{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T01:11:04Z","timestamp":1777338664917,"version":"3.51.4"},"reference-count":54,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,6,16]],"date-time":"2022-06-16T00:00:00Z","timestamp":1655337600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Israeli Innovation Authority","award":["55926"],"award-info":[{"award-number":["55926"]}]},{"name":"Dahlia Greidinger anti-cancer fund","award":["55926"],"award-info":[{"award-number":["55926"]}]},{"name":"Israel Cancer Association","award":["55926"],"award-info":[{"award-number":["55926"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>The initial discovery phase of protein modulators, which consists of filtering molecular libraries and in vitro direct binding validation, is central in drug discovery. Thus, virtual screening of large molecular libraries, together with the evaluation of binding affinity by isothermal calorimetry, generates an efficient experimental setup. Herein, we applied virtual screening for discovering small molecule inhibitors of MDM2, a major negative regulator of the tumor suppressor p53, and thus a promising therapeutic target. A library of 20 million small molecules was screened against an averaged model derived from multiple structural conformations of MDM2 based on published structures. Selected molecules originating from the computational filtering were tested in vitro for their direct binding to MDM2 via isothermal titration calorimetry. Three new molecules, representing distinct chemical scaffolds, showed binding to MDM2. These were further evaluated by exploring structure-similar chemical analogues. Two scaffolds were further evaluated by de novo synthesis of molecules derived from the initial molecules that bound MDM2, one with a central oxoazetidine acetamide and one with benzene sulfonamide. Several molecules derived from these scaffolds increased wild-type p53 activity in MCF7 cancer cells. These set a basis for further chemical optimization and the development of new chemical entities as anticancer drugs.<\/jats:p>","DOI":"10.3390\/ph15060752","type":"journal-article","created":{"date-parts":[[2022,6,16]],"date-time":"2022-06-16T03:01:22Z","timestamp":1655348482000},"page":"752","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Application of In Silico Filtering and Isothermal Titration Calorimetry for the Discovery of Small Molecule Inhibitors of MDM2"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6246-6023","authenticated-orcid":false,"given":"Hen","family":"Alali","sequence":"first","affiliation":[{"name":"Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Itai","family":"Bloch","sequence":"additional","affiliation":[{"name":"Migal\u2014Galilee Research Institute, Tel-Hai Academic College, Upper Galilee 12210, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irena","family":"Rapaport","sequence":"additional","affiliation":[{"name":"Migal\u2014Galilee Research Institute, Tel-Hai Academic College, Upper Galilee 12210, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luisa","family":"Rodrigues","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Inbal","family":"Sher","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tamar","family":"Ansbacher","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"Hadassah Academic College, Jerusalem 91010, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3668-8647","authenticated-orcid":false,"given":"Maayan","family":"Gal","sequence":"additional","affiliation":[{"name":"Department of Oral Biology, Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,16]]},"reference":[{"key":"ref_1","unstructured":"Donev, R. (2020). Protein Interactions as Targets in Drug Discovery, Academic Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/j.tips.2012.03.006","article-title":"Structural biology and drug discovery for protein\u2013protein interactions","volume":"33","author":"Jubb","year":"2012","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1002\/bip.20608","article-title":"Protein-protein interactions as targets for small molecule drug discovery","volume":"84","author":"Fry","year":"2006","journal-title":"Biopolymers"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1016\/j.cell.2011.02.016","article-title":"Interactome networks and human disease","volume":"144","author":"Vidal","year":"2011","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1257601","DOI":"10.1126\/science.1257601","article-title":"Uncovering disease-disease relationships through the incomplete interactome","volume":"347","author":"Menche","year":"2015","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6959","DOI":"10.1073\/pnas.0708078105","article-title":"Estimating the size of the human interactome","volume":"105","author":"Stumpf","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","first-page":"5762","article-title":"Therapeutic opportunities in cancer therapy: Targeting the p53-MDM2\/MDMX interactions","volume":"11","author":"Munisamy","year":"2021","journal-title":"Am. J. Cancer Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"a026245","DOI":"10.1101\/cshperspect.a026245","article-title":"Targeting the MDM2-p53 Protein-Protein Interaction for New Cancer Therapy: Progress and Challenges","volume":"7","author":"Wang","year":"2017","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.ejmech.2019.05.018","article-title":"The past, present and future of potential small-molecule drugs targeting p53-MDM2\/MDMX for cancer therapy","volume":"176","author":"Liu","year":"2019","journal-title":"Eur. J. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1415","DOI":"10.1038\/sj.bjc.6602164","article-title":"Targeting the p53-MDM2 interaction to treat cancer","volume":"91","author":"Klein","year":"2004","journal-title":"Br. J. Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/nrm2147","article-title":"p53 in health and disease","volume":"8","author":"Vousden","year":"2007","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"994","DOI":"10.3390\/cancers3010994","article-title":"Role of p53 in Cell Death and Human Cancers","volume":"3","author":"Ozaki","year":"2011","journal-title":"Cancers"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1038\/nrc2723","article-title":"The first 30 years of p53: Growing ever more complex","volume":"9","author":"Levine","year":"2009","journal-title":"Nat. Rev. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1093\/jmcb\/mjy070","article-title":"Tumor suppressor p53 and metabolism","volume":"11","author":"Liu","year":"2019","journal-title":"J. Mol. Cell Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1038\/nrc2693","article-title":"When mutants gain new powers: News from the mutant p53 field","volume":"9","author":"Brosh","year":"2009","journal-title":"Nat. Rev. Cancer"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1007\/978-94-017-9211-0_4","article-title":"p53: Its mutations and their impact on transcription","volume":"85","author":"Vaughan","year":"2014","journal-title":"Subcell. Biochem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1002\/(SICI)1097-4644(19970301)64:3<343::AID-JCB1>3.0.CO;2-V","article-title":"Mdm-2: \u201cbig brother\u201d of p53","volume":"64","author":"Momand","year":"1997","journal-title":"J. Cell. Biochem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3","DOI":"10.2174\/1568009053332627","article-title":"MDM2 is a central node in the p53 pathway: 12 years and counting","volume":"5","author":"Bond","year":"2005","journal-title":"Curr. Cancer Drug Targets"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/S0959-437X(01)00264-7","article-title":"The p53 and Mdm2 families in cancer","volume":"12","author":"Michael","year":"2002","journal-title":"Curr. Opin. Genet. Dev."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Beloglazkina, A., Zyk, N., Majouga, A., and Beloglazkina, E. (2020). Recent Small-Molecule Inhibitors of the p53-MDM2 Protein-Protein Interaction. Molecules, 25.","DOI":"10.3390\/molecules25051211"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1080\/13543776.2019.1582645","article-title":"A therapeutic patent overview of MDM2\/X-targeted therapies (2014\u20132018)","volume":"29","author":"Skalniak","year":"2019","journal-title":"Expert Opin. Ther. Pat."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"324","DOI":"10.2174\/1574892814666191022163540","article-title":"MDM2-p53 Interaction Inhibitors: The Current State-of-Art and Updated Patent Review (2010-Present)","volume":"14","author":"Rusiecki","year":"2019","journal-title":"Recent Pat. Anticancer Drug Discov."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1186\/s13045-017-0500-5","article-title":"MDM2\/X inhibitors under clinical evaluation: Perspectives for the management of hematological malignancies and pediatric cancer","volume":"10","author":"Tisato","year":"2017","journal-title":"J. Hematol. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1126\/science.1092472","article-title":"In vivo activation of the p53 pathway by small-molecule antagonists of MDM2","volume":"303","author":"Vassilev","year":"2004","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6332","DOI":"10.1021\/jm500627s","article-title":"Discovery of a small molecule MDM2 inhibitor (AMG 232) for treating cancer","volume":"57","author":"Rew","year":"2014","journal-title":"J. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1021\/ci100113p","article-title":"Molecular interaction fields and 3D-QSAR studies of p53-MDM2 inhibitors suggest additional features of ligand-target interaction","volume":"50","author":"Dezi","year":"2010","journal-title":"J. Chem. Inf. Model."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"12809","DOI":"10.1021\/ja073687x","article-title":"Small molecule inhibitors of the MDM2-p53 interaction discovered by ensemble-based receptor models","volume":"129","author":"Bowman","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3759","DOI":"10.1021\/jm060023+","article-title":"Discovery of a nanomolar inhibitor of the human murine double minute 2 (MDM2)-p53 interaction through an integrated, virtual database screening strategy","volume":"49","author":"Lu","year":"2006","journal-title":"J. Med. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1021\/cc050142v","article-title":"Proteomimetic libraries: Design, synthesis, and evaluation of p53-MDM2 interaction inhibitors","volume":"8","author":"Lu","year":"2006","journal-title":"J. Comb. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2504","DOI":"10.1021\/jm200045v","article-title":"Small-molecule ligands of methyl-lysine binding proteins","volume":"54","author":"Herold","year":"2011","journal-title":"J. Med. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2417","DOI":"10.1021\/ci200280m","article-title":"Discovery of novel selective serotonin reuptake inhibitors through development of a protein-based pharmacophore","volume":"51","author":"Manepalli","year":"2011","journal-title":"J. Chem. Inf. Model."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1021\/ml4000657","article-title":"Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development","volume":"4","author":"Vu","year":"2013","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4934","DOI":"10.1021\/jm3007068","article-title":"AM-8553: A novel MDM2 inhibitor with a promising outlook for potential clinical development","volume":"55","author":"Bernard","year":"2012","journal-title":"J. Med. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1021\/cc9000218","article-title":"Rapid and efficient hydrophilicity tuning of p53\/mdm2 antagonists","volume":"11","author":"Srivastava","year":"2009","journal-title":"J. Comb. