Biological characterization of 10-(2-propynyl) estr-4-ene-3, 17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase

MDL 18,962 was shown to be a highly specific, potent (Ki = 3-4 nM), enzyme-activated inhibitor of aromatase with minimal intrinsic endocrine properties. The affinity of MDL 18,962 was higher for human and baboon placental aromatase than for rhesus placental or rodent ovarian aromatase. These species differences necessitated the development of a novel model of peripheral aromatase utilizing human enzyme. Human choriocarcinoma trophoblast xenografts in athymic nude mice were used for pharmacologic and pharmacokinetic evaluation of MDL 18,962. The ED50 for inhibition of aromatase activity in these trophoblast tumors at 6 h post-treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, oral. Preliminary results indicated that the ED50 for inhibition of peripheral aromatization of androgen by MDL 18,962 in female baboons was 0.01 mg/kg, i.v. and 4 mg/kg, oral.     

Effects of 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962)--a mechanism-based irreversible inhibitor of aromatase--in cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37 degrees C in the absence and presence of 1.0-10 nM inhibitor. The IC50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent Ki of 2.75 nM for the inhibitor. Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a Ki of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2-20 microM) did not influence the 5 alpha-reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2 nM) to androgen receptors was not affected by MDL 18,962 (25-1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.     

Enzyme inactivation and inhibition by Gossypol

Summary Three lactate dehydrogenase isozymes and malate dehydrogenase purified from mouse tissues were inactivated with time by low concentration of gossypol. The degree of enzyme inactivation is both gossypoland enzyme-concentration-dependent. Under the same experimental conditions, lactate dehydrogenase-X and lactate dehydrogenase-5 were inactivated faster than lactate dehydrogenase-1. NADH was shown to partially protect the enzymes against inactivation by gossypol. The results of this study suggest that the enzymes are inactivated by the minor components in gossypol preparations. Isozymes of glutathione S-transferases were reversibly inhibited by gossypol. The inhibition of transferases by gossypol was shown to be competitive with respect to the 1-chloro-2,4-dinitrobenzene. It is proposed that the male antifertility effect of gossypol may be related to the selective inactivation of sperm-specific lactate dehydrogenase-X.     

Enzyme inactivation and stabilization studies in an ultrafiltration reactor

Summary An ultrafiltration membrane enzymatic reactor is used in connection with different reacting systems.The experimental conditions are such that the enzyme, which operates at fairly high concentration levels because of the concentration polarization phenomena taking place in the reactor, is still in soluble form.The analysis of the system unsteady-state response enables the identification of the mechanism of enzyme deactivation and the extraction of the kinetic parameters of both the deactivation and the main reaction.The stabilizing effect observed in connection with enzyme entrapment within an inert gel deposited onto the U.F. membrane active surface is also discussed.List of Symbols A U.F. membrane cross sectional area cm² - C_E Enzyme concentration mg/ml - C_EI Enzyme concentration at the active membrane surface mg/ml - C_E Mean enzyme concentration mg/ml - c_s^o Substrate concentration in the feed m moles/ml - c_s^u Substrate concentration in the outlet m moles/ml - D_E Enzyme diffusivity cm²/s - Km michaelis constant mM - k₂ Kinetic constant of the enzymatic reaction m moles/mg s - k_d Kinetic constant of the enzyme deactivation reaction s^–1 - N^o Initial amount of active enzyme mg - N(t) Active enzyme amount at reaction time t mg - Q Flow rate ml/s - r Rate of the main reaction m moles/ml s - t Reaction time s - t^* Reaction time at which product concentration in the outlet is within ± 2% of the steady-state value s - v Fluid velocity cm/s - V Cell volume ml - V_B Volume within which 99% of the enzyme fed is contained at the steady-state ml - V_S Volume within which 99% of the total substrate concentration drop occurs at the steady-state ml - x Distance upstream the membrane measured from the membrane surface cm     

(4S)-4-amino-5,6-heptadienoic acid (MDL 72483): A potent anticonvulsant GABA-T inhibitor

(4S)-4-Amino-5,6-heptadienoic acid ((S)--allenyl-GABA; MDL 72483) is a potent inactivator of brain GABA-T in mice; (ED₅₀ (i.p.)=60 mg·kg^–1; ED₅₀ (oral)=70 mg·kg^–1). Its anticonvulsant effects against 3-mercaptopropionic acid (MPA)-induced seizures in mice is related to the elevation of whole brain GABA concentrations: The mentioned doses of MDL 72483 which cause a decrease of GABA-T activity by 50%, produce within 5 h after dosing an increase of GABA concentration by about 3 mol·g^–1, and protect 50% of the mice against seizures in this model of presynaptic GABA deficit. When given orally MDL 72483 is about five times more potent than vigabatrin ((4R/S)-4-amino-5-hexenoic acid) a known antiepileptic GABA-T inhibitor. Complete protection was achieved with a dose of 150 mg·kg^–1. Similar to vigabatrin, MDL 72483 does not protect significantly against metrazol-induced convulsions. However, at a dose of 300 mg·kg^–1, the time elapsing between metrazol administration and onset of convulsions was prolonged by a factor of 3.4. Oral administration of MDL 72483 for up to 19 days at a daily dose of 91–96 mg·kg^–1 did not produce any obvious behavioral changes in mice, nor was the ED₅₀ of the drug in MPA-seizure tests significantly altered by the pretreatment. These observations indicate that MDL 72483 is a promising drug for the treatment of certain epilepsies.Special issue dedicated to Dr. Eugene Roberts.     

Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.     

Search for potential Angiotensin Converting Enzyme (ACE)-inhibitors from plants

MeOH extracts, fractions and pure substances from Musanga cecropioides, Cecropia species and Crataegus oxyacantha /C. monogyna were screened by using an in vitro bio-assay based on the inhibition of Angiotensin Converting Enzyme (ACE), as measured from the enzymatic cleavage of the chromophore-fluorophore-labelled substrate dansyltriglycine into dansylglycine and diglycine. Phenolic acids showed no significant ACE-inhibition whereas flavonoids and proanthocyanidins demonstrated inhibitory activity at 0.33 mg/ml using this test system.     

Computational finding of potential inhibitor for Cytochrome P450 Mono-oxygenases Enzyme of Mycobacterium tuberculosis

Cytochrome P450 mono-oxygenases (2UUQ) enzyme from Mycobacterium tuberculosis catalyzes oxidation of organic compounds such as lipids and steroidal hormones therefore remain as potential drug target. Currently available first line anti-tuberculosis drugs have been caused several side effects in the body as well as resistance development by mycobacterium against these drugs, necessitates the considerable need for finding new drugs. Therefore, we propose a structure based computational method to find a new potential inhibitor for cytochrome P450 mono-oxygenases enzyme. Compounds from several ligand databases were docked against the functional sites of 2UUQ (A) through the standard GEMDOCK v2.0 and AUTODOCK4.0 molecular docking tools. Commercially available chemical compound ZINC00004165 (5-[3-(2-nitroimidazol-1-yl) propyl] phenanthridine) has produced top rank with lowest interaction energy of -113.2 (via GEMDOCK) and lowest docking energy of -9.80 kcal/mol (via AUTODOCK) as compared to first line anti TB compounds. Z score and normal distribution analysis verified that the ZINC00004165 compound has more affinity towards 2UUQ in comparison to large number of random population of compounds. ZINC00004165 is also in agreement with the drug likeness properties of Lipinski rule of five without any violation. Therefore, our finding concludes that the commercial compound ZINC00004165 can act as a potential inhibitor against cytochrome P450 mono-oxygenases enzyme of Mycobacterium tuberculosis.     

Alpha 2-plasmin inhibitor inactivation by human granulocyte elastase

Plasminogen-binding human alpha 2-plasmin inhibitor is converted by human granulocyte elastase into its non-plasminogen-binding and finally into the inactive form of the inhibitor. This degradation of the plasmin inhibitor, described earlier as "spontaneously" occurring conversion, is shown in dodecyl sulfate polyacrylamide gel electrophoresis, in two-dimensional immunoelectrophoresis and by measuring the kinetics of plasmin inhibition. Experiments in the presence of normal human plasma required unphysiologically high concentrations of elastase to inactivate alpha 2-plasmin inhibitor, suggesting a role of elastase in this type of indirect fibrinolysis in a microenvironment only and not in systemic events.     

Kinetics of irreversible enzyme inhibition by an unstable inhibitor (Short Communication)

A mathematical treatment for the general case of enzyme inactivation by an inhibitor that breaks down in solution in a first-order reaction is presented. Cathepsin D was inactivated by fluorescein isothiocyanate with a K(i) of 4.47mum. Kinetic constants were also determined for the inactivation of cathepsin D by 1,1-bis(diazoacetyl)-2-phenylethane, and the inactivation of pepsin C by diazoacetyl-dl-norleucine methyl ester.     

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Green tea catechin induced phagocytosis can be blocked by catalase and an inhibitor of transient receptor potential melastatin 2 (TRPM2)

The major polyphenols in green tea, (−)-epigallocatechin and (−)-epigallocatechin gallate, have been shown to enhance the phagocytic activity of macrophage-like cells; however, the mechanism involved was not clarified. In this study, we have identified that the catechins induced phagocytosis can be blocked by catalase and an inhibitor of transient receptor potential melastatin 2.     

Mechanism of heparin inactivation by thromboplastin (factor III)

Thromboplastin (a commercial one and that obtained from different tissues) is shown to inactivate heparin in proportion to the quantity of thromboplastin or to the heparin:thromboplastin ratio. A degree of inactivation of heparin changes after the modification of protein component of thromboplastin by proteases, however there is no dependence between the protein amount in the preparation and its antiheparin activity. Inactivation of heparin by thromboplastin is stipulated by the formation of associations due to electrostatic interactions between the clusters of amino acid protein residues (which dissociate under physiological conditions as bases) and heparin sulphogroups. It is suggested that factor III circulating in blood flow participates in the creation of hemostatic potential not only as a result of its ability to catalyze thrombinogenesis, but also due to the decrease of the anticoagulant blood activity.     

CU(II)-dependent inactivation of Mn-catalase by hydroxylamine

Hydroxylamine is a strong inhibitor of the Mn-catalase of Lactobacillus plantarum in the presence of hydrogen peroxide [Kono, Y., and Fridovich, I. (1983) J. Biol. Chem. 258, 13646-13648]. In the presence of CuCl2 the Mn-catalase was rapidly inactivated by hydroxylamine without the addition of hydrogen peroxide. FeSO4 and MnCl2 were approximately 10% and 4% as effective as was CuCl2. Under anaerobic conditions, the inactivation did not occur. The chelating agents such as EDTA and histidine completely prevent the inactivation. These results indicate that the hydrogen peroxide produced during the autooxidation of hydroxylamine catalyzed by CuCl2 participates in the CuCl2-dependent inactivation by hydroxylamine.