{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T03:40:33Z","timestamp":1791776433441,"version":"4.3.4"},"reference-count":31,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>NOS1AP (CAPON) is a signaling adaptor implicated in glioblastoma (GBM) proliferation, but its chemical tractability remains largely unexplored. Here, affinity selection\u2013mass spectrometry screening of 10,000 drug-like compounds identified NGM1, a small molecule scaffold that directly binds NOS1AP. Binding was confirmed by microscale thermophoresis (MST), yielding a dissociation constant of 11.9 \u00b1 7.0 \u03bcM. Focused structure\u2013activity analysis and molecular modeling identified structural features associated with NOS1AP recognition and provided a framework for compound optimization. In living cells, NGM1 produced a concentration-dependent reduction in the NanoBRET signal generated by the NOS1AP-NOS1 reporter pair, consistent with perturbation of the complex. NGM1 reduced viability in U87 and U251 GBM cells, with IC50 values of 12.7 \u00b1 0.91 and 17.3 \u00b1 1.04 \u03bcM, respectively, while normal human astrocyte viability remained above 50% at 150 \u03bcM. Mechanistic studies in U87 cells demonstrated reduced DNA synthesis, G0\/G1 cell-cycle accumulation, and induction of apoptosis, accompanied by increased p53 and CDKN1A and decreased CDK6. Collectively, these findings establish the initial chemical tractability of NOS1AP and identify NGM1 as a chemical scaffold for investigating NOS1AP-associated signaling in GBM. The findings also provide a foundation for optimizing NOS1AP-directed compounds.<\/jats:p>","DOI":"10.3390\/life16091519","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:14:59Z","timestamp":1789146899000},"page":"1519","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Small Molecule Modulation of NOS1AP for Molecularly Targeted Glioblastoma Therapy"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8977-0495","authenticated-orcid":false,"given":"Ashraf N.","family":"Abdo","sequence":"first","affiliation":[{"name":"Department of Radiology, Columbia University Irving Medical Center, New York, NY 10032, USA"},{"name":"Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sungwoo","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Radiology, Columbia University Irving Medical Center, New York, NY 10032, USA"},{"name":"Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9074-3331","authenticated-orcid":false,"given":"Moustafa","family":"Gabr","sequence":"additional","affiliation":[{"name":"Department of Radiology, Columbia University Irving Medical Center, New York, NY 10032, USA"},{"name":"Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"94","DOI":"10.3892\/ijo.2026.5907","article-title":"Radiotherapy resistance in glioblastoma: Mechanistic insights and novel therapeutic approaches (Review)","volume":"69","author":"Zhang","year":"2026","journal-title":"Int. J. Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Yang, J., Li, J., Jin, M., Lu, Y., and Zhang, L. (2026). The immunosuppressive tumor microenvironment in glioblastoma. Front. Immunol., 17.","DOI":"10.3389\/fimmu.2026.1806996"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"He, Y., Song, W., Li, S., and Guo, J. (2026). The glioblastoma ecosystem: Clonal evolution, heterogeneity, and therapeutic resistance. Front. Cell Dev. Biol., 14.","DOI":"10.3389\/fcell.2026.1834106"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"242","DOI":"10.3892\/mmr.2026.13952","article-title":"TREM2 in glioma: Reprogramming the immune microenvironment from mechanistic understanding to clinical translation (Review)","volume":"34","author":"Fan","year":"2026","journal-title":"Mol. Med. Rep."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Abuliezi, N., Zhao, J., Sun, Y., Tuersong, T., Dun, X., and Wu, Q. (2026). Reconceptualizing glioblastoma immunotherapy: A four-pillar framework to overcome multidimensional resistance. Front. Med., 13.","DOI":"10.3389\/fmed.2026.1848364"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Miko\u0142ajczak, K., Chmiel, J., and Leszek, J. (2026). Neuroinflammation in Alzheimer\u2019s Disease (AD) and Glioblastoma (GBM): Shared Mechanisms and Therapeutic Insights. Cells, 15.","DOI":"10.3390\/cells15121111"},{"key":"ref_7","first-page":"110","article-title":"Glioblastoma Multiforme: Current Developments in Molecular Pathways, Magnetic Field-Based Interventions, and Personalized Therapy","volume":"48","author":"Tosunbayraktar","year":"2026","journal-title":"J. Clin. Pract. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2297","DOI":"10.62347\/RNUV4829","article-title":"METTL14\/IGF2BP-mediated m6A methylation of circSLIT2 promotes malignant phenotypes of glioblastoma via the miR-127-5p\/SH3GLB1 axis","volume":"16","author":"Wu","year":"2026","journal-title":"Am. J. Cancer Res."},{"key":"ref_9","first-page":"vdaf255","article-title":"Innovative radiotherapies for the treatment of glioblastoma","volume":"8","author":"Waked","year":"2025","journal-title":"Neurooncol. Adv."},{"key":"ref_10","first-page":"422","article-title":"Efficacy of tepotinib in patients with high-grade glioma with MET alterations: A case series","volume":"13","author":"Rodriguez","year":"2025","journal-title":"Neurooncol. Pract."},{"key":"ref_11","first-page":"vdaf252","article-title":"Nuclear and membrane-bound hormone receptors in glioblastoma: Expression, functionality, and therapeutic implications","volume":"8","author":"Gantner","year":"2025","journal-title":"Neurooncol. Adv."