Design and synthesis of selective IRAK4/1 inhibitors guided by water-mediated interaction and topological water networks

The interleukin-1 receptor-associated kinases (IRAKs) mediate immune and inflammatory signaling through Toll-like and interleukin-1 receptors. Among the four IRAK isoforms, IRAK4 and IRAK1 have been reported to play distinct pathological roles in autoimmune inflammatory disorders and epithelial ovarian cancer, respectively, underscoring the need for isoform-selective inhibitors. However, the high sequence similarity within their binding sites poses significant challenges for selective inhibitor design. In this study, we performed a computational analysis on a series of inhibitors with differential activities against IRAK4 and IRAK1 to identify previously unrecognized determinants of isoform selectivity. Structural analysis revealed a distinct conformation of R273 near the solvent-exposed region in IRAK4 that supports a conserved water-mediated interaction involving D272, an interaction that is absent in IRAK1. Molecular dynamics (MD) simulations confirmed the stability of this water bridge, and fragment molecular orbital (FMO) calculations quantified its energetic contribution to ligand binding. In contrast, IRAK1 lacks this interaction but features a solvent-exposed hydrophobic pocket characterized by a unique Topological Water Network (TWN) distribution. Guided by these insights, we designed and synthesized two novel compounds with improved selectivity for IRAK4 and IRAK1, respectively, and validated their activities through in vitro assays.