Abstract 4494: Cryo-EM structure-based small molecule inhibitor drugs targeting CSPG4/NG2 in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most malignant primary brain tumor with a median survival of 15 months despite aggressive multimodal treatment. There is an urgent need for novel drug discovery for GBM. CSPG4/NG2 transmembrane proteoglycan is a validated target, upregulated in 50% of GBM patient tumours, drives malignant progression and is independently prognostic for poor survival. We have recently identified a cancer specific 13 bp frameshift deletion in the CSPG4/NG2 gene (CSPG4/NG2del13), which leads to reduced protein expression and slowed cell growth in vitro and tumor take in vivo. The 3D structure of CSPG4/NG2 has not yet been solved, and unsuited for structure prediction by AlphaFold2 due to its size and PTMs. The structure of CSPG4/NG2 domain 1 (residues 411-547), containing the site of the 13 bp deletion, has been solved using cryo-EM and can be useful for new drug design. We hypothesize that a small-molecule inhibitor that binds to this 13 bp region of the CSPG4/NG2 proteoglycan, might induce the same phenotype as the 13 bp deletion. Methods: Using the published structure of domain 1, we performed in silico analysis to predict binding sites and docked 1280 compounds from the Prestwick Chemical library’s database of FDA-approved drugs to these sites. The high ranked hits were further tested in vitro in GBM cells. Clonogenic survival, cell proliferation and scratch-wound assays were performed in patient derived P3, A172 and in CSPG4/NG2del13 heterozygous (HZ) GBM cells, that served as controls. Results: Three binding sites were found in the close proximity to the 13 bp deletion region within the N-terminal domain 1 of CSPG4/NG2. Docking analysis revealed multiple drug candidates bound to the predicted binding site, three of which were further analyzed. Dopamine is found to closely interact with CSPG4/NG2 domain 1, near the 13 bp deletion site, forming hydrogen bonds with Glu413 and Glu521. Pyrazinamide forms two hydrogen bonds with Glu521. 5-Azacytidine fits well into active site 1, establishing hydrogen bonds with key residues Gly475, Glu482, Val472 and Thr473. The in vitro functional assays showed that the top hits have a dose dependent negative effect on colony formation and cell proliferation in P3 and A172 GBM cells (P < 0.0001). The treatment with the same drug IC50 doses led to a growth rescue in the CSPG4/NG2del13 HZ cells compared to the WT cells (P < 0.001). 5-Azacytidine showed a significant inhibitory effect (P < 0.0001) on cell migration in P3, A172 GBM cells and also in HZ cells, (P < 0.001). Conclusion: The residues spanning the site of 13 bp deletion have chemical motifs that are salient for docking small molecule inhibitors that disrupt CSPG4/NG2 function. N-terminus domain 1 and full-length CSPG4/NG2 proteins for Cryo-EM 3D structure determination are being used to interrogate biochemical kinetics and identify new target regions. Citation Format: Lasse Neset, Mia Kristin Nilsen, Victoria Smith Arnesen, Kazi Asraful Alam, Martha Chekenya, Mohummad Aminur Rahman. Cryo-EM structure-based small molecule inhibitor drugs targeting CSPG4/NG2 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4494.