hymecromone and 4-aminobenzamidine

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.

Topics: Benzamidines; Binding Sites; Binding, Competitive; Cross-Linking Reagents; Dioxoles; Fluorescent Dyes; Guanidines; Humans; Hymecromone; Kinetics; Osmolar Concentration; Protein Binding; Recombinant Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Spectrometry, Fluorescence; Sulfones; Thermodynamics; Tryptases

Low molecular weight, synthetic proteinase inhibitors that inhibit sperm-associated acrosin, were released systemically in female mice at a constant rate from minipumps. The release was timed so that, after mating, the minipumps were depleted of inhibitor before blastocyst implantation took place. Three of the inhibitors: 4-aminobenzamidine (ABD), 4-nitrophenyl-4-guanidino-benzoate (NPGB) and 4-methylumbelliferone-4-guanidinobenzoate (MUGB) caused a 50% decrease in fertility, the last two at very low concentrations. The fourth inhibitor, benzamidine (BD), which is also the weakest inhibitor of mouse acrosin and in vitro fertilization, had no effect. These results show that at least one of the processes leading to fertilization or early blastogenesis, is dependent on proteolytic activity and that the systemic application of proteinase inhibitors inhibits conception. MUGB possessed low toxicity and a high margin of safety, encouraging the development of phenol derivatives of guanidinobenzoic acid as contraceptive agents.

Topics: Acrosin; Amidines; Animals; Benzamidines; Benzoates; Contraceptive Agents, Female; Drug Implants; Female; Hymecromone; Mice; Protease Inhibitors; Umbelliferones

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.

Topics: Acrosin; Animals; Benzamidines; Benzoates; Dose-Response Relationship, Drug; Fertilization in Vitro; Hymecromone; Kinetics; Mice; Protease Inhibitors; Trypsin Inhibitors