Obtaining a family of high-affinity,high-specificity protein inhibitors of plasmin and plasma kallikrein

Summary Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had K_D=2 nM. Using information from the first selection, we made a PLA-biased library containing 500 000 proteins and selected from these a protein, EPI-P302, having a K_D for PLA of 87 pM. EPI-P302 inhibits pKAL with K_D=250 nM (2800-fold higher than for PLA) and K_D values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a K_D for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a K_D for pKAL of 40 pM. EPI-K502 inhibits PLA with K_D20 nM (500-fold higher than for pKAL); K_D values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.Abbreviations APP-I-D1 Kunitz domain of amyloid precursor protein-I - BPTI bovine pancreatic trypsin inhibitor - F.VII_a Factor VII_a - F.XI_a Factor XI_a - FIR fraction of input recovered - hNE human neutrophil elastase - LACI-D1 lipoprotein-associated coagulation inhibitor, first Kunitz domain - pKAL human plasma kallikrein - PLA human plasmin - RT room temperature - THBN human thrombin - Vg variegated - w.t. wild-type - %RIA percent residual inhibitory activity - :: fusion (gene or protein)