Tight binding inhibitors—IX: Kinetic parameters of dihydrofolate reductase inhibited by methotrexate,an example of equilibrium study

Equilibrium studies in the presence of methotrexate (MTX), based on the new theories of tight-binding inhibitors and on classical initial velocity analysis, indicated that the reaction mechanism of dihydrofolate reductase Lactobacillus casei MTX/R is consistent with a rapid equilibrium random bi-bi and that MTX inhibits the enzyme competitively with respect to dihydrofolate but noncompetitively with respect to NADPH. The kinetic parameters determined at pH 7.3 and 23° were: K_m for DHF, 9.8 ± 1.3 μM; K_m for NADPH, 6.0 ± 1.2 μM; K_d for E·DHF, 5.7 ± 0.7 μM; K_d for E·NADPH, 0.037 ± 0.028 μM; K_d for E·MTX, 1.20 ± 0.15 nM; K_d for E·NADPH·MTX → E·NADPH + MTX, 0.19 ± 0.04 nM; and K_d for E·NADPH·MTX → E·MTX + NADPH, 7.6 ± 5.9 nM; the molar equivalency factor was 3.33 ± 0.44 nM per unit/liter of the enzyme, and the catalytic number was 300 min^?.     

Detection by high-performance liquid chromatography of methotrexate and its metabolites in tumor tissue from osteosarcoma patients treated with high-dose methotrexate/leucovorin rescue

Methotrexate (MTX) polyglutamates were detected in osteogenic sarcoma tumor samples obtained from patients 24 or 48 h after receiving high-dose MTX/leucovorin rescue therapy. Tumor samples were assayed by high-performance liquid chromatography, and polyglutamyl metabolites, along with MTX, were quantitated using both direct u.v. absorption at 313 nm and an enzyme titration assay. Good agreement between these two methods was found although the more sensitive enzyme assay detected peaks in some samples not detected by u.v. absorbance. A wide variation in MTX:MTX polyglutamate levels (1:1 to 25:1) was found among the six clinical samples studied. Also, no correlation between the extent of polyglutamate formation and plasma levels (determined at the time of tumor sampling) was observed. High intracellular levels of a derivative which appears to be the 7-hydroxy metabolite of MTX were also detected in four of six samples. This material coeluted with authentic standard, showed spectral properties like standard 7-OH-MTX, and did not inhibit dihydrofolate reductase.     

Differentiation of the cardiac and pulmonary toxicity of monocrotaline, a pyrrolizidine alkaloid

Monocrotaline, given to rats as a 20 mg/l solution in drinking water for 3 weeks, doubled the mass of the right heart and lung. The rise in lung mass preceded that of the heart. These increases were accompanied by increases in the absolute protein content of the two organs, together with increases in the rates of both protein and RNA syntheses. The increase in lung mass was not accompanied by a change in total collagen content, as measured by two independent methods: 4-hydroxyproline content and detergent fractionation. In contrast, the right ventricle showed more than a 4-fold increase in total collagen content. Total pulmonary lipids increased by 86%, but the lipid: protein ratio was unchanged. Right ventricular lipids were unchanged in amount but the lipid: protein ratio fell by 29%. Lung DNA:RNA ratio decreased 49% and right ventricle DNA:RNA ratio decreased 69%, indicating that both of these organs were responding to monocrotaline with hypertrophy. These results suggest that the processes of hypertrophy differ in the two organs: in the lung, there was no fibrosis despite a marked increase in dry weight, while right ventricular hypertrophy was characterized by increased collagen deposition. There was no alteration in the left ventricle in any of the parameters investigated.     

Inhibition of rabbit liver monoamine oxidase by nitro aromatic compounds

Nitrobenzoid, nitroheterocyclic and cyanobenzoid compounds inhibit type B monoamine oxidase. A partially purified enzyme preparation from rabbit liver mitochondria, oxidizing rho-dimethyl-aminobenzylamine as the substrate, was competitively inhibited by nitrobenzoid compounds with K1 values in the range of 0.28 muM for rho-dinitrobenzene to 0.56 muM for rho-nitrobenzoic acid. The potencies of nitrobenzoid compounds were positively correlated with the Hammett sigma value for each substituent on nitrobenzene. Dinitro derivatives were slightly more potent than the corresponding mononitro compounds but not as potent as would be expected from their sigma values. For the nitroheterocyclic compounds, inhibition was also competitive; the lowest K1 was 1.3 muM for 5-nitrofurfural semicarbazone (nitrofurazone). Compounds with cyano groups in place of nitro groups were also inhibitory; the most potent was rho-acetobenzonitrile with a K1 of 1.3 muM. The results of this study indicate that, in addition to nitrobenzoid compounds, other compounds with planar, electron-deficient nuclei are effective inhibitors of type B monoamine oxidase. Although hydrophobic and steric parameters may play some role in inhibition, the predominant factor is the electron-withdrawing power of the ring substituents.     

