DOI
https://doi.org/10.25772/0ENK-0909
Author ORCID Identifier
https://orcid.org/0009-0008-1473-6170
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Martin K Safo
Second Advisor
Julian Zhu
Third Advisor
Aaron May
Fourth Advisor
Yana Cen
Fifth Advisor
Abdelsattar Mansour
Abstract
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 nucleocapsid (N) protein emerges as a crucial target due to its role in viral replication and pathogenesis.
The SARS-CoV-2 N protein, 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). Among these, the NTD stands out for its conserved electropositive pocket, crucial for viral RNA binding during packaging stages, making it a validated target for small molecule intervention. This highlights the multifunctionality of the N protein and its potential as a therapeutic target due to its essential roles and conserved features across diverse coronavirus species. Our collaborators initiated an intriguing drug repurposing screen pointing to certain β-lactam antibiotics as SARS-CoV-2 N protein inhibitors in vitro. Complementing this, the current study employed ensemble computational methodologies biophysical and biochemical assays to discover novel chemotype hits against this target. 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 procured for experimental evaluation. These included MY1, MY2, MY3, MY4, NP6, NP7, NP1, NP2, NP3, NP4 and NP5, which demonstrated favorable binding orientations and affinity scores from the virtual screening studies. Additionally, one supplementary compound denoted CE provided by Dr. Cen’s collaborating laboratory was assessed in parallel. These hits were further evaluated for their in vitro activity using various biophysical and biochemical techniques including DSF, MST, FP and EMSA. DSF revealed native NTD had a baseline thermal melting temperature (Tm) of 43.82°C. Compounds NP3, NP6 and NP7 notably increased this 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 revealed that compounds NP1, NP3, and NP7 exhibited the strongest interactions 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, which displayed dissociation constants of 8.35 μM and 5.24 μM. In contrast, while showing weaker interactions than NP1, NP3, and NP7, compounds NP2, NP6, and CE still demonstrated better binding affinities than the control compounds PJ34 and Suramin, with dissociation constants of 4.1 μM, 2.50 μM, and 1.81 μM, respectively. These results substantiate the potential of these scaffolds as modulators of NTD activity. In FP assays, NP1 and NP3 exhibited the lowest half-maximal inhibitory concentrations (IC50) of 5.18 μM and 5.66 μM, respectively, indicating the highest binding affinities among the tested compounds. These values are significantly lower than those of controls PJ34 and Suramin, which had IC50 values of 21.72 μM and 17.03 μM, respectively, as well as lower than those of other tested compounds (NP6, NP7, CE, and NP2) whose IC50 values ranged from 7.00 μM to 10.13 μM. EMSA confirmed this potency, with NP1 showing the most significant disruption of NTD-ssRNA complex at an IC50 of 2.70 μM. NP3, NP7, CE, NP6, and NP2 followed with IC50s ranging from 3.31 μM to 7.61 μM. These consistent results from both FP and EMSA highlight the superior effectiveness of NP1 and NP3 in disrupting NTD-ssRNA interactions, showcasing their potential as particularly powerful antiviral agents. Additional EMSA monitored the formation of complexes between the full-length SARS-CoV-2 nucleocapsid protein and single-stranded RNA (ssRNA). The gradual addition of the control inhibitor Suramin and previously identified hits NP1 and NP3 resulted in a concentration-dependent disruption of this complex. Quantified by IC50 values, NP1 (1.67 μM) and NP3 (1.95 μM) exhibited superior potency over Suramin (3.24 μM) in abolishing the shift representing nucleocapsid-RNA binding. Extensive crystallization trials were conducted to elucidate the structure of the SARS-CoV-2 N-NTD, assessing over 8000 unique conditions. Ultimately, only a PJ34-bound structure could be resolved, albeit with weak ligand density potentially owing to tight crystal packing impeding binding site access. The crystal structure was determined to 2.2Å by molecular replacement using the published apo N-NTD (PDB 7CDZ) coordinates as a search model. Iterative rounds of refinement lowered R-factors to 0.193 (Rwork) and 0.234 (Rfree).
In conclusion, a combination of computational screening and experimental validation identified novel lead compounds against SARS-CoV-2 nucleocapsid N-terminal domain. Binding assays proved NP1 and NP3 as superior hits disrupting both N-NTD-RNA and full length nucleocapsid-RNA interactions with low micromolar IC50s. Structural efforts elucidated partial binding modes, set the stage for further advancement of these scaffolds through synergistic structure
Rights
© Mona AlKhairi
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2024