title: Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display.
abstract: Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Dennis
authors.forename: A
authors.initials: A
authors.lastname: Herzka
authors.forename: R A
authors.initials: RA
authors.lastname: Lazarus
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000494:pharmacology*; D007611:Aprotinin / Q000235:genetics / Q000494:pharmacology*; D001435:Bacteriophages / Q000235:genetics; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D015949:Factor IXa / Q000187:drug effects; D015723:Gene Library; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin; D007610:Kallikreins; D015949:Factor IXa