Inactivation of dopamine beta-hydroxylase by p-cresol: evidence for a second, minor site of covalent modification at tyrosine 357.

p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.

Sequence similarity between dopamine beta-hydroxylase and peptide alpha-amidating enzyme: evidence for a conserved catalytic domain.

A comparison of human dopamine beta-hydroxylase (EC 1.14.17.1) with bovine peptide C-terminal alpha-amidating enzyme (EC 1.14.17.3), revealed a 28% identity extending throughout a common catalytic domain of approximately 270 residues. The shared biochemical properties of these two enzymes from neurosecretory granules suggests that the sequence similarity reflects a genuine homology and provides a structural basis for a new family of copper type II, ascorbate-dependent monooxygenases.

Design and synthesis of 14 alpha-methyl-15-aza-D-homosterols as novel antimycotics.

A novel series of 14 alpha-methyl-15-aza-D-homosterols 3-7 has been synthesized. These compounds display significant antimycotic activity in vitro (MIC = 0.8-3.1 micrograms/mL) that compares quite favorably to the activity observed for fluconazole (MIC = 0.8 micrograms/mL). Azasterols 3 and 4 were active in vivo as reflected in the increased survival time of Candida albicans infected mice. The antimycotic activity of 3-7 is hypothesized to be a consequence of the inhibition of fungal 14,15-sterol reductase.

6,7-Dichloro-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline . A structurally novel beta-adrenergic receptor blocking agent.

Replacement of the catecholic hydroxyl groups of the beta-adrenergic receptor agonist 6,7-dihydroxy-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (trimetoquinol) with chloro substituents results in a compound with marked beta-adrenoceptor antagonist properties. This, therefore, parallels the similar transformation of the beta-adrenoreceptor agonist isoproterenol into the antagonist dichloroisoproterenol. In a test for inhibition of isoproterenol-induced enhancement of the rate of contraction of spontaneously beating guinea pig atrial pairs the resultant 6,7-dichloro-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (6b) had a KB value of (6.7 +/- 2.3) X 10(-8) M. Although this is nearly 2 orders of magnitude less potent than propranolol (KB = 6.2 X 10(-10) M in this test), this compound represents the prototype of a new class of beta-adrenergic receptor blockers, and unlike dichloroisoproterenol it is not a partial agonist. It has physicochemical properties, e.g., pKa and distribution and partition coefficients, that differ from the prototypic beta-blockers. These altered properties might impart advantageous tissue distribution and altered pharmacological properties to the new molecule. This new beta-adrenoreceptor antagonist is suggested to merit further study.

Synthesis, tubulin binding, antineoplastic evaluation, and structure-activity relationship of oncodazole analogues.

In an attempt to identify a soluble oncodazole analogue that could be easily formulated, a series of substituted oncodazoles was synthesized and evaluated for tubulin binding affinity, in vitro cytotoxicity against cultured mouse B-16 cells, and ability to prolong lifespan at the maximally tolerated dose in the P388 mouse leukemia model. Biological evaluation of all the isomeric methyloncodazoles demonstrated the thiophene 4'-position to be the only site of significant bulk tolerance, although substitution of this position with polar or charged functional groups abolished biological activity. Simple esters of the 4'-carboxymethyloncodazole were shown to have enhanced antitumor activity and tubulin binding affinity relative to oncodazole. Despite a failure of this study to identify a water-soluble oncodazole with antitumor activity, the structure-activity relationship developed led to a derivative with enhanced activity in the P388 leukemia model and facilitated the preparation of a biologically active photolabile analogue.

Inhibitors of dopamine beta-hydroxylase. 3. Some 1-(pyridylmethyl)imidazole-2-thiones.

The 1-benzylimidazole-2-thione moiety has been previously shown by Kruse et al. to be broadly associated with dopamine beta-hydroxylase (DBH) inhibitory activity both in vitro and in vivo in spontaneously hypertensive rats (SHR). An extension of structure-activity studies to 1-(pyridylmethyl)- and 1-(oxypyridylmethyl)imidazole-2-thiones is reported here in an attempt to exploit the pH differential that exists across the chromaffin vesicle membrane. We hypothesized that the weakly basic pyridyl compounds would diffuse into the acidic vesicles in their neutral forms where protonation and concentration would occur to enhance their in vivo effectiveness as inhibitors. To test this hypothesis, isomeric 2-, 3- and 4-(1-pyridylmethyl)imidazole-2-thiones were synthesized from the appropriate pyridinecarboxaldehydes by reductive alkylation of aminoacetaldehyde dialkyl acetal followed by imidazole-2-thione formation using acidic potassium thiocyanate. Related oxypyridyl compounds were synthesized by first preparing the appropriate aldehyde intermediate followed by conversion to the imidazole-2-thione by the same procedure. The unsubstituted pyridylmethyl compounds showed modest DBH inhibition in vitro but, consistent with a transport-mediated increase in observed potency, showed significant effects in vivo to increase the vascular ratio of dopamine to norepinephrine and to lower blood pressure.