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"bgag008","DOI":"10.1093\/carcin\/bgag008","article-title":"A dual approach to glioblastoma treatment with epigenetic reprogramming and neurogenetic modulation","volume":"47","author":"Yuncu","year":"2025","journal-title":"Carcinogenesis"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"101841","DOI":"10.1016\/j.gendis.2025.101841","article-title":"Targeting SUMOylation in glioblastoma: A novel avenue for therapy and biomarker discovery","volume":"13","author":"Dubanosow","year":"2025","journal-title":"Genes Dis."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Meleiro, M., and Henrique, R. (2025). Epigenetic Alterations in Glioblastoma Multiforme as Novel Therapeutic Targets: A Scoping Review. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26125634"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1007\/s10238-025-01631-0","article-title":"Emerging therapeutic strategies in glioblastoma: Drug repurposing, mechanisms of resistance, precision medicine, and technological innovations","volume":"25","author":"Anwer","year":"2025","journal-title":"Clin. Exp. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/S0896-6273(00)80439-0","article-title":"CAPON: A protein associated with neuronal nitric oxide synthase that regulates its interactions with PSD95","volume":"20","author":"Jaffrey","year":"1998","journal-title":"Neuron"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.1007\/s12264-023-01060-7","article-title":"nNOS and Neurological, Neuropsychiatric Disorders: A 20-Year Story","volume":"39","author":"Zhu","year":"2023","journal-title":"Neurosci. Bull."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Courtney, M.J., Li, L.L., and Lai, Y.Y. (2014). Mechanisms of NOS1AP action on NMDA receptor\u2013nNOS signaling. Front. Cell. Neurosci., 8.","DOI":"10.3389\/fncel.2014.00252"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/S0896-6273(00)00095-7","article-title":"Dexras1: A G protein specifically coupled to neuronal nitric oxide synthase via CAPON","volume":"28","author":"Fang","year":"2000","journal-title":"Neuron"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Xie, W., Xing, N., Qu, J., Liu, D., and Pang, Q. (2023). The Physiological Function of nNOS-Associated CAPON Proteins and the Roles of CAPON in Diseases. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242115808"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"614","DOI":"10.7150\/ijms.31579","article-title":"Effects of Long Form of CAPON Overexpression on Glioma Cell Proliferation are Dependent on AKT\/mTOR\/P53 Signaling","volume":"16","author":"Liang","year":"2019","journal-title":"Int. J. Med. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"152793","DOI":"10.1016\/j.bbrc.2025.152793","article-title":"Affinity selection\u2013mass spectrometry coupled with biophysical validation enables proof-of-concept discovery of CHI3L1 binders","volume":"787","author":"Kaur","year":"2025","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"25112","DOI":"10.1021\/acs.jmedchem.5c02136","article-title":"Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-Stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation","volume":"68","author":"Upadhyay","year":"2025","journal-title":"J. Med. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e202400545","DOI":"10.1002\/cmdc.202400545","article-title":"Discovery of ICOS-Targeted Small Molecules Using Affinity Selection Mass Spectrometry Screening","volume":"19","author":"Zhang","year":"2024","journal-title":"ChemMedChem"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.ddtec.2021.10.005","article-title":"Drug discovery from natural products using affinity selection-mass spectrometry","volume":"40","author":"Muchiri","year":"2021","journal-title":"Drug Discov. Today Technol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1080\/17460441.2026.2622373","article-title":"Affinity selection mass spectrometry (AS-MS) as a tool to accelerate drug discovery efforts","volume":"21","author":"Pandey","year":"2026","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3855","DOI":"10.1021\/acs.analchem.4c04445","article-title":"pyBinder: Quantitation to Advance Affinity Selection-Mass Spectrometry","volume":"97","author":"Brown","year":"2025","journal-title":"Anal. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"100174","DOI":"10.1016\/j.slast.2024.100174","article-title":"Optimizing the affinity selection mass spectrometry workflow for efficient identification and ranking of potent USP1 inhibitors","volume":"29","author":"Zhao","year":"2024","journal-title":"SLAS Technol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"103760","DOI":"10.1016\/j.drudis.2023.103760","article-title":"Affinity selection mass spectrometry speeding drug discovery","volume":"28","author":"Prudent","year":"2023","journal-title":"Drug Discov. Today"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1002\/mas.21800","article-title":"Affinity selection-mass spectrometry in the discovery of anti-SARS-CoV-2 compounds","volume":"43","author":"Muchiri","year":"2024","journal-title":"Mass Spectrom. Rev."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"W582","DOI":"10.1093\/nar\/gkq383","article-title":"fpocket: Online tools for protein ensemble pocket detection and tracking","volume":"38","author":"Schmidtke","year":"2010","journal-title":"Nucleic Acids Res."}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/9\/1519\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T04:15:37Z","timestamp":1789532137000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/16\/9\/1519"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,11]]},"references-count":31,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["life16091519"],"URL":"https:\/\/doi.org\/10.3390\/life16091519","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,11]]}}}