Inhibiting Immune Evasion : Drug Discovery Screening Strategies Against the Staphylococcal Superantigen-like Proteins and Zika Virus NS2B-NS3 Protease

The rise of drug-resistant pathogens has rendered many existing treatments ineffective, and despite the significant advances in drug development, some pathogens still lack targeted therapies. As conventional antimicrobial strategies lose efficacy, alternative approaches to combat infectious diseases are urgently needed.
This thesis focuses on two clinically significant pathogens: the bacterium Staphylococcus aureus, known for causing a broad spectrum of infections ranging from superficial skin and soft tissue infections to life-threatening systemic conditions such as sepsis, and Zika virus, a mosquito-borne virus associated with congenital abnormalities and neurological complications. Both pathogens have evolved sophisticated mechanisms to evade the host immune system, enabling them to establish infections and resist clearance. S. aureus secretes various immune evasion molecules, including the staphylococcal superantigen-like proteins (SSLs), which interfere with multiple host immune functions and thereby promote bacterial survival. Immunoevasive proteins such as the SSLs are promising drug targets, as their inhibition could partly disarm the bacteria and give the host a better opportunity to mount an effective immune response. Similarly, Zika virus relies on its nonstructural protein NS2B-NS3 protease for both viral replication and suppression of host immunity. As no specific treatments currently exist against Zika virus, targeting this protease presents a particularly attractive dual-purpose strategy to inhibit both viral propagation and immune evasion. The aim of this thesis was to apply two high-throughput screening strategies, structure-based virtual screening and phage display, to identify novel inhibitors against these immune-modulating proteins of S. aureus and Zika virus.
To identify inhibitors of the Zika virus NS2B-NS3 protease, a virtual screening workflow was conducted. A compound library containing 1.2 million small molecules was docked against the protease active site. The library was gradually filtered based on docking scores, binding free energy estimates, and the docking complex stability evaluated by molecular dynamics (MD) simulations. The top-ranked hit compounds were subsequently tested in vitro, resulting in the identification of a small-molecule inhibitor with moderate affinity for the protease and demonstrated antiviral activity in a cell-based assay.
For S. aureus, two screening approaches were used to identify inhibitors of SSL1, SSL5, SSL7, and SSL10. The experimental approach, phage display was employed to screen for single-chain variable fragment (scFv) antibodies capable of recognizing SSL1, SSL5, and SSL10 and blocking the interactions of these pathogen proteins with their respective immune targets in the host. Functional analyses revealed several scFvs that could bind to the SSLs, including one that completely blocked SSL1-mediated matrix metalloproteinase 9 inhibition in a concentration-dependent manner. Additionally, the computational approach, structure-based virtual screening was used to screen peptide and macrocycle libraries against SSL5 and SSL7. The compounds were ranked based on their docking scores against each SSL, after which MD simulations and free energy calculations were used to further guide the hit selection. In vitro assays confirmed that some peptides inhibited SSL5 at high concentrations and showed moderate affinity. The compounds screened against SSL7 showed only weak inhibition and non-specific binding.
These findings demonstrate that both computational screening and phage display are effective approaches for identifying inhibitors targeting bacterial and viral immune evasion mechanisms. While promising inhibitors were identified, further optimization is needed to improve their efficacy, stability, and specificity. This research contributes to the growing fields of antivirulence and antiviral drug development, offering an alternative strategy to traditional antimicrobials in the fight against drug-resistant bacterial infections and emerging viral threats.