Developing Antiviral Drugs for COVID-19 and Hepatitis C: Targeting Key Viral Proteases

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Virginia Commonwealth University
Viruses are submicroscopic infectious agents causing immense global disease burdens. Propagation of viral particles relies on proteolytic cleavage of polyprotein precursors by host or virally-encoded proteases to liberate functional components necessary for replication and infection cycles. These processing events present vulnerable intervention points for antiviral targeting. This thesis focused on two indispensable viral proteases - the SARS-CoV-2 main protease and the NS3 protease domain from hepatitis C virus. The first project centered on the discovery of small molecule inhibitors against the SARS-CoV-2 main protease (Mpro). As a cysteine protease, Mpro plays an indispensable role in processing the virally-encoded replicase polyproteins through specific cleavages to liberate functional non-structural proteins that regulate virion maturation and assembly pathways. Owing to such critical involvement, Mpro offered an attractive target for coronavirus pathogenesis intervention. Its near-identical architecture with the SARS-CoV strain enabled rapid knowledge transfer for drug design using prior scaffolds. Therefore, an ensemble small molecule discovery platform consolidating computational screening, synthetic chemistry, enzymology and biophysical characterization was constructed to systematically retrieve inhibitors against this important drug target. Three virtual screening protocols using complementary in silico techniques – ligand-based 3D pharmacophore searches, protein structure-centric molecular docking, and artificial intelligence models employed deep neural networks. This triaged computational workflow efficiently narrowed a search space of millions to selectively cherry pick prospective hit candidates. In parallel, quantitative structure-activity examinations of a small, focused library of 168 synthetically derived α-ketoamide compounds revealed a reactive Michael acceptor warhead amenable for covalently targeting the key catalytic cysteine residue. Downstream characterization in a tiered cascade of biochemical and biophysical techniques validated the tandem computational-experimental screening approach. Fluorescence resonance energy transfer (FRET) enzyme assays confirmed dose-dependent SARS CoV-2 Mpro inhibition for 10 ligands – 7 from virtual screening pipelines and 3 α-ketoamide derivatives – with low micromolar half maximal inhibitory concentrations between 1.7-55 μM. Direct binding quantification via label-free biophysical methods like microscale thermophoresis and isothermal titration calorimetry supplemented functional data. The tightest-binder, compound MA4, achieved a binding affinity of around 5 μM. Attempts to co-crystallize Mpro with ligands for atomic perspectives encountered technical limitations likely owing to poor aqueous solubility, nevertheless yielding 1.8 Å resolution apo-enzyme insight into plasticity elements lining the substrate binding cleft. Microsecond timescale explicit-solvent molecular dynamics simulations tracked long-term dynamic stabilities of inhibitor-bound complexes, corroborated through rigorously computed binding free energy predictions. Lastly, objective hit enrichment and success rate metrics evaluated relative virtual screening performances, demonstrating superior early retrieval rates for the deep learning technique that leveraged biochemical data patterns. The second collaborative project expanded targeting scope beyond conventionally exploited catalytic sites to explore an allosteric regulatory protein-protein interface on the hepatitis C NS3 protease domain. NS3 requires binding of a co-factor NS4A peptide to achieve sufficient catalytic activity essential for mediating downstream viral polyprotein processing events linked to replication competency. NS4A triggers key structural rearrangements in otherwise natively disordered NS3 that enable organization of the catalytic triad into a configuration competent for catalyzing substrates. This activation paradigm presented possibilities for blocking the interaction site with engineered variants retaining affinity but subtly distorting functional geometries through strategic mutations. Results validated this, revealing a designed nanomolar-binding NS4A variant with a single cyclohexylglycine substitution that associated with NS3 but eliminated enzyme activity. Microscale thermophoresis quantifications revealed PEP15 associated with the NS3 protease domain target with remarkably high, low nanomolar binding affinity exhibiting a dissociation constant (KD) of 22.23 ± 0.297 nM. This was approximately two orders of magnitude stronger binding compared to the native NS4A cofactor peptide, which achieved a KD of 2.595 ± 0.0015 μM in the same assay configuration. The exceptionally improved affinity despite a single residue substitution substantiates the significant energetics contributions of the engineered glycine mutation and validates the allosteric targeting rationale underlying the inhibitor design. Differential scanning fluorimetry indicated unexpected reductions in thermal stability relative to native complex or isolated protein controls. Metadynamics simulations provided insights into the unexpected biophysical findings by modeling dynamics and stability of the PEP15-NS3 complex. The trajectories revealed favorable occupying of the deep hydrophobic environment lining the NS3 allosteric pocket by the engineered glycine substitution. Notably, the modelling also captured shifting of the key SER139 hydroxyl moiety away from the organized catalytic triad geometric center. Displacement of this nucleophilic residue plausibly misaligns other proximal components due to intricate hydrogen bonding networks. Structural rearrangement of active site elements likely contributes to the abolished enzymatic activity despite high affinity binding of the strategic PEP15 peptide.
COVID-19, Virus, Antiviral, Drug Discovery, Hepatitis C, SARS-CoV-2, Proteases