title: Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein.
abstract: 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 KD = 2 nM. Using information from the first selection, we made a PLA-biased library containing approximately 500,000 proteins and selected from these a protein, EPI-P302, having a KD for PLA of 87 pM. EPI-P302 inhibits pKAL with KD approximately 250 nM (approximately 2800-fold higher than for PLA) and KD values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a KD 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 KD for pKAL of 40 pM. EPI-K502 inhibits PLA with KD approximately 20 nM (500-fold higher than for pKAL); KD 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.
authors.affiliation: Protein Engineering Corporation, Cambridge, MA 02138, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Ley
authors.forename: W
authors.initials: W
authors.lastname: Markland
authors.forename: R C
authors.initials: RC
authors.lastname: Ladner
mesh_terms: D000595:Amino Acid Sequence; D005341:Fibrinolysin / Q000037:antagonists & inhibitors*; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors*; D008074:Lipoproteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000302:isolation & purification
chemical_list: D008074:Lipoproteins; D019151:Peptide Library; D015842:Serine Proteinase Inhibitors; C051928:lipoprotein-associated coagulation inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D007610:Kallikreins; D005341:Fibrinolysin