Benzamidine Sepharose resin specifically interacts with S1 of trypsin-like serine proteases (Fig. ), and we previously showed it can immobilize TMPRSS2 and be specifically eluted with buffer containing benzamidine hydrochloride . Furthermore, TMPRSS2 S441A mutant protein can also be immobilized and eluted from Benzamidine Sepharose (Extended Data Fig. ), demonstrating that the resin's interaction with TMPRSS2 does not depend on the TMPRSS2 catalytic serine residue. '2222 and '2602 competitively eluted TMPRSS2 from Benzamidine Sepharose, producing a TMPRSS2 protein band at its expected molecular weight (Fig. ). In contrast, the cathepsin inhibitor E-64D was incapable of eluting TMPRSS2 from the resin. Interestingly, TMPRSS2 S441A was not competitively eluted from Benzamidine Sepharose resin using '2222 or '2602, which suggested that these ligands could not bind the TMPRSS2 S1 binding site with high affinity in the absence of S441 (Fig. ).