Synthesis, specificity, and antifungal activity of inhibitors of the Candida albicans delta 24-sterol methyltransferase.

A series of side chain modified analogues of cholesterol and lanosterol (1-10) have been synthesized and evaluated as inhibitors of the Candida albicans delta 24-sterol methyltransferase. Two sterol substrate analogues 1 and 2 which contained a 24-thia substituent were relatively modest inhibitors of the enzyme (Ki = 1.5-72 microM). Compounds which mimic the carbocation intermediates proposed for the methyltransferase reaction, including sulfonium salts 4-6, amidines 7 and 8, and imidazoles 9 and 10 were substantially more potent inhibitors (Ki = 5-500 nM). All of the sterol analogues examined displayed less than 10-fold selectivity for inhibition of the methyltransferase versus the rat liver delta 24-sterol reductase. The sterol analogues were tested for in vitro antifungal activity against C. albicans, Candida tropicalis, and Torulopsis glabrata. The minimum inhibitory concentrations versus C. albicans correlated well with the Ki values for methyltransferase inhibition, and the potency of several compounds approached that of amphotericin B, although only modest fungicidal activity was observed.

ATP-citrate lyase as a target for hypolipidemic intervention. Sulfoximine and 3-hydroxy-beta-lactam containing analogues of citric acid as potential tight-binding inhibitors.

Citric acid analogues (+/-)-12a,b and (+/-)-17a,b, where one of the primary carboxylates has been replaced by a sulfoximinoyl and a 3-(3-hydroxy-beta-lactamyl) moiety, respectively, have been synthesized and evaluated as inhibitors of ATP-citrate lyase. The design of these inhibitors was based on methionine sulfoximine and tabtoxinine beta-lactam, potent, tight-binding inhibitors of glutamine synthetase. Both ATP-citrate lyase and glutamine synthetase employ phosphate-carboxylate anhydrides as a method for carboxylate activation during catalysis. Only one diastereomer, (+/-)-12a, displayed weak, reversible inhibition, while the remaining citrate analogues (+/-)-12b and (+/-)-17a,b were inactive against the lyase. No time-dependent inactivation of the enzyme was observed.

Synthesis of novel thiol-containing citric acid analogues. Kinetic evaluation of these and other potential active-site-directed and mechanism-based inhibitors of ATP citrate lyase.

ATP citrate lyase is an enzyme involved in mammalian lipogenesis and cholesterogenesis. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agents. Citric acid analogues 5-16 bearing electrophilic and latent electrophilic substituents were synthesized and evaluated as irreversible inhibitors of the enzyme. The design of these agents was based on the classical enzymatic mechanism where an active-site nucleophile (thiol) was believed to be critically involved in catalysis. Reversible inhibition (Ki's ranging from ca. 20 to 500 microM) was observed for compounds 5, 10, and 12-16. Compounds 6-9 and 11 had no appreciable affinity for enzyme (Ki > 1 mM). Time-dependent inactivation of the enzyme by 5-16 was not detected following long incubation times (> 1 h, 37 degrees C) at 2 mM inhibitor concentrations.

Multisubstrate inhibitors of dopamine beta-hydroxylase. 2. Structure-activity relationships at the phenethylamine binding site.

1-Aralkylimidazole-2-thiones have been shown to be potent multisubstrate inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1). In the present study, a series of 1-benzylimidazole-2-thiones was prepared to explore the effects of substitution in the benzyl ring on the inhibition of DBH. A detailed structure-activity relationship for in vitro activity was discovered and this was shown by a modified Hansch analysis to correlate (r = 0.91) with four key structural features of the benzyl ring: the presence of a hydroxyl at the 4-position, molar refractivity at the 3-, 4-, and 5-positions, inductive effects of the substituents at the 3-, 4-, and 5-positions, and pi-electron density. The affinity (Kis) of eight substituted inhibitors for DBH was shown to correlate (r = 0.75) with the affinity (KD) of comparably substituted tyramines for the ternary DBH-oxygen-tyramine complex. This correlate is used to support the hypothesis that binding of inhibitor to DBH occurs in a fashion that mimics the binding of tyramine substrates. The most potent inhibitors were selected for study in vivo in the spontaneously hypertensive rat model of hypertension. The changes in vascular dopamine and norepinephrine levels that resulted from oral administration of the inhibitors corresponded to the observed reduction in mean arterial blood pressure. A divergence between in vitro potency and in vivo efficacy upon oral dosing was noted and is suggested to result from an in vivo metabolic conjugation of the phenolic group of inhibitor.

Some benzyl-substituted imidazoles, triazoles, tetrazoles, pyridinethiones, and structural relatives as multisubstrate inhibitors of dopamine beta-hydroxylase. 4. Structure-activity relationships at the copper binding site.

