Conformational and steric aspects of phenylethanolamine and phenylethylamine analogues as substrates or inhibitors of phenylethanolamine N-methyltransferase

The conformational and steric aspects of binding to phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) for phenylethanolamine substrates and phenylethylamine inhibitors were probed with three conformationally defined analogues (11, 12, and 13) of phenylethylamine (1) and phenylethanolamine (6) containing the benzobicyclo[3.2.1]octane skeleton. The 2-aminotetralin (2AT) moiety in conformationally defined analogues 11, 12, and 13 exists in a half-chair conformation with an equatorial amino group. Although conformationally restricted phenylethylamine analogue 2AT (3, Ki = 6.8 microM) and conformationally restricted phenylethanolamine analogues (cis)- and (trans)-2-amino-1-tetralol (9, Km = 22 microM; Vmax = 0.15; 100 X Vmax/Km = 0.68; 10, Ki = 9.4 microM) are good ligands for PNMT, none of the analogues 11, 12, and 13 showed activity as a substrate of PNMT. The fact that 11 (Ki = 206 microM) is more potent than analogues 4 (Ki = 1296 microM) and 5 (Ki = 479 microM), with a half-boat 2AT moiety, suggests that PNMT preferentially binds the half-chair conformation of 2AT at the active site. This is consistent with previous findings that a fully extended conformation for the aminoethyl side chain of phenylethylamine inhibitors is optimal for PNMT binding. The reduced activity of 11, 12 (Ki = 1246 microM), and 13 (Ki = 3000 microM), compared with 2AT and (cis)- and (trans)-2-amino-1-tetralol (9 and 10) is consistent with a negative steric interference from the extra ethano bridge in 11, 12, and 13. The results from 11, 12, and 13, combined with previous findings, suggest that PNMT interacts better with relatively planar ligands.     

Conformational requirements of substrates for activity with phenylethanolamine N-methyltransferase

beta-Phenylethanolamines have long been known to be substrates for the enzyme that converts norepinephrine to epinephrine (phenylethanolamine N-methyltransferase, PNMT, EC 2.1.1.28). In an effort to determine which, if any, particular conformation of the aminoethyl side chain of phenylethanolamines is required for PNMT active site binding and catalysis, we have prepared and evaluated conformationally restricted phenylethanolamine analogues 8-10. The folded phenylethanolamine derivative 4-hydroxy-1,2,3,4-tetrahydroisoquinoline (8) is not a substrate and does not interact with the enzyme active site as an inhibitor as well as 1,2,3,4-tetrahydroisoquinoline (6). In the cyclic 2-aminotetralol systems, only cis-phenylethanolamine derivative 9 demonstrates activity as a PNMT substrate. The corresponding trans isomer 10 is not a substrate, in spite of enhanced active site interactions with respect to the parent analogue (2-aminotetralin, 4). Comparison of the inhibition constants for the folded (8,Ki = 175 microM) and extended (10,Ki = 9 microM) phenylethanolamine analogues strongly suggests that simultaneous binding of both the amino and hydroxyl functionalities to the PNMT active site requires an extended aminoethyl side chain conformation.     

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.     

Enzyme adaptation to inhibitor binding: a cryptic binding site in phenylethanolamine N-methyltransferase

Shape complementarity is a fundamental principle of inhibitor design. Here we show that an enzyme for which the crystal structure has been determined (phenylethanolamine N-methyltransferase, PNMT) conceals a cryptic binding site. This site is revealed upon binding of inhibitors that are double the size of the physiological substrate. These large inhibitors are not predicted to bind in that they protrude through the accessible surface calculated from a PNMT/7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (SK&F 29661) crystal structure, yet they are potent inhibitors of PNMT. We determined structures of the enzyme complexed with large inhibitors and found that the volume of the active site increases by 140 A3 upon binding. Changes in active site size and shape are brought about by unfavorable side chain conformations and rigid body helix motions. The energetic cost is modest, estimated at 2-3 kcal/mol from mutational analyses. Our findings further underline the importance of protein flexibility in structure-based inhibitor design studies.     

