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.

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Citric acid analogues (+/-)-12a,b and (+/-)-17a,b, where one of the primary carboxylates has been replaced by a sulfoximinoyl and a 3-(3-hydroxy-beta-lactamyl) moiety, respectively, have been synthesized and evaluated as inhibitors of ATP-citrate lyase.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, 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.

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing.

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.

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.

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.

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.

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)

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.

Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides.

Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) [Goodhart, P. J., DeWolf, W. E., Jr., & Kruse, L. I. (1987) Biochemistry 26, 2576-2583]. This inactivation was suggested to result from alkylation of an active site residue by an aberrant 4-hydroxybenzyl radical intermediate. In support of this hypothesis, we report here the isolation and characterization of two modified tryptic peptides from DBH inactivated by p-cresol. Using a combination of automated Edman sequencing, mass spectroscopy (MS), and tandem MS, we have determined the sequence of the putative active site peptides, identified the site of attachment of p-cresol, and defined the chemical nature of the adduct formed. Both modified peptides are the same primary sequence: Ala-Pro-Asp-Val-Leu-Ile-Pro-Gly-Gln-Gln-Thr-Thr-Tyc-Trp-Cys-Tyr-Va l-Thr-Glu- Leu-Pro-Asp-Gly-Phe-Pro-Arg, where Tyc is an amino acid residue with the in-chain mass of a cresol-Tyr adduct (106 + 163 Da). Gas-phase deuterium exchange studies (employing N2H3-DCI MS) of the isolated phenylthiohydantoin (Pth) derivatives of modified residue 13 demonstrate that p-cresol forms two chemically distinct covalent adducts and support the hypothesis that a (4-hydroxyphenyl)methyl radical is generated during catalysis. Rearrangement to a (4-methylphenyl)oxy radical may also occur prior to inactivation.

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.

The copper sites of dopamine beta-hydroxylase: an X-ray absorption spectroscopic study.

X-ray absorption edge and extended X-ray absorption fine structure (EXAFS) spectra are reported for the Cu(I) and Cu(II) forms of bovine dopamine beta-hydroxylase (DBH; EC 1.14.17.1) and for the Cu(I) form of DBH bound either to tyramine substrate or to a multisubstrate inhibitor [Kruse, L. I., DeWolf, W. E., Jr., Chambers, P. A., & Goodhart, P. J. (1986) Biochemistry 25, 7271-7278]. A significant change in the structure of the copper sites occurs upon ascorbate-mediated reduction of Cu(II) DBH to the Cu(I) form. While the average Cu(II) site most likely consists of a square-planar array of four (N,O)-containing ligands at 1.98 A, the average Cu(I) site shows a reduction in (N,O) coordination number (from approximately 4 to approximately 2) and the addition of a S-containing ligand at 2.30 A. No change in the average Cu(I) ligand environment accompanies binding of tyramine substrate, whereas binding of a multisubstrate inhibitor, 1-(3,5-difluoro-4-hydroxybenzyl)-1H-imidazole-2(3H)-thione, causes an increase in the Cu-S coordination, consistent with inhibitor binding to the Cu(I) site through the S atom. Although excellent signal-to-noise ratio in the EXAFS spectra of ascorbate-reduced DBH facilitated analysis of outer-shell scattering for a Cu..Cu interaction, the presence of a binuclear site could not be proven or disproven due to interference from Cu...C scattering involving the carbons of imidazole ligands.

Effects of the novel dopamine beta-hydroxylase inhibitor SK&F 102698 on catecholamines and blood pressure in spontaneously hypertensive rats.

