Development of a proteomic framework for sheddome-wide assessment of iRhom2 inhibitors

A disintegrin and metalloproteinase 17 (ADAM17) releases tumor necrosis factor α (TNF), a central mediator of inflammation. Despite its therapeutic relevance, targeting ADAM17 has proven challenging due to its broad substrate repertoire (the ADAM17 sheddome) and widespread expression. ADAM17 activity depends on the cofactors iRhom1 and iRhom2, which promote its maturation and regulate shedding at the plasma membrane. Although iRhom1 and iRhom2 are largely redundant in supporting ADAM17 function, iRhom2—but not iRhom1—is expressed in macrophages, the major source of TNF, making it an attractive target for immune-selective ADAM17 inhibition. Importantly, iRhom2 also exhibits unique properties, including the ability to support stimulated ADAM17-mediated shedding that iRhom1 cannot compensate, raising the possibility that the two cofactors differentially shape the ADAM17 sheddome and that iRhom2 inhibition could entail unintended effects beyond TNF suppression. Here, using high-resolution mass spectrometry, we systematically compared the ADAM17 sheddome associated with iRhom1 or iRhom2 and applied this workflow to evaluate iRhom2 inhibitors. We identified novel ADAM17 substrates and found that iRhom1 and iRhom2 shape highly similar sheddomes overall. By screening a panel of compounds, we further established a proteomic platform to study iRhom function and to assess, in a sheddome-wide manner, the effectiveness and specificity of iRhom2 inhibitors.