Alternate recognition by dengue protease: Proteolytic and binding assays provide functional evidence beyond an induced-fit

Abstract

Proteases are key enzymes in viral replication, and interfering with these targets is the basis for therapeutic interventions. We previously introduced a hypothesis about conformational selection in the protease of dengue virus and related flaviviruses, based on conformational plasticity noted in X-ray structures. The present work presents the first functional evidence for alternate recognition by the dengue protease, in a mechanism based primarily on conformational selection rather than induced-fit. Recognition of distinct substrates and inhibitors in proteolytic and binding assays varies to a different extent, depending on factors known to influence the dengue protease structure such as pH and salinity. Furthermore, the buffer type and temperature cause a change in binding, proteolysis, or inhibition behavior. Using representative inhibitors with distinct structural scaffolds, we identify two contrasting binding profiles to dengue protease. Noticeable effects are observed in the binding assay upon inclusion of a non-ionic detergent in comparison to the proteolytic assay. The findings highlight the impact of the selection of testing conditions on the observed ligand affinity or inhibitory potency. From a broader scope, the dengue protease presents an example, where the induced-fit paradigm appears insufficient to explain binding events with the biological target. Furthermore, this protein reveals the complexity of comparing or combining biochemical assay data obtained under different conditions. This can be particularly critical for artificial intelligence (AI) approaches in drug discovery that rely on large datasets compiled from different sources.

Antiviral Drug Discovery with an Optimized Biochemical Dengue Protease Assay: Improved Predictive Power for Antiviral Efficacy

“…Besides, it does not cover cis -cleavage of NS3 which was proposed to leave a gap in the development of anti-DENV compounds. 8 Recent work from our group was therefore aimed at fundamental aspects of the substrate processing and ligand binding properties of DENV protease, with a particular emphasis on induced-fit vs. conformational selection mechanisms (Behnam & Klein, in revision, preprint available 25 ). One result of this work was the identification of a bithiophene sensor tag to study ligand binding, irrespective of catalytic processing, to DENV protease under in vitro conditions.…”