Differential modification of opiate receptor activity by arylsulfatase treatment

Treatment with the enzyme arylsulfatase in vivo selectively attenuated the effect of analgesia induced by morphine, beta-endorphin or ethylketocyclazocine but not that induced by Sandoz FK33824 or D-ala2-D-leu5-enkephalin. The effect on morphine analgesia was indicated both by an increased morphine ED50 in the presence of a fixed dose of naloxone and by a decreased naloxone ED50 in the presence of a fixed dose of morphine. Arylsulfatase treatment in vivo also selectively affected in vitro ligand binding; Bmax values of the low affinity binding site of dihydromorphine, naloxone, D-ala2-D-leu5-enkephalin, D-ala2-met5-enkephalinamide and ethylketocyclazocine were decreased significantly while the Bmax values of the high affinity sites as well as the KD values of both the high and low affinity sites were affected little or not at all. The data suggest that the change induced by the enzyme may have been due to the alteration of certain constituents of the low affinity opiate binding site.     

Inhibition of human elastase from polymorphonuclear leucocytes by gold sodium thiomalate and pentosan polysulfate (SP-54)

Human lysosoma) elastase, the serine proteinase from the azurophil granules of polymorphonuclear leucocytes, is inhibited by gold thiomalate and pentosan polysulfate (SP-54®). The kinetic mechanism of the inhibition was studied using succinyl-alanyl-alanyl-prolyl-valyl-4-methyl-7-coumarylamide and (t-butyloxycarbonyl-alanyl-p-nitrophenylester as subsrates. The degree of inhibition was also tested using insoluble elastin as substrate. Independent of the substrate, the maximal inhibition of elastase by gold thiomalate and pentosan polysulfate was 40% and 60%, respectively. Pentosan polysulfate behaved as a simple intersecting, hyperbolic, non-competitive inhibitor and k′_i. the dissociation constant of the E-S-I complex, was 1.8 × 10^?7M. The interaction between inhibitor and enzyme is driven by electrostatic forces. Gold thiomalate showed a hyperbolic mixed type inhibition (intersecting, slope-hyperbolic, intercept-hyperbolic, non-competitive inhibition) with k′_i = 5.4 × 10^?5M.The overall kinetic mechanism of lysosomal elastase conforms to that of other serine proteinases. With both the ester and peptide substrates the rate limiting step of the reaction has been identified with the formation of the acyl-enzyme.     

Inhibition of dog kidney Na+, K+-ATPase activity by procaine, tetracaine and dibucaine

The potency of local anesthetics as inhibitors of Na+, K+-ATPase and K+- NPPase activities correlated with lipid solubility. The order of potencies was: dibucaine greater than tetracaine much greater than procaine. Na+-ATPase activity was remarkably more sensitive to inhibition by tetracaine and procaine, and inhibitory potency did not correlate with lipid solubility. The order of potencies for inhibition of Na+-ATPase activity was: tetracaine greater than dibucaine greater than procaine. We examined interactions between the local anesthetics and monovalent cations in an attempt to explain this observation. Inhibition of Na+-K+-ATPase by tetracaine and dibucaine was competitive with respect to Na+, and inhibition of Na+-ATPase activity by all three agents was competitive with respect to Na+. Inhibition of Na+, K+-ATPase activity by procaine and tetracaine was competitive with respect to K+, and inhibition of K+- NPPase activity by all three agents was competitive with respect to K+. Dibucaine, the most lipid soluble agent, was equipotent as an inhibitor of all three activities and was generally less effective as a competitor with respect to activation by monovalent cations. These results suggest that dibucaine may interact nonspecifically with membrane lipids to inhibit enzyme activity whereas less lipid soluble agents, such as tetracaine and procaine, may interact more selectively with cation binding sites. It appears that the presence of K+ in the assay medium specifically decreases the inhibitory potency of tetracaine and procaine. Direct competition between these agents and K+ may prevent inhibition or, alternately, the presence of K+ may convert the enzyme to a conformation less susceptible to inhibition by agents of low to intermediate lipid solubility.     

Inhibition of prolyl hydroxylase activity and collagen biosynthesis by the anthraquinone glycoside, P-1894B, an inhibitor produced by Streptomyces albogriseolus

P-1894B, a potent prolyl hydroxylase inhibitor produced by Streptomyces albogriseolus subsp. No. 1894, inhibited about 50% of the activity of purified chick embryo prolyl hydroxylase at a concentration of 2.2 x 10(-6) M. The inhibition was noncompetitive with respect to (Pro-Pro-Gly)5 with a Ki of 1.8 x 10(-6)M. When excess amounts of ferrous ions or ascorbate were added to the reaction mixture, the inhibition was slightly reversed. P-1894B at a dose of 0.15 mg/kg reduced the hydroxylation of peptidyl proline and caused a significant inhibition of collagen biosynthesis in the uterus of the immature rat stimulated by the administration of estradiol-17 beta.     

