<i>De novo</i> design of proteinaceous binders targeting the <scp>LEDGF</scp> <scp>PWWP</scp> domain

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin reader that recognizes di- or trimethylated Lys36 of histone H3 (H3K36me2/3)-modified nucleosomes and is implicated in diverse diseases, including cancer and human immunodeficiency virus (HIV) infection. Inhibiting the interaction between the Pro-Trp-Trp-Pro (PWWP) domain of LEDGF and chromatin through a small-molecule 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 de novo computational design and structural validation of proteinaceous binders that engage both this canonical pocket and adjacent DNA-interacting surfaces of the PWWP domain. Using a hotspot-driven 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-micromolar 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-crystallized with the PWWP domain. The resulting atomic structure at 2.1 Å 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-generation small-molecule or peptide-based inhibitors targeting the LEDGF PWWP domain and related epigenetic readers. These results demonstrate that modern in silico design pipelines can directly yield functional proteinaceous binders without the need for additional experimental screening using phage display or related technologies.