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Inhibiting the interaction between the Pro\u2010Trp\u2010Trp\u2010Pro (PWWP) domain of LEDGF and chromatin through a small\u2010molecule drug presents an attractive therapeutic opportunity, but the compounds developed to date bind entirely within the small H3K36me2\/3 pocket and achieve only modest affinity. Here, we report\n                    <jats:italic>de novo<\/jats:italic>\n                    computational design and structural validation of proteinaceous binders that engage both this canonical pocket and adjacent DNA\u2010interacting surfaces of the PWWP domain. Using a hotspot\u2010driven workflow integrating RFdiffusion, ProteinMPNN, AlphaFold, molecular dynamics simulations and manual structural assessment, we generated four protein designs that were subjected to experimental validation. Biophysical analysis confirmed that one designed binder had a low\u2010micromolar affinity for the LEDGF PWWP domain. Another designed binder revealed unexpected homodimerization which apparently interfered with its binding to the target in solution. Nevertheless, this binder could be co\u2010crystallized with the PWWP domain. The resulting atomic structure at 2.1\u2009\u00c5 resolution confirms correct engagement of the intended binding interface. This crystal structure enabled the construction of an expanded pharmacophore model that can instruct the design of next\u2010generation small\u2010molecule or peptide\u2010based inhibitors targeting the LEDGF PWWP domain and related epigenetic readers. These results demonstrate that modern\n                    <jats:italic>in silico<\/jats:italic>\n                    design pipelines can directly yield functional proteinaceous binders without the need for additional experimental screening using phage display or related technologies.\n                  <\/jats:p>","DOI":"10.1002\/pro.70726","type":"journal-article","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T10:37:22Z","timestamp":1784803042000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<i>De novo<\/i>\n                    design of proteinaceous binders targeting the\n                    <scp>LEDGF<\/scp>\n                    <scp>PWWP<\/scp>\n                    domain"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1452-9584","authenticated-orcid":false,"given":"Thibault","family":"Vantieghem","sequence":"first","affiliation":[{"name":"Biocrystallography KU Leuven  Leuven Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-8476-6901","authenticated-orcid":false,"given":"Julie","family":"Delepine","sequence":"additional","affiliation":[{"name":"Biocrystallography KU Leuven  Leuven Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2831-5042","authenticated-orcid":false,"given":"Sam","family":"Noppen","sequence":"additional","affiliation":[{"name":"Molecular, Structural and Translational Virology, Rega Institute for Medical Research KU Leuven  Leuven Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9687-0891","authenticated-orcid":false,"given":"Steven","family":"Beelen","sequence":"additional","affiliation":[{"name":"Biocrystallography KU Leuven  Leuven Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9111-3509","authenticated-orcid":false,"given":"Mat\u00fa\u0161","family":"Drexler","sequence":"additional","affiliation":[{"name":"Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3421-1813","authenticated-orcid":false,"given":"Jitka","family":"Holkov\u00e1","sequence":"additional","affiliation":[{"name":"Central European Institute of Technology Masaryk University  Brno Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4504-3891","authenticated-orcid":false,"given":"Josef","family":"Houser","sequence":"additional","affiliation":[{"name":"Central European Institute of Technology Masaryk University  Brno Czech Republic"},{"name":"National Centre for Biomolecular Research, Faculty of Science Masaryk University  Brno Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3782-5279","authenticated-orcid":false,"given":"V\u00e1clav","family":"Veverka","sequence":"additional","affiliation":[{"name":"Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences  Prague Czech Republic"},{"name":"Department of Cell Biology, Faculty of Science Charles University  Prague Czech 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