Conformational preference for the binding of biaryl substrates and inhibitors to the active site of phenylethanolamine N-methyltransferase

We have previously described regions of steric bulk tolerance in the aromatic-ring binding site of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) for phenylethanolamine substrates and alpha-methylbenzylamine inhibitors. For bound substrates, this region is located in the vicinity of the para position of the aromatic ring, while for bound alpha-methylbenzylamine inhibitors, it is located in the region complementary to the meta position. In the present study, we sought to determine the preferred conformation of the biaryl portion of (m-phenylphenyl)- and (p-phenylphenyl)ethanolamine (4 and 5, respectively) as well as for m-phenyl- and p-phenyl-alpha-methylbenzylamine (7 and 8, respectively) for PNMT active site interactions. Planar derivatives of 4, 5, 7, and 8 were obtained through the synthesis of 2-(1-fluorenyl)-2-hydroxyethylamine (9), 2-(2-fluorenyl)-2-hydroxyethylamine (10), 1-(1-fluorenyl)ethylamine (11), and 1-(2-fluorenyl)ethylamine (12). The four fluorene derivatives were examined for in vitro activity as substrates and inhibitors of the PNMT-catalyzed reaction. As in the case of 4, 5, 7, and 8, we have observed a positional preference for the alkylamine side chain with respect to the biphenyl skeleton present in 9-12. Thus, fluorenylethanolamine 10 ("p-biphenyl") displays a Michaelis constant (Km = 26 microM) that is approximately 10 times lower than that for 9 ("m-biphenyl", Km = 297 microM); in the alpha-methylbenzylamine inhibitors, fluorenyl derivative 11 ("m-biphenyl", Ki = 4.14 microM) is approximately 40 times better than 12 ("p-biphenyl", Ki = 185 microM) for in vitro inhibition of PNMT. In each case, conformational restriction of the biaryl system present in 4, 5, 7, and 8, such that the aromatic rings are coplanar, resulted in enhanced affinity for the PNMT active site. Thus, conformational restriction of ethanolamine 5 (Km = 82 microM) as in 10 (Km = 26 microM) and alpha-methylbenzylamine 7 (Ki = 89 microM) as in 11 (Ki = 4.14 microM) leads, in each case, to a stronger enzyme-ligand dissociable complex. These results, in conjunction with others from these laboratories, indicate that the PNMT active site beyond the zone that interacts with the central aromatic ring portion of phenylethanolamine substrates and alpha-methylbenzylamine inhibitors is essentially a flat, hydrophobic pocket.     

Activation in young rats induced by LY134046, an inhibitor of phenylethanolamine N-methyltransferase

LY134046, a potent, selective inhibitor of rat brain phenylethanolamine N-methyltransferase, was shown to increase activity in 18- and 19-day-old rats. The effects on day 25 were different, with LY134046 causing a decrease in activity. The effects of yohimbine, a selective antagonist of the alpha-2 adrenergic receptor, were markedly different from LY134046, causing a decrease in activity on day 19. These data suggest that epinephrine synthesis may play an inhibitory role in the regulation of activity in young rats.     

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH- could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH- with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the alpha2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT Ki = 1.3 microM) is the most potent compound in this series and is quite selective for PNMT versus the alpha2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT Ki = 0.13 microM). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH- is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.     

The long term effects of an inhibitor of phenylethanolamine N-methyltransferase upon adrenal epinephrine biosynthesis

Summary Chronic administration of SK & F 64139, an inhibitor of phenylethanolamine N-methyltransferase (PNMT), initially resulted in a lowered adrenal epinephrine content in both the rat and squirrel monkey adrenal gland. With continued dosing, however, these levels returned toward control. The latter changes were accompanied by increased adrenal levels of norepinephrine and PNMT, but not by decreased plasma drug levels. These results suggest that long-term pharmacological PNMT inhibition may evoke compensatory mechanisms to maintain adrenal epinephrine biosynthesis under basal laboratory conditions.     

