency across the different analogs. These structural data further enable structure-based drug design against SARS-CoV-2 3CLpro for the treatment of COVID-19.

<p> Over 40 years since the emergence of the AIDS epidemic and still no cure exists for AIDS or its causative HIV-1 infection. Protease inhibitors are an integral part of the most effective treatment regimen for HIV-1 infected patients known as combination antiretroviral therapy (cART), which is extremely effective at decreasing viral loads to nearly undetectable levels. One of the most alarming issues with current treatments is the emergence of multi-drug resistant strains. Even darunavir, which has shown exceptional activity against drug resistant strains, has experienced this issue. Herein we designed a novel series of heterocyclic based P2 ligand HIV-1 protease inhibitors based on kinase inhibitors such as imatinib and dasatinib. These inhibitors were designed to promote hydrogen bonding with the peptide backbone atoms of HIV-1 protease. Compounds were synthesized, biologically evaluated, and underwent X-ray structural studies. Inhibitors displayed activity as low as sub-nanomolar potency and low nanomolar antiviral activity. Important ligand-binding site interactions were determined through two X-ray crystal structures.</p> <p>Emergence of SARS-CoV-2 at the end of 2019 resulted in a global pandemic that has affected millions. Researchers all over the world turned their attention to developing drug therapies aimed at preventing and treating the viral infection. One such target became the main viral protease, or 3-chymotrypsin like protease (3CLpro). 3CLpro is an essential viral enzyme responsible for polypeptide cleavage during the viral replication cycle to produce 16 nonstructural proteins (nsps). Thus, it has been a highly researched area for effective SARS-CoV-2 drug therapies. Therefore, we designed, synthesized, and biologically evaluated a series of competitive reversible SARS-CoV-2 3CLpro inhibitors. </p>