{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T23:24:22Z","timestamp":1790897062421,"version":"4.1.0"},"reference-count":35,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T00:00:00Z","timestamp":1742774400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The incorporation of disease-relevant targets into ternary complexes in a compound-dependent manner by utilizing an assisting chaperone has become a common modality as far as bifunctional ternary complex-forming compounds are concerned. In contrast, examples of ternary complexes formed by molecular glues are much rarer. Due to their lack of significant binary (independent) target affinity, their identification cannot yet be achieved by rational methods and is, therefore, much more challenging. However, it is precisely for that reason (given the associated advantages) that their systematic identification and application in drug discovery has recently attracted particular interest. In contrast to bifunctional ternary complex-forming compounds, molecular glues retrieve a significant part of their thermodynamic stability through newly induced chaperone\u2013target or glue\u2013target interactions that occur only in the ternary complex. These interactions lead to enhanced ligand binding\u2014termed intrinsic cooperativity \u03b1\u2014which can be retrieved via the apparent cooperativity either by monitoring ligand binding through the chaperone or through the target protein. In this publication, the advantage of measuring the apparent cooperativity (to determine the cooperativity \u03b1) by the weaker binding protein is discussed and illustrated using the example of ternary complexes between FKBP12, MAPRE1 and macrocyclic molecular glues derived from the rapamycin binding motif for FKBP12. Furthermore, the impact of the following three parameters on the apparent cooperativity is illustrated: (1) the concentration of the monitoring protein, (2) the excess of the counter protein, and (3) the affinity of the glue to the weaker binding protein in combination with the degree of intrinsic cooperativity \u03b1. From this, experimental conditions to determine the intrinsic cooperativity \u03b1 with only one binding assay and without the need for a comprehensive mathematical model covering all simultaneous events under non-saturating conditions are highlighted. However, this framework requires a binding assay capable of measuring or at least estimating very weak binary affinities. If this is not possible for experimental reasons, but binding assays for both proteins are available within a normal bandwidth and the affinity to the stronger binding protein is not too high, it is discussed how the binding curve for the weaker binding protein in the presence of an excess of the weaker binding protein can be used to overcome the missing binary Kd for the weakly binding protein.<\/jats:p>","DOI":"10.3390\/ijms26072936","type":"journal-article","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T12:01:50Z","timestamp":1742817710000},"page":"2936","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Experimental Conditions to Retrieve Intrinsic Cooperativity \u03b1 Directly from Single Binding Assay Data Exemplified by the Ternary Complex Formation of FKBP12, MAPRE1 and Macrocyclic Molecular Glues"],"prefix":"10.3390","volume":"26","author":[{"given":"Jan","family":"Schnatwinkel","sequence":"first","affiliation":[{"name":"NanoTemper Technologies, 81379 M\u00fcnchen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard R.","family":"Stein","sequence":"additional","affiliation":[{"name":"Novartis, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6695-6513","authenticated-orcid":false,"given":"Michael","family":"Salcius","sequence":"additional","affiliation":[{"name":"Novartis, Cambridge, MA 02139, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian L.","family":"Wong","sequence":"additional","affiliation":[{"name":"Novartis, Emeryville, CA 94608, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1762-0269","authenticated-orcid":false,"given":"Shu-Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Applied Biosciences, ETH Zurich, 8092 Zurich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marianne","family":"Fouch\u00e9","sequence":"additional","affiliation":[{"name":"Novartis, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5517-775X","authenticated-orcid":false,"given":"Hans-Joerg","family":"Roth","sequence":"additional","affiliation":[{"name":"Novartis, 4056 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1032","DOI":"10.1016\/j.chembiol.2021.04.009","article-title":"Haven\u2019t got a glue: Protein surface variation for the design of molecular glue degraders","volume":"28","author":"Kozicka","year":"2021","journal-title":"Cell Chem. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Li, F., Aljahdali, I.A.M., and Ling, X. (2022). Molecular Glues: Capable Protein-Binding Small Molecules That Can Change Protein-Protein Interactions and Interactomes for the Potential Treatment of Human Cancer and Neurodegenerative Diseases. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23116206"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3548","DOI":"10.1016\/j.apsb.2022.03.019","article-title":"Molecular glues modulate protein functions by inducing protein aggregation: A promising therapeutic strategy of small molecules for disease treatment","volume":"12","author":"Wu","year":"2022","journal-title":"Acta Pharm. Sin. B"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1007\/s00044-022-02882-2","article-title":"Molecular glues: Enhanced protein-protein interactions and cell proteome editing","volume":"31","author":"Weagel","year":"2022","journal-title":"Med. Chem. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"100018","DOI":"10.1016\/j.crchbi.2021.100018","article-title":"Clues to molecular glues","volume":"2","author":"Geiger","year":"2022","journal-title":"Curr. Res. Chem. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"10680","DOI":"10.1021\/acs.jmedchem.1c01126","article-title":"The Future of Medicinal Chemistry, PROTAC, and Undruggable Drug Targets","volume":"64","author":"Poso","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"8902","DOI":"10.1021\/jacs.1c03852","article-title":"TF-PROTACs Enable Targeted Degradation of Transcription Factors","volume":"143","author":"Liu","year":"2021","journal-title":"J. Am. Chem. Soc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9278","DOI":"10.1021\/acs.jmedchem.3c00449","article-title":"Molecular Glue Discovery: Current and Future Approaches","volume":"66","author":"Dewey","year":"2023","journal-title":"J. Med. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2807","DOI":"10.1021\/acs.jmedchem.9b01167","article-title":"Heterobifunctional Molecules Induce Dephosphorylation of Kinases\u2014A Proof of Concept Study","volume":"63","author":"Yamazoe","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8091","DOI":"10.1021\/acs.jmedchem.2c00316","article-title":"Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors","volume":"65","author":"Hua","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Lai, S., Modell, A.E., and Choudhary, A. (2022). Proximity-Inducing Bifunctional Molecules Beyond PROTACs. Protein Homeostasis in Drug Discovery: A Chemical Biology Perspective, Wiley.","DOI":"10.1002\/9781119774198.ch12"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"14052","DOI":"10.1021\/jacs.0c05537","article-title":"Phosphorylation-Inducing Chimeric Small Molecules","volume":"142","author":"Siriwardena","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"26028","DOI":"10.1021\/jacs.3c06179","article-title":"Bifunctional Small Molecules That Induce Nuclear Localization and Targeted Transcriptional Regulation","volume":"145","author":"Gibson","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1039\/D3CB00084B","article-title":"Bringing enzymes to the proximity party","volume":"4","author":"Tender","year":"2023","journal-title":"RSC Chem. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1021\/acscentsci.3c00003","article-title":"What Makes a Good Protein-Protein Interaction Stabilizer: Analysis and Application of the Dual-Binding Mechanism","volume":"9","author":"Chen","year":"2023","journal-title":"ACS Cent. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2869","DOI":"10.1039\/C8SC05242E","article-title":"Cooperativity basis for small-molecule stabilization of protein\u2013protein interactions","volume":"10","author":"Andrei","year":"2019","journal-title":"Chem. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1016\/j.chembiol.2024.04.002","article-title":"Molecular glues for protein-protein interactions: Progressing toward a new dream","volume":"31","author":"Konstantinidou","year":"2024","journal-title":"Cell Chem. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1038\/s41467-022-28507-1","article-title":"Defining molecular glues with a dual-nanobody cannabidiol sensor","volume":"13","author":"Cao","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e202300163","DOI":"10.1002\/cbic.202300163","article-title":"Biophysical and Computational Approaches to Study Ternary Complexes: A \u2018Cooperative Relationship\u2019 to Rationalize Targeted Protein Degradation","volume":"24","author":"Ward","year":"2023","journal-title":"Chembiochem"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1038\/s41589-021-00765-y","article-title":"Discovery and resistance mechanism of a selective CDK12 degrader","volume":"17","author":"Jiang","year":"2021","journal-title":"Nat. Chem. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1038\/s41589-018-0055-y","article-title":"Plasticity in binding confers selectivity in ligand-induced protein degradation","volume":"14","author":"Nowak","year":"2018","journal-title":"Nat. Chem. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1021\/acschembio.2c00747","article-title":"CRISPR Screen Reveals BRD2\/4 Molecular Glue-like Degrader via Recruitment of DCAF16","volume":"18","author":"Shergalis","year":"2023","journal-title":"ACS Chem. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1629","DOI":"10.1021\/acsmedchemlett.1c00543","article-title":"Degradation of Protein Kinases: Ternary Complex, Cooperativity, and Selectivity","volume":"12","author":"Grigglestone","year":"2021","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.chembiol.2018.11.006","article-title":"Homolog-Selective Degradation as a Strategy to Probe the Function of CDK6 in AML","volume":"26","author":"Brand","year":"2019","journal-title":"Cell Chem. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"8113","DOI":"10.1021\/acs.jmedchem.2c00397","article-title":"Selectivity through Targeted Protein Degradation (TPD)","volume":"65","author":"Gopalsamy","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1042\/EBC20170041","article-title":"Molecular recognition of ternary complexes: A new dimension in the structure-guided design of chemical degraders","volume":"61","author":"Hughes","year":"2017","journal-title":"Essays Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"11772","DOI":"10.1021\/jacs.0c02151","article-title":"Selectivity via Cooperativity: Preferential Stabilization of the p65\/14-3-3 Interaction with Semisynthetic Natural Products","volume":"142","author":"Wolter","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1038\/nchembio.2329","article-title":"Structural basis of PROTAC cooperative recognition for selective protein degradation","volume":"13","author":"Gadd","year":"2017","journal-title":"Nat. Chem. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1039\/D2CB00216G","article-title":"A model-informed method to retrieve intrinsic from apparent cooperativity and project cellular target occupancy for ternary complex-forming compounds","volume":"4","author":"Stein","year":"2023","journal-title":"RSC Chem. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"15533","DOI":"10.1038\/s41598-017-15502-6","article-title":"Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites","volume":"7","author":"Almeida","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Salcius, M., Tutter, A., Fouch\u00e9, M., King, D., Dhembi, A., Koc, H., Golosov, A., Jahnke, W., Henry, C., and Argoti, D. (2024). Identification and characterization of ternary complexes consisting of FKBP12, MAPRE1 and macrocyclic molecular glues. RSC Chem. Biol., in preparation.","DOI":"10.1039\/D4CB00279B"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1089\/adt.2021.133","article-title":"A New Spectral Shift-Based Method to Characterize Molecular Interactions","volume":"20","author":"Langer","year":"2022","journal-title":"Assay Drug Dev. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1002\/pro.2520","article-title":"Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments","volume":"23","author":"Majorek","year":"2014","journal-title":"Protein Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1002\/pro.3280","article-title":"Improvements to the APBS biomolecular solvation software suite","volume":"27","author":"Jurrus","year":"2018","journal-title":"Protein Sci."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/2936\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:59:15Z","timestamp":1760029155000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/7\/2936"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,24]]},"references-count":35,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["ijms26072936"],"URL":"https:\/\/doi.org\/10.3390\/ijms26072936","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2025.01.15.631501","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,24]]}}}