Binding requirements of phenolic phenylethylamines in the benzonorbornene skeleton at the active site of phenylethanolamine N-methyltransferase

In order to determine the active site binding orientation of norepinephrine, a series of conformationally defined analogues of the tyramines, in which the ethylamine side chain is held fixed by incorporation into a benzonorbornene skeleton, were prepared and evaluated for phenylethanolamine N-methyltransferase (PNMT) activity. While exo-2-amino-5- and exo-2-amino-8-hydroxybenzonorbornene (7 and 10, respectively) were prepared from 5-methoxybenzonorbornadiene by azidomercuration/demercuration and reduction, it was necessary to employ both normal (inversion of configuration) and abnormal (retention of configuration) Mitsunobu reactions to prepare, stereoselectively, exo-2-amino-6- and exo-2-amino-7-hydroxybenzonorbornene (8 and 9, respectively) from 6- and 7-methoxybenzonorbornen-2-ol. None of the six analogues were substrates. However, exo-2-amino-6-hydroxybenzonorbornene (8) and anti-9-amino-6-hydroxybenzonorbornene (12) displayed significant activity as inhibitors toward PNMT. The greater potency of 8 and 12, as compared to the parent unsubstituted analogues exo-2-amino- and anti-9-amino-benzonorbornene (4 and 5, respectively), indicates the presence of a spatially compact hydrophilic pocket within the aromatic ring binding region of the active site of the enzyme. Furthermore, the greater activity of 12, relative to 8, is consistent with an active site binding preference for molecules in which a more coplanar relationship exists between the aromatic ring and the amine nitrogen. From the findings of this study, it appears that norepinephrine has a different active site binding orientation than most known substrates and competitive inhibitors of PNMT.     

The effects of an inhibitor of phenylethanolamine N-methyltransferase upon stimulated adrenal catecholamine release and excretion in the rat

Summary SK&F 64139, an inhibitor of adrenal phenylethanolamine N-methyltransferase (PNMT), was found to significantly decrease 2-deoxy-d-glucose (2-DG) induced epinephrine excretion in the conscious rat under conditions where the former agent was administered chronically at 50 and 200 mg/kg/day over a 12-day period and 2-DG was administered after 3, 7 and 11 days of treatment. The reduced epinephrine output caused by SK&F 64139 in response to 2-DG was accompanied by an increased norepinephrine excretion pattern at 200 mg/kg/day of the compound. The reductions in epinephrine excretion were also associated with significant decreases in adrenal epinephrine and increases in the norepinephrine content.     

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase

The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed with its product, S-adenosyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Val53, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased Km values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its kcat value. Both WT hPNMT and D267A had similar kcat values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The Ki values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenosyl-L-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.     

Inhibition of phenylethanolamine N-methyltransferase (PNMT) by aromatic hydroxy-substituted 1,2,3,4,-tetrahydroisoquinolines: further studies on the hydrophilic pocket of the aromatic ring binding region of the active site

In a continuation of studies directed toward characterizing the hydrophilic pocket within the aromatic ring binding region of the active site of phenylethanolamine N-methyltransferase (PNMT), 5-, 6-, 7-, and 8-hydroxy-1,2,3,4-tetrahydroisoquinoline were prepared and evaluated as substrates and inhibitors of PNMT. In order to discern the necessity of an acidic hydrogen for interaction at this pocket the corresponding methyl ethers were also evaluated. The enhanced affinity of 7-hydroxy-1,2,3,4-tetrahydroisoquinoline (16) versus tetrahydroisoquinoline (13) itself indicates that a hydrophilic pocket exists off of carbon C7 in bound tetrahydroisoquinolines. The diminished affinity of the corresponding methyl ether is consistent with a requirement for the acidic hydrogen of 16 for interaction of the aromatic hydroxyl at this site. From the relative activities of the other regioisomeric aromatic hydroxyl-substituted tetrahydroisoquinolines, their corresponding methyl ethers, and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, it appears that the hydrophilic pocket is spatially compact with respect to bound tetrahydroisoquinolines and is surrounded by larger areas of lipophilic character. To allow a comparison of the results of this study with previous data on bound beta-phenylethylamines, the methyl ethers of 5-, 6-, 7-, and 8-hydroxy-exo-2-aminobenzonorbornene and of 5- and 6-hydroxy-anti-9-aminobenzonorbornene were also evaluated for their activity as substrates and inhibitors for PNMT. The results of this study are in agreement with previous findings for bound beta-phenylethylamines and support the conclusion that the natural substrate for PNMT, norepinephrine, has a different active site binding orientation than most known substrates and competitive inhibitors of the enzyme.     