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.4161\/cc.9.6.10956","article-title":"Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal new approaches for p53-MDMX\/MDM2 antagonist drug discovery","volume":"9","author":"Popowicz","year":"2010","journal-title":"Cell Cycle"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2441","DOI":"10.4161\/cc.6365","article-title":"Structure of the human Mdmx protein bound to the p53 tumor suppressor transactivation domain","volume":"7","author":"Popowicz","year":"2008","journal-title":"Cell Cycle"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3498","DOI":"10.1016\/j.bmcl.2012.03.083","article-title":"The central valine concept provides an entry in a new class of non peptide inhibitors of the p53-MDM2 interaction","volume":"22","author":"Furet","year":"2012","journal-title":"Bioorganic Med. Chem. Lett."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1126\/science.274.5289.948","article-title":"Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain","volume":"274","author":"Kussie","year":"1996","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1016\/j.jmb.2010.03.005","article-title":"Systematic mutational analysis of peptide inhibition of the p53-MDM2\/MDMX interactions","volume":"398","author":"Li","year":"2010","journal-title":"J. Mol. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"31a","DOI":"10.1016\/j.bpj.2017.11.217","article-title":"Repeatability of enthalpies and Gibbs energies of a protein-ligand binding reaction measured by ITC","volume":"114","author":"Paketuryte","year":"2018","journal-title":"Biophys. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.3389\/fphar.2018.01133","article-title":"Application of ITC-Based Characterization of Thermodynamic and Kinetic Association of Ligands With Proteins in Drug Design","volume":"9","author":"Su","year":"2018","journal-title":"Front. Pharmacol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"4","DOI":"10.2174\/1386207319666151203001529","article-title":"Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein","volume":"19","author":"Gal","year":"2016","journal-title":"Comb. Chem. High Throughput Screen."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"4398","DOI":"10.1021\/acsomega.7b00576","article-title":"A small molecule inhibitor of Bruton\u2019s tyrosine kinase involved in B-cell signaling","volume":"2","author":"Ratzon","year":"2017","journal-title":"ACS Omega"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2216","DOI":"10.1007\/s00044-021-02802-w","article-title":"Sulfonamide derivatives of cis-imidazolines as potent p53-MDM2\/MDMX protein-protein interaction inhibitors","volume":"30","author":"Bazanov","year":"2021","journal-title":"Med. Chem. Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"F971","DOI":"10.1152\/ajprenal.00294.2009","article-title":"The Ste20-like kinase SLK promotes p53 transactivation and apoptosis","volume":"297","author":"Cybulsky","year":"2009","journal-title":"Am. J. Physiol. Renal. Physiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1177\/1087057112465184","article-title":"Affordable luciferase reporter assay for cell-based high-throughput screening","volume":"18","author":"Vermeulen","year":"2013","journal-title":"J. Biomol. Screen."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4478","DOI":"10.1038\/sj.onc.1206777","article-title":"Rescue of mutant p53 transcription function by ellipticine","volume":"22","author":"Peng","year":"2003","journal-title":"Oncogene"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"724","DOI":"10.1016\/j.bcp.2010.04.031","article-title":"Current strategies to target p53 in cancer","volume":"80","author":"Chen","year":"2010","journal-title":"Biochem. Pharm."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4163","DOI":"10.1021\/jm034182u","article-title":"A nonpeptidic sulfonamide inhibits the p53-mdm2 interaction and activates p53-dependent transcription in mdm2-overexpressing cells","volume":"47","author":"Galatin","year":"2004","journal-title":"J. Med. Chem."},{"key":"ref_50","first-page":"11","article-title":"Current Challenges and Opportunities in Designing Protein\u2013Protein Interaction Targeted Drugs","volume":"13","author":"Shin","year":"2020","journal-title":"Bioinform. Chem."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"811190","DOI":"10.3389\/fchem.2021.811190","article-title":"Editorial: Protein-Protein Interactions: Drug Discovery for the Future","volume":"9","author":"Rapposelli","year":"2021","journal-title":"Front. Chem."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1545","DOI":"10.1002\/jcc.21287","article-title":"CHARMM: The biomolecular simulation program","volume":"30","author":"Brooks","year":"2009","journal-title":"J. Comput. Chem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1021\/ci00024a018","article-title":"Analysis of Conformational Coverage. 1. Validation and Estimation of Coverage","volume":"35","author":"Smellie","year":"1995","journal-title":"J. Chem. Inf. Comput. Sci."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"6504","DOI":"10.1021\/jm050543p","article-title":"Modeling water molecules in protein-ligand docking using GOLD","volume":"48","author":"Verdonk","year":"2005","journal-title":"J. Med. Chem."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/15\/6\/752\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:32:39Z","timestamp":1760139159000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/15\/6\/752"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,16]]},"references-count":54,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["ph15060752"],"URL":"https:\/\/doi.org\/10.3390\/ph15060752","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,16]]}}}