Mechanism for potentiation of warfarin by phenylbutazone. Inhibition of vitamin K-dependent carboxylation and prothrombin synthesis by phenylbutazone in preparations from rat liver

Phenylbutazone potentiated the anticoagulant effects of racemic warfarin and of the individual enantiomers to similar extents in the rat. This indicates that the phenylbutazone did not act stereospecifically on the enantiomers, as it does in humans. Phenylbutazone doubled the turnover rate of warfarin in plasma, but it did not increase the amount of the anticoagulant in liver or the amount excreted in urine. The drug had no effect on plasma disappearance of [3H] or on hepatic levels of [3H] vitamin K1 or of its chief metabolite, [3H] vitamin K1 epoxide, after injection of [3H] vitamin K1. Phenylbutazone, however, at concentrations of 0.5 to 2.8 mM inhibited vitamin K-dependent carboxylation of a synthetic pentapeptide substrate in liver microsomes by 40-88 per cent. Vitamin K-dependent protein carboxylation was also inhibited by about 40 per cent in microsomes and post-mitochondrial supernatant fluid at drug concentrations of 2.8 to 4.8 mM. Most importantly, prothrombin synthesis was inhibited in post-mitochondrial supernatant fractions by 19 and 39 per cent at drug concentrations of 2.8 and 4.8 mM respectively. The inhibition of both carboxylation and prothrombin synthesis appears to have been of sufficient magnitude to account for the potentiation by phenylbutazone observed in vivo. The calculated hepatic level of phenylbutazone during potentiation was around 3 mM, a concentration that produced inhibition in vitro.     

Methotrexate analogues--XVII. Antitumor activity of 4-amino-4-deoxy-N10-methylpteroyl-D,L-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and D,L-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase (IC50 = 1 nM), inhibited L1210 cell proliferation in culture (IC50 = 0.3 microM), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA (K1 = 190 +/- 70 microM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.     

Some properties of monoamine oxidase and a semicarbazide sensitive amine oxidase capable of the deamination of 5-hydroxytryptamine from porcine dental pulp

The deamination of 5-hydroxytryptamine, tryptamine and benzylamine by porcine dental pulp membrane preparations is brought about not only by monoamine oxidase, but also by a clorgyline (and deprenyl) resistant, semicarbazide sensitive enzyme. The semicarbazide sensitive enzyme was also inhibited by aminoguanidine, hydroxylamine and phenylhydrazine, but was not affected to any significant extent by incubation at 50° for up to 100 min. There was, on the other hand, considerable inhibition of monoamine oxidase activity after incubation at this temperature. The semicarbazide sensitive enzyme neither metabolised, nor was inhibited by putrescine or cadaverine. Mixed substrate experiments indicated that 5-hydroxytryptamine and tryptamine interacted at the same catalytic centre on the semicarbazide sensitive enzyme.     

Inhibition of iron-stimulated catecholamine degradation by the iron-chelators DETAFAC and Desferal: Potentially useful laboratory agents

The addition of ferrous sulfate to phosphate buffer at pH 7.4 brought about a large increase in the rate of autoxidation of dopamine or norepinephrine. The iron-chelating agents diethylenetri-aminepentaacetic acid (DETAPAC) and desferroxamine (Desferal) inhibited both the baseline as well as the iron-stimulated autoxidation of these catecholamines. Lesser inhibitory effects were observed with another iron-chelating agent, namely EDTA. In other experiments, DETAPAC or Desferal had little effect on the autoxidation of 6-hydroxydopamine in the absence of added ferrous sulfate, but both inhibited the ferrous sulfate-stimulated autoxidation. In contrast, both the baseline and ferrous sulfate-catalyzed rate of oxidation of 6-hydroxydopamine were greatly stimulated by EDTA addition. DETAPAC and Desferal appear to be useful experimental tools that may be added to solutions containing catecholamines to prevent their degradation.     

Effects of vinca alkaloids on calcium-calmodulin regulated cyclic adenosine 3' ,5'-monophosphatase phosphodiesterase activity from brain

The effects of a series of vinca alkaloids on calcium-calmodulin regulated brain cyclic adenosine 3',5'-monophosphate phosphodiesterase (PDE) activity were examined. The alkaloids tested included the dimeric indole alkaloids, vinblastine, vincristine and desacetylvinblastine amide, and the monomeric alkaloids, catharanthine and vindoline. The order of magnitude of the inhibitory effects on the calcium-calmodulin stimulated activity of phosphodiesterase was vinblastine > desacetylvinblastine amide s > vincristine = catharanthine > vindoline. In contrast, both catharanthine and vindoline were more potent inhibitors than the dimeric vinca alkaloids of the basal unstimulated phosphodiesterase activity. Vinblastine and vincristine inhibition of calcium-calmodulin activated partially purified PDE was non-competitive with substrate. In contrast, the inhibitory actions of vinblastine, desacetylvinblastine amide, vincristine and catharanthine, but not of vindoline, were competitive with calcium-calmodulin. Colchicine inhibited both activated and basal phosphodiesterase activity. The inhibitory effect of colchicine was not reversed by the addition of either calmodulin or calcium. These results suggest that the calcium-calmodulin dependent inhibition of PDE activity by the dimeric vinca alkaloids had the greater specificity, and that this inhibitory action required the catharanthine moiety. In view of these results, dimeric vinca alkaloids may provide a useful tool for the elucidation of the physiological role of calcium-calmodulin.