Structure-Guided Optimization of Selective Covalent Reversible Peptidomimetic Inhibitors Targeting TMPRSS6

Developing selective protease inhibitors is a challenging task due to the high structural resemblance of their catalytic pockets. Here, we aimed to develop selective inhibitors targeting TMPRSS6, a protease involved in regulating iron homeostasis. By exploiting structural differences in the catalytic subpockets between TMPRSS6 and matriptase, we optimized ketobenzothiazole-based peptidomimetics using the P4–P3–P2-Arg-Kbt scaffold. We found that a combination of bulky residues at P4 and P3, along with polar amino acids at P2, enhance selectivity while preserving high potency. Notably, WGU55 showed exceptional selectivity toward TMPRSS6 over matriptase and minimal off-target inhibition of coagulation serine proteases such as Factor Xa and Thrombin, representing, to our knowledge, the most selective TMPRSS6 inhibitor identified to date. Cell-based assays confirmed the inhibitor’s high potency and selectivity. These findings validate a rational design strategy for the selective inhibition of TMPRSS6, paving the way for the development of targeted therapeutics based on peptidomimetics.