The novel dopamine beta-hydroxylase (D beta H) inhibitor SK&F 102698 was characterized in vitro with soluble enzyme from bovine adrenal medulla and in vivo by measuring the dopamine/norepinephrine (DA/NE) ratio in the mesenteric artery, heart and brains of spontaneously hypertensive rats (SHR). SK&F 102698 was a potent D beta H inhibitor with an IC50 of 1.2 microM on crude enzyme and had a Ki value of 40 nM on purified enzyme. SK&F 102698 produced a dose-dependent fall in NE and a dose-dependent increase in DA in the vasculature of SHR after p.o. administration. Elevation of the vascular DA/NE ratio was observed within 0.5 hr after administration. Peak effects were observed at 12 hr and values were still significantly increased at 18 hr. The rise in the DA/NE ratio of the blood vessels correlated with the fall in blood pressure following the first 4 hr after SK&F 102698. SK&F 102698 inhibited SHR heart D beta H and elevated the myocardial DA/NE ratio approximately 2.4-fold. SK&F 102698 also caused a dose-dependent increase in the whole brain DA/NE ratio of SHR. Catecholamine levels were also studied in six discrete brain regions and SK&F 102698 produced the greatest increase in the DA/NE ratio in the cerebellum, brain stem and midbrain regions, whereas the striatum was the region least affected. No overt sedation was observed in the rats. Further study with SK&F 102698 is warranted to better explore the role of DA and D beta H in pathophysiology, and to determine whether this drug or a congener D beta H inhibitor will be a useful therapeutic agent in humans.

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.

Immuno cross-reactivity suggests that catecholamine biosynthesis enzymes and beta-adrenergic receptors may be related.

Turkey red blood cell, beta 1-adrenergic receptors (BARs) were prepared to electrophoretic homogeneity by affinity chromatography, size exclusion high performance liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare rabbit polyclonal anti-BAR antibodies. Anti-BAR activity was confirmed by immunoadsorption of [125I]cyanopindolol-labeled BAR to a protein A affinity column using the anti-BAR antibodies. BAR was compared to the catecholamine biosynthetic enzyme dopamine B-hydroxylase (DBH) by anti-BAR antibody cross-reactivity. DBH was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion, and concanavalin A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 units/mg of protein. Homogeneity was confirmed by nondenaturing polyacrylamide gel electrophoresis. Both DBH and BAR were recognized by the anti-BAR antibodies on Western transfer and immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH, suggesting that the anti-BAR antibodies recognize specific portions of DBH amino acid sequence and not associated carbohydrate. DBH-cross-reactive antibodies were also purified by affinity chromatography using immobilized DBH and shown to immunoadsorb [125I]cyanopindolol-labeled BAR by protein A affinity chromatography. These results suggest that the catecholamine biosynthetic enzyme DBH and BAR may be related in structure.

Cardiovascular effects of a new potent dopamine beta-hydroxylase inhibitor in spontaneously hypertensive rats.

The cardiovascular effects of a new class of potent inhibitors of dopamine beta-hydroxylase (DBH) were evaluated in spontaneously hypertensive rats (SHR). SK&F 102698 [1-(3,5-difluorobenzyl)imidazole-2-thiol] is the prototype molecule of this class of substituted 1-benzylimidazole-2-thiols and is one of the most potent inhibitors of DBH yet described. After acute p.o. administration in conscious unrestrained SHR, SK&F 102698 elicited a dose-dependent decrease in mean arterial blood pressure. The antihypertensive effect was marked by a gradual onset with long duration of activity. The antihypertensive effect produced by SK&F 102698 was accompanied by bradycardia. SK&F 102698 inhibited DBH in vivo as demonstrated by its ability to increase vascular levels of dopamine (DA) while concomitantly decreasing vascular levels of norepinephrine (NE), thus increasing the overall DA/NE ratio. The chronic cardiovascular effects of SK&F 102698 were evaluated in developing SHR. SHR were administered SK&F 102698 p.o. once daily for 9 weeks beginning when animals were 4 weeks of age. SK&F 102698 (50 mg/kg) significantly attenuated the development of hypertension of these SHR. Tolerance to the chronic effects of DBH inhibition was not observed and blood pressures in drug-treated animals were still reduced significantly 20 hr after drug administration. Vascular catecholamine levels were determined in the mesenteric artery of these chronically treated animals. Vascular DA levels were increased 290%, vascular NE levels were decreased 36% and the DA/NE ratio was increased 520%, as compared to controls. Furthermore, hearts weights of SHR receiving SK&F 102698 were approximately 10% lower than controls. The present study demonstrates that in SHR SK&F 102698 is an effective antihypertensive whose effects are mediated by the novel mechanism of DBH inhibition.

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.