Discovery of small-molecule modulators of ppGalNAc-T2 targeting the metastable states during its loop-closing process

The polypeptide N-acetyl-galactosaminyltransferase 2 (ppGalNAc-T2) is a promising therapeutic target for metabolic and neurodevelopmental disorders in humans. The atomic-scale insights into metastable states of ppGalNAc-T2 during the loop-closing process had been revealed by the Markov state model previously. Here, we further applied the metastable conformations of ppGalNAc-T2 for the discovery of novel small-molecule modulators. A coexistent pocket with high druggability was detected and applied in the structure-based virtual screening against five metastable conformations. Based on a weighted scoring strategy considering the conformational population of metastable states, chemical entities of 29 candidate compounds were selected for experimental validation. Two inhibitors (compounds 10 and 15) with IC₅₀ values of 9.25 ± 3.83 μM and 7.11 ± 2.58 μM respectively, and an activator (compound 12) with the maximal activity of threefold of the baseline were identified for the first time. Further molecular modeling studies revealed that compound 12 and 15 interacted with metastable conformations in the similar binding modes to luteolin in the crystal structure, and differed in dynamic stabilities when binding to metastable conformations. These findings not only provide a feasible strategy for the virtual screening against multiple metastable states but also expand the structural diversity of small-molecule modulators of ppGalNAc-T2.