Abstract
Background: Inosine monophosphate dehydrogenase 2 (IMPDH2) is a critical rate-limiting enzyme in the de novo guanine nucleotide biosynthesis pathway and is essential for rapidly proliferating cells. IMPDH2 expression is upregulated in hematological malignancies and solid tumors. In contrast, the expression of its isoform IMPDH1 remains low. Current drugs, such as mycophenolic acid (MPA) and its prodrug mycophenolate mofetil (MMF), inhibit both isoforms and cause IMPDH filament formation, which may lead to metabolic imbalance and side effects. Therefore, we need more targeted methods to identify allosteric IMPDH inhibitors that overcome those issues. Furthermore, the activity of many metabolic enzymes is regulated by their post-translational modification (PTM). The role of IMPDH2 PTM in regulating IMPDH2 activity remains understudied. This study aims to investigate the mechanisms by which tyrosine phosphorylation at Y233 in the allosteric regulatory domain and phosphoinositide interactions, specifically phosphatidylinositol 3-phosphate (PI(3)P), regulate IMPDH2, and to identify novel peptides and small molecules that can selectively inhibit IMPDH2.
Methods: We investigated IMPDH2 tyrosine phosphorylation using in vitro kinase assays with recombinant ALK and SRC kinases, followed by LC-MS/MS phosphoproteomics. IMPDH2-phospholipid binding was assessed via PIP strips and lipid overlay assays. Enzymatic activity assays performed on purified IMPDH1 and IMPDH2, tested the effects of PI(3)P, peptides, and inhibitors on IMPDH activity. Peptides derived from the IMPDH2 Y233 region in allosteric domain motifs were evaluated for their inhibitory effects. Structure-based computational molecular docking of the peptide binding to IMPDH2's allosteric site and in silico screening were performed. The effects of inhibitors on lymphoma cell lines, including ALK-inhibitor-resistant variants, were assessed through proliferation, IMPDH activity, and colony formation assays.
Results: IMPDH2 was significantly overexpressed in hematologic malignancies, such as anaplastic large cell lymphoma (ALCL), mantle cell lymphoma (MCL), and diffuse large B-cell Lymphoma (DLBCL), whereas IMPDH1 showed minimal expression. Our phosphoproteomic analysis revealed multiple phosphorylation sites on IMPDH2, notably Y110 and Y233, both of which are located within the cystathionine-β-synthase (CBS domain), also known as a Bateman domain, which is part of its allosteric regulatory domain. In vitro kinase assays confirmed phosphorylation of IMPDH2 at Y110 and Y233 by SRC and ALK kinases. Phosphorylation at Y233 disrupted IMPDH2 binding to PI(3)P. A synthetic peptide derived from the Y233 region selectively inhibited IMPDH2 enzymatic activity in vitro, supporting the allosteric regulatory model. Structure-based screening led to the identification of a novel compound, Comp-10, which binds on the GTP allosteric domain of IMPDH2 and mimics the inhibitory function of PI(3)P. Comp-10 exhibited potent enzymatic inhibition (IC₅₀ ~260 nM) of human IMPDH2, selectively reduced IMPDH2 protein levels, and disrupted IMPDH2 filament formation (rods and rings), unlike MPA, which stabilizes these structures. Comp-10 significantly reduced cell growth in ALCL, MCL, and DLBCL cell lines, including ALK inhibitor–resistant variants.
Conclusion: This study reveals novel mechanisms by which IMPDH2 is regulated through isoform-specific phosphorylation and phosphoinositide binding. These regulatory events directly affect the enzymatic activity and structural dynamics of IMPDH2, providing insights into cancer-specific metabolic control. Our discovery indicates that PI(3)P selectively binds to and inhibits IMPDH2, and this interaction is modulated by tyrosine phosphorylation, introducing a unique phospholipid-mediated regulatory axis. Additionally, we identify peptides and a small molecule, Comp-10, that selectively inhibit IMPDH2 through allosteric mechanisms without promoting filament formation. This work advances the understanding of isoform-specific IMPDH regulation and opens new avenues for precision oncology targeting metabolic vulnerabilities.
Disclosures: NP and JB are listed as inventors on a provisional patent application filed by Fox Chase Cancer Center for the discovery of Comp-10, a novel IMPDH inhibitor. This patent application relates to the content discussed or presented in this work.
