Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric...

Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP⁺-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP⁺ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.