OBTAINING A FAMILY OF HIGH-AFFINITY, HIGH-SPECIFICITY PROTEIN INHIBITORS OF PLASMIN AND PLASMA KALLIKREIN

Human lipoprotein-associated coagulation inhibitor (LACI) is a serum p rotein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this d omain. We iteratively varied 13 residues in the region corresponding t o the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round be st binder, EPI-P211, had K-D = 2 nM. Using information from the first selection, we made a PLA-biased library containing similar to 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 approximate to 250 nM ( similar to 2800-fold higher than for PLA) and K-D values for other pro teases are higher yet. From the same initial LACI-D1 library, we selec ted 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 librar y from which we selected EPI-K502, which has a K-D for pKAL of 40 pM. EPI-K502 inhibits PLA with K-D similar to 20 nM (500-fold higher than for pKAL); K-D values for other proteases are much higher. For both ta rgets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These pr oteins are candidate drugs and imaging agents for indications involvin g excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for th ese targets.