SACM - United States of America
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Item Restricted Developing Novel Antiviral Agents: Targeting the N-Terminal Domain of SARS-CoV-2 Nucleocapsid Protein with Small Molecule Inhibitors(Virginia Commonwealth University, 2024-05-13) Alkhairi, Mona A.; Safo, Martin K.The COVID-19 pandemic, caused by SARS-CoV-2, persists globally with over 7 million deaths and 774 million infections. Urgent research is needed to understand virus behavior, especially considering the limited availability of approved medications. Despite vaccination efforts, the virus continues to pose a significant threat, highlighting the need for innovative approaches to combat it. The SARS-CoV-2 nucleocapsid protein (NP) emerges as a crucial target due to its role in viral replication and pathogenesis. The SARS-CoV-2 NP, essential for various stages of the viral life cycle, including genomic replication, virion assembly, and evasion of host immune defenses, comprises three critical domains: the N-terminal domain (NTD), C-terminal domain (CTD), and the central linker region (LKR). Notably, the NTD is characterized by a conserved electropositive pocket, which is crucial for viral RNA binding during packaging stages. This highlights the multifunctionality of the nucleocapsid protein and its potential as a therapeutic target due to its essential roles and conserved features across diverse pathogenic coronavirus species. Our collaborators previously initiated an intriguing drug repurposing screen, identifying certain β-lactam antibiotics as potential SARS-CoV-2 NP-NTD protein inhibitors in vitro. The current study employed ensemble of computational methodologies, biophysical, biochemical and X-ray crystallographic studies to discover novel chemotype hits against NP-NTD. Utilizing a combination of traditional molecular docking tools such as AutoDock Vina, alongside AI-enhanced techniques including Gnina and DiffDock for enhanced performance, eleven structurally diverse hit compounds predicted to target the SARS-CoV-2 NP-NTD were identified from the virtual screening (VS) studies. The hits include MY1, MY2, MY3, MY4, NP6, NP7, NP1, NP2, NP3, NP4 and NP5, which demonstrated favorable binding orientations and affinity scores. Additionally, one supplementary compound provided by Dr. Cen’s laboratory (denoted as CE) was assessed in parallel. These hits were further evaluated for their in vitro activity using various biophysical and biochemical techniques including differential scanning fluorimetry (DSF), microscale thermophoresis (MST), fluorescence polarization (FP), and electrophoretic mobility shift assay (EMSA). DSF revealed native NTD had a baseline thermal melting temperature (Tm) of 43.82°C. The compounds NP3, NP6 and NP7 notably increased the Tm by 2.55°C, 2.47°C and 2.93°C respectively, indicating strong thermal stabilization over the native protein. In contrast, NP4 and NP5 only achieved marginal Tm increases. MST studies showed NP1, NP3, and NP7 exhibited the strongest affinity with low micromolar dissociation constants (KD) of 0.32 μM, 0.57 μM, and 0.87 μM, respectively, significantly outperforming the control compounds PJ34 and Suramin, with dissociation constants of 8.35 μM and 5.24 μM, respectively. Although NP2, NP6, and CE showed relatively weaker affinity, these compounds still demonstrated better binding affinities with dissociation constants of 4.1 μM, 2.50 μM, and 1.81 μM, respectively than the control compounds PJ34 and Suramin. These results substantiate the potential of these scaffolds as modulators of NTD activity. In FP competition assays, NP1 and NP3 exhibited the lowest half-maximal inhibitory concentrations (IC50) of 5.18 μM and 5.66 μM, respectively, indicating the highest potency at disrupting the NTD-ssRNA complex among the compounds, outperforming the positive controls PJ34 and Suramin, with IC50 of 21.72 μM and 17.03 μM, respectively. The compounds NP6, NP7, CE, and NP2 also showed significant IC50 values that ranged from 7.00 μM to 10.13 μM. EMSA studies confirmed the NTD-ssRNA complex disruptive abilities of the compounds, with NP1 and NP3 as the most potent with IC50 of 2.70 μM and 3.31 μM, respectively. These values compare to IC50 of 8.64 μM and 3.61 μM of the positive controls PJ34 and Suramin, respectively. NP7, CE, NP6, and NP2 also showed IC50 ranging from 4.31 μM to 7.61 μM. The use of full-length nucleocapsid protein also showed that NP1 and NP3 disrupted the NP-ssRNA binding with IC50 of 1.67 μM and 1.95 μM, which was better than Suramin with IC50 of 3.24 μM. These consistent results from both FP and EMSA highlight the superior effectiveness of NP1 and NP3 in disrupting nucleocapsid protein-ssRNA binding, showcasing their potential as particularly powerful antiviral agents. Extensive crystallization trials were conducted to elucidate the atomic structures of SARS-CoV-2 NP-NTD in complex with selected hit compounds, assessing over 8000 unique crystallization conditions. Ultimately, only a PJ34-bound structure could be determined, albeit with weak ligand density, likely due to tight crystal packing impeding binding site access. The crystal structure was determined to 2.2 Å by molecular replacement using the published apo NP-NTD (PDB 7CDZ) coordinates as a search model, and refined to R-factors of 0.193 (Rwork) and 0.234 (Rfree). The refined NP-NTD structure showed conserved intermolecular interactions with PJ34 at the RNA binding pocket as observed in the previously reported HCoV-OC43 NP-NTD-PJ34 complex (PDB 4KXJ). This multi-faceted drug discovery endeavor, combining computational screening and in vitro assays resulted in successful identification of novel compounds inhibiting the SARS-CoV-2 nucleocapsid N-terminal domain. Biophysical and biochemical studies established compounds NP1 and NP3 as superior hits with low micromolar binding affinities, as well as low micromolar potency superior to standard inhibitors at disrupting both isolated N-NTD-RNA and full-length nucleocapsid-RNA complex formation. Though crystallographic efforts encountered challenges, important validation was achieved through a resolved crystal structure of PJ34 in complex with NP-NTD. Future effort will be to obtain co-crystals of NP-NTD with our compounds to allow for targeted structure modification to improve on the potency of the compounds.6 0Item Restricted Developing Antiviral Drugs for COVID-19 and Hepatitis C: Targeting Key Viral Proteases(Virginia Commonwealth University, 2024-05-14) AlAwadh, Mohammed; Safo, Martin K.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.33 0