title: Approaches to selective peptidic inhibitors of factor Xa.
abstract: Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site.
authors.affiliation: Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Bromfield
authors.forename: Noelene S
authors.initials: NS
authors.lastname: Quinsey
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Duggan
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
mesh_terms: D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000737:chemistry; D020134:Catalytic Domain; D015195:Drug Design; D015951:Factor Xa / Q000737:chemistry; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D013917:Thrombin; D015951:Factor Xa