Objectives

O.1 Characterization of hγD-p23T:αB-Crys interaction

In order to better understand how structural changes in the P23T variant of hγD-Crys lead to congenital cataract, we carry out comparative protein-protein interaction studies between wild-type hγD-Crys, and hγD-P23T, with small heat shock chaperone αB-Crys. The interaction differences will provide a molecular framework for understanding whether the protein aggregation associated with the cataract is a direct property of the mutation, or may reflect an inability to be recognized by the α-Crys chaperone. In order to gain new insights whether αB-Crys is able to suppress to the aggregation of hγD-p23T mutant, we use solution NMR. Suppression levels of hγD-p23T aggregation in the presence of αB-Crys will be monitored as a function of increasing chaperone: mutant molar ratio.

 O.2 In silico inhibitors screening of hγD-P23T aggregation

We aim to identify novel suppression inhibitors against hγD-P23T aggregation, using in silico compounds screening. The potential inhibitors of hγD-P23T aggregation will be searched through docking subset of compound found in ZINC15, a public access database of commercially available compounds. Subsequently, the best performing ones will be further analyzed by molecular dynamics simulations to improve docking results. A molecular docking based on shape complementarity will be carried out to subset rank in term of free energy score, and predicted affinity to the binding cleft of the protein.

 O.3 Suppression bioassay of hγD-P23T aggregation

Inhibitors aggregation suppression in vitro will be performed using turbidity bioassay, protein-ligand binding NMR studies in solution, ITC, and molecular docking. Combining in silico tools, with aggregation suppression bioassays, will reduce time and cost afferent to high-throughput library screening.