Stereochemical aspects of phenylethanolamine analogues as substrates of phenylethanolamine N-methyltransferase

Phenylethylamines and phenylethanolamines represent two major classes of ligands for the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT;EC 2.1.1.28). Phenylethylamines are usually competitive inhibitors and the isomers with the relative configuration as in (2S)-amphetamine (1) and (2S)-2-aminotetralin (3) are better inhibitors than their enantiomers. Phenylethanolamines are usually substrates of PNMT and the enzyme prefers the 1R isomers, such as (1R)-phenylethanolamine (5), in this class. Optically active norephedrines (7 and 8), norpseudoephedrines (9 and 10), and 2-amino-1-tetralols (13-16) were used to study the stereochemical requirements of phenylethanolamines for PNMT active site binding. Although the norephedrines (7 and 8) and the norpseudoephedrines (9 and 10) were poorer ligands for PNMT than were the 2-amino-1-tetralols (13-16), (1R,2S)-(-)-norephedrine (7) showed some activity as a PNMT substrate (Km = 1310 microM, Vmax = 0.22, 100 x Vmax/Km = 0.017). In the 2-amino-1-tetralols (13-16), the isomers with the 2S configuration (13 and 15) showed higher affinity to PNMT (13, Km = 4.5 microM; 15, Ki = 4.6 microM) and those with the 1R configuration (13 and 16) were substrates for the PNMT-catalyzed methyl transfer (13, Km = 4.5 microM, Vmax = 0.16, 100 x Vmax/Km = 3.6; 16, Km = 195 microM, Vmax = 0.12, 100 x Vmax/Km = 0.062); the combination of 1R and 2S configurations, such as in (1R,2S)-2-amino-1-tetralol (13), was required for a good substrate. These stereochemical requirements derived from the norephedrines (7 and 8), the norpseudoephedrines (9 and 10), and the 2-amino-1-tetralols (13-16) complement those for phenylethylamines (1-4) and for phenylethanolamines (5 and 6) and strongly suggest that phenylethylamine inhibitors bind to PNMT in the same orientation as do phenylethanolamine substrates.     

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.     

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.     

Molecular recognition of sub-micromolar inhibitors by the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.     

Conformational analysis and active site modelling of angiotensin-converting enzyme inhibitors

The discovery of captopril as a potent, orally active inhibitor of angiotensin-converting enzyme (ACE) led to the recent development of many series of novel structures with similar biological activity. To date, however, all of these inhibitors are flexible or semiflexible molecules, and there is therefore no clear definition of the conformational requirements for ACE inhibition. In an effort to solve this problem, we have carried out conformational energy calculations on a series of eight structurally diverse ACE inhibitors. Comparison of the low-energy conformations available to these molecules leads to the conclusion that there is a common low-energy conformation throughout the series. The calculations thus define the structural and conformational requirements for ACE inhibition. Expansion of this model to the receptor level has been achieved by considering possible alternative receptor sites for each of the molecules in its proposed biologically active conformation and leads to an active-site model for ACE which may be useful for the design of further inhibitors.     

Conformationally restricted and conformationally defined tyramine analogues as inhibitors of phenylethanolamine N-methyltransferase

In a search for a selective inhibitor for the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28), phenolic 2-aminotetralins (12-15 as conformationally restricted analogues of tyramine) and phenolic benzobicyclo[3.2.1]octylamines (22-24 as conformationally defined analogues of tyramine) were used to gain information about the binding interactions of the catecholic hydroxyl groups in the natural substrate norepinephrine at the active site of PNMT. In addition, these analogues provided information about the effects of conformational flexibility on active-site interaction of the aminoethyl side chain in phenolic phenylethylamines that may aid in learning the manner in which norepinephrine binds at the active site of PNMT. Analogues 22-24 were synthesized by a nine-step sequence, in which a Friedel-Crafts type intramolecular cyclization was the key step in the construction of the benzobicyclo[3.2.1]octane skeleton. p-Tyramine (10, Ki = 294 microM) was more potent than phenylethylamine (1, Ki = 854 microM) but m-tyramine (9, Ki = 1250 microM) was less potent than phenylethylamine as an inhibitor of PNMT. Similarly, in the conformationally restricted and conformationally defined tyramine analogues (12-15 and 22-24, respectively), the analogues with the p-tyramine moiety (14, Ki = 4.7 microM; 23, Ki = 111 microM) bind to PNMT better than do the corresponding unsubstituted compounds (16, Ki = 6.8 microM; 25, Ki = 206 microM) while the analogues with the m-tyramine moiety (13, 15, 22, and 24) have a lower binding affinity than do 16 and 25. The greatly enhanced activity of the phenolic 2-aminotetralins (12-15) compared