title: Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor.
abstract: Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.
authors.affiliation: University of Pennsylvania, Department of Dermatology, Philadelphia, PA 19104, USA.
authors.forename: Trevor
authors.initials: T
authors.lastname: Selwood
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Elrod
authors.forename: Norman M
authors.initials: NM
authors.lastname: Schechter
mesh_terms: D001550:Benzamidines / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004149:Dioxoles / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006146:Guanidines / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013450:Sulfones / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D053802:Tryptases
chemical_list: C017532:4-methylumbelliferylguanidinobenzoate; D001550:Benzamidines; C490162:CRA 2059; D003432:Cross-Linking Reagents; D004149:Dioxoles; D005456:Fluorescent Dyes; D006146:Guanidines; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D006923:Hymecromone; C006500:4-aminobenzamidine; D012697:Serine Endopeptidases; D053802:Tryptases