Towards a mechanistic understanding of Von Hippel-Lindau syndrome in different tissues. Computational simulation and analysis of enzymatic systems to characterize the anti-cancer effect of α-ketoglutarate mimetic molecules

Von Hippel-Lindau (VHL) syndrome is a hereditary cancer disorder characterized by the development of benign and malignant tumors, caused by mutations in the VHL gene. This gene encodes for the tumor suppressor protein pVHL, which is crucial in regulating oxygen sensing. Under normal conditions, prolyl hydroxylase domain (PHD) enzymes hydroxylate hypoxia-inducible factors (HIFs-α), allowing pVHL to bind HIFs-α, driving their degradation. Hypoxia inhibits PHD activity, leading to HIFs-α stabilization and cell adaptation to hypoxia. As a hallmark of cancer, hypoxia promotes neo-angiogenesis and cell proliferation. In VHL syndrome, the loss of pVHL function results in abnormal HIF-α activation, driving tumor growth. PHD isoforms (PHD1,2,3) hydroxylate HIF-α subunits (HIF-1,2,3α) with different affinities and effects on tumor progression. However, the mechanisms determining these differences poorly understood. In this thesis, molecular dynamics (MD) simulations were employed to the molecular details governing PHDs/HIFs-α interactions focusing on PHD2 and PHD3, in complexes with HIF-1α and HIF-2α. Conservation analysis and binding free energy calculations were also performed to better understand PHDs substrate affinity. The results showed distinct affinities of PHD2 and PHD3 for HIFs-α substrates, with the PHD2 C-terminus playing a critical role in substrate binding and stabilization. MD simulations revealed that the PHD2 C-terminus exists in an equilibrium between folded and unfolded states. In its unfolded state, PHD2 enhances interactions with HIF-1α, while the folded state binds more strongly to HIF-2α. These findings challenge earlier assumptions that PHD2 preferentially binds HIF-1α, showing that under certain conditions, it binds more strongly to HIF-2α. Phosphorylation of the PHD2 residue Thr405, located in the C-terminus, influences binding energy without having a significant structural impact on PHD2/HIFs-α complexes. For PHD3, the study revealed that interactions with HIF-1α are primarily stabilized by the β2β3-loop, while its C-terminal region primarily binds HIF-2α. Despite this, binding free energy calculations suggest that PHD3 has a stronger affinity for HIF-1α, contrasting with reports that emphasize its role in regulating HIF-2α in hypoxic conditions. The study also identified cancer-associated mutations at specific interaction sites in the PHD2/HIFs-α and PHD3/HIFs-α complexes, with potential implications for drug development. Targeting specific residues could selectively modulate PHDs substrate specificity in tumors without affecting overall enzyme function. In addition, the effects of several PHDs inhibitors were examined, including N-oxalylglycine (NOG), dimethyloxalylglycine (DMOG), fumarate (FUM), Molidustat, and Vadadustat. NOG and Vadadustat were the most effective at stabilizing HIF-1α and HIF-2α complexes, while DMOG and Molidustat showed instability, even exiting the binding pocket during simulations. Overall, these findings advance our understanding of VHL syndrome’s molecular mechanisms and highlights the C-terminal region as a potential regulator of PHDs activity and suggest avenues for developing targeted therapies to modulate PHDs function in cancer and hypoxia-related diseases.