Pharmacological analysis of the neurotransmitter mechanisms regulating phenylethanolamine N-methyltransferase in the adrenal gland

The i.p. administration of reserpine daily for 4 days to rats brought about an increase of adrenal phenylethanolamine N-methyltransferase (PNMT) activity. However, the combination of the systemic administration of p-chlorophenylalanine (PCPA) and reserpine for 3 days produced an earlier increase in this adrenal enzyme. The effect was reduced significantly in the denervated gland. Prior administration of 5,7-dihydroxytryptamine (DHT) i.c.v. to rats greatly potentiated the inducing effect of reserpine. On the other hand, the depletion of catecholamines by giving rats alpha-methyl-p-tyrosine (AMPT) i.p. or 6-hydroxydopamine (6-OHDA) i.c.v. did not alter the action of reserpine on adrenal PNMT. PCPA, DHT, AMPT and 6-OHDA did not have any effect by themselves on adrenal PNMT, but the combination of PCPA and AMPT, each given i.p., caused increased adrenal PNMT activity. The administration of dopamine agonists, a treatment that increases adrenal TH, did not modify adrenal PNMT. We conclude that the induction of PNMT by reserpine involves depletion of catecholamines and serotonin, the depletion of serotonin having the more powerful effect. A monoaminergic (serotonergic) inhibitory pathway is involved in the central regulation of adrenal PNMT activity.     

Multi-target-directed drug design strategy: from a dual binding site acetylcholinesterase inhibitor to a trifunctional compound against Alzheimer's disease

A design strategy to convert a dual-binding site AChE inhibitor into triple functional compounds with promising in vitro profile against multifactorial syndromes, such as Alzheimer's disease, is proposed. The lead compound bis(7)-tacrine (2) was properly modified to confer to the new molecules the ability of chelating metals, involved in the neurodegenerative process. The multifunctional compounds show activity against human AChE, are able to inhibit the AChE-induced amyloid-beta aggregation, and chelate metals, such as iron and copper.     

9-Amino-1,2,3,4-tetrahydroacridine is a potent inhibitor of histamine N-methyltransferase

The effect of 9-amino-1,2,3,4-tetrahydroacridine (THA) on histamine N-methyltransferase (HMT), an enzyme catalyzing the methylation of histamine to form tele-methylhistamine in the brain, was studied in vitro using a partially purified enzyme preparation from bovine brain and in vivo in the mouse brain. THA inhibited the HMT activity in competitive and non-competitive mixed type manners with respect to histamine. The Ki and Ki' values were 75 nM and 1.2 microM, respectively. The IC50 values for THA, 9-aminoacridine and physostigmine in the inhibition of HMT determined at fixed concentrations of histamine (20 microM) and S-adenosylmethionine (50 microM) were 0.2, 0.37 and 20 microM, respectively. Neostigmine exhibited only 15% inhibition even at a concentration of 100 microM. THA (2-10 mg/kg, s.c.) dose-dependently inhibited HMT in the mouse brain. The inhibition of HMT by THA (10 mg/kg) was marked at 30 and 60 min after treatment, but disappeared by 120 min after. THA (10 mg/kg) significantly increased the histamine level and decreased the tele-methylhistamine level in the mouse brain. These results indicate that THA is a potent inhibitor of HMT.     

Possible mechanism of action of SKF 64139 in vivo on rat adrenal and brain phenylethanolamine N-methyltransferase activity

SKF 64139, a specific inhibitor of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), has been widely used as a pharmacological tool for studying the characteristics of epinephrine-containing neurons. However, the mechanism of action of this drug on PNMT in vivo has not been fully elucidated. In the present study, we traced changes of PNMT activity in rat adrenal glands and medulla oblongata between 1 and 48 hr after intraperitoneal injection of SKF 64139 (50 mg/kg body wt). Within 1 hr, enzyme activity in both tissues decreased to 10% of the respective control value. However, starting at 4 hr, activity gradually recovered from the inhibition and completely returned to the respective control level by 48 hr. Removal of the inhibitor by dialysis substantially restored the adrenal enzyme activity in 1, 2 and 4 hr groups and completely returned it to control levels in 18 and 48 hr groups. A similar pattern also seemed to hold with brain extracts. The profiles of immunotitration curves, using dialyzed tissue extracts and specific antibodies to bovine adrenal PNMT, clearly indicate that, even after dialysis, a substantial amount of inactive enzyme was present in tissue extracts from 1, 2 and 4 hr groups. In contrast, by 18 hr a very small amount of inactive enzyme was present. Throughout the experimental periods there was no noticeable differences among the control and the experimental groups in the number or intensity of immunocytochemical stained neurons with PNMT antibodies of the C1 area of ventrolateral medulla. Judging from the data obtained by dialysis, immunochemical titration and immunocytochemical staining, recovery of PNMT activity following its inhibition by SKF 64139 was not due to irreversible inhibition of the enzyme followed by new enzyme synthesis. Instead, reversible binding of inhibitor to PNMT and its release were responsible for recovery. PNMT from the 1, 2 and 4 hr groups resisted further in vitro inhibition by SKF 64139 because the residual inhibitor was probably still bound to the enzyme.