Optimizing Conditions for Mpox Holliday Junction Resolvase Small Molecule Inhibitor Assays
The pox virus mpox replicates its genome using an enzyme called the mpox Holliday junction resolvase (MPHJR). A leading contender for treating mpox infection is to inhibit this enzyme using small molecule inhibitors. Before potential inhibitors can be screened for their effectiveness in preventing replication of the mpox genome and their compatibility with human cellular mechanisms, the optimal reaction conditions of MPHJR first need to be determined. The conditions optimized in this study include pH, metal ligand concentration, metal ligand identity, reaction time, and reaction temperature. Using a combination of fluorescence and gel-based assays, as well as an in-house developed enzyme kinetics calculator, the conditions of small molecule inhibitor assays involving MPHJR were able to be optimized. By completion of these optimizations, preliminary small molecule inhibitor assays can be carried out, which can eventually yield novel drug treatments that can be used to treat human mpox infections.