Structure-activity relationships (SAR) were determined for novel multisubstrate inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by examining the effects upon in vitro inhibitory potencies resulting from structural changes at the copper-binding region of inhibitor. Attempts were made to determine replacement groups for the thione sulfur atom of the prototypical inhibitor 1-(4-hydroxybenzyl)imidazole-2-thione described previously. The synthesis and evaluation of oxygen and nitrogen analogues of the soft thione group demonstrated the sulfur atom to be necessary for optimal activity. An additional series of imidazole-2-thione relatives was prepared in an effort to probe the relationship between the pKa of the ligand group and inhibitory potency. In vitro inhibitory potency was shown not to correlate with ligand pKa over a range of approximately 10 pKa units, and a rationale for this is advanced. Additional ligand modifications were prepared in order to explore bulk tolerance at the enzyme oxygen binding site and to determine the effects of substituting a six-membered ligand group for the five-membered imidazole-2-thione ligand.

Multisubstrate inhibitors of dopamine beta-hydroxylase. 1. Some 1-phenyl and 1-phenyl-bridged derivatives of imidazole-2-thione.

The synthesis and characterization of some 1-(phenylalkyl)imidazole-2-thiones as a novel class of "multisubstrate" inhibitors of dopamine beta-hydroxylase (DBH) are described. These inhibitors incorporate structural features that resemble both tyramine and oxygen substrates, and as evidenced by steady-state kinetics, they appear to bind both the phenethylamine binding site and the active site copper atom(s) in DBH. A series of structural congeners that incorporate different bridging chain lengths between the phenyl ring (dopamine mimic) and the imidazole-2-thione group (oxygen mimic) define the optimum distance for inhibitory potency and the likely intersite distance in the DBH active site. Additional bridging analogues were prepared to determine the active site bulk tolerance and the effects of heteroatom replacement.

Differential effects of manoalide on secreted and intracellular phospholipases.

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A2's. Phospholipase A2 isolated from bee venom (Apis mellifera) was the most sensitive to inactivation by manoalide (IC50 approximately equal to 0.12 microM). Extracellular phospholipase A2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide (IC50 values of 0.7 and 1.9 microM respectively). Porcine pancreatic phospholipase A2 was relatively resistant to inactivation by manoalide (IC50 approximately equal to 30 microM). The phospholipase A2 assayed in crude cytosol fractions from four mammalian tissues exhibited IC50 values of 30 microM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine (Mr = 57,000) protected purified bee venom phospholipase A2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A2 from inactivation by manoalide. Because cellular proteins protect PLA2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.

Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols.

The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside.

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide.

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the cardiovascular effects of the dopamine beta-hydroxylase inhibitor SK&F 102698 in conscious hypertensive rats.

The dopamine beta-hydroxylase inhibitor SK&F 102698 was characterized by studying its cardiovascular effects in hypertensive rats. The antihypertensive effects of SK&F 102698 (50 mg/kg p.o.) were studied in three different rat models of hypertension. In spontaneously hypertensive and deoxycorticosterone acetate-salt rats SK&F 102698 produced blood pressure reductions of approximately 21 and 23%, respectively. In contrast, SK&F 102698 did not produce a significant decrease in blood pressure in 2-kidney, 1-clip Goldblatt hypertensive rats. The antihypertensive mechanism of action of dopamine beta-hydroxylase inhibition was probed with the selective DA1-receptor antagonist SCH 23390, which produced an attenuation of the antihypertensive effects of SK&F 102698. Experiments were designed to separate the peripheral from the central components of the cardiovascular effects of SK&F 102698. In spinal cord stimulated pithed spontaneously hypertensive rats, SK&F 102698 reduced blood pressure but not heart rate, indicating a peripherally mediated vasodilation and a centrally mediated heart rate effect. Furthermore, when SK&F 102698 was administered directly into the fourth ventricle of conscious spontaneously hypertensive rats, a pronounced bradycardia and lowering of blood pressure was observed. SCH 23390 (200 micrograms/kg i.v.) and l-sulpiride (1 mg/kg i.v.) inhibited the cardiovascular effects of SK&F 102698 administered into the fourth ventricle. These data indicate that inhibition of dopamine beta-hydroxylase with SK&F 102698 results in both peripherally and centrally mediated cardiovascular effects and suggest that central dopamine receptors contribute to the control of systemic blood pressure in hypertensive models associated with an increased sympathetic outflow.

Design and kinetic characterization of multisubstrate inhibitors of dopamine beta-hydroxylase.

The synthesis and kinetics characterization of a new class of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) inhibitor, 1-(4-hydroxybenzyl)imidazole-2-thiol, is reported. These inhibitors, which incorporate a phenethylamine substrate mimic and an oxygen mimic into a single molecule, exhibit both the kinetic properties and the potency (Kis approximately 10(-9) M) expected for a multisubstrate inhibitor and are therefore classified as such. Steady-state kinetic experiments with these multisubstrate inhibitors and their substructural analogues support the recently proposed pH-dependent changes in substrate binding order [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3106] and a mechanism whereby the inhibitor binds specifically to the reduced Cu+ form of enzyme at both the phenethylamine substrate site and the active-site copper atom(s). A Yonetani-Theorell double-inhibition experiments indicates mutually exclusive binding of the inhibitor substructures p-cresol and 1-methylimidazole-2-thiol to suggest an extremely short intersite distance between the phenethylamine binding site and the active-site copper atom(s).