Dengue Protease Substrate Recognition: Binding of the Prime Side

Area of Science:

• Virology
• Structural Biology
• Drug Discovery

Background:

• Dengue virus (DENV) infection is a significant global health concern, causing millions of cases and thousands of deaths annually.
• Current treatments for DENV are limited, highlighting the urgent need for direct-acting antiviral therapies.
• The dengue NS2B/NS3 protease is a critical target for antiviral development due to its essential role in viral replication.

Purpose of the Study:

• To investigate the molecular mechanisms of substrate recognition by the dengue NS2B/NS3 protease.
• To identify optimal substrate residues, particularly at the P' side, for enhanced protease inhibition.
• To guide the development of novel direct-acting antivirals against DENV.

Main Methods:

• Engineered the serine protease inhibitor aprotinin by modifying its binding loop to mimic DENV substrate P' side sequences.
• Assessed protease-substrate interactions using inhibition constant measurements, structural analysis, and dynamic studies.
• Employed isothermal titration calorimetry to determine the thermodynamic basis of inhibitor binding.

Main Results:

• Engineered P' residues significantly modulated substrate affinity, with inhibition constants ranging from nanomolar to sub-millimolar.
• Structural and dynamic analyses elucidated the molecular basis for P' residue-mediated affinity modulation.
• Identified optimal P' residues and confirmed that binding is primarily entropy-driven.

Conclusions:

• The P' side of DENV substrates plays a crucial role in protease recognition and binding affinity.
• Mimicking optimal P' residues, incorporating hydrophobicity and rigidity, can enhance inhibitor potency.
• Findings provide a molecular basis for designing potent flaviviral P' side inhibitors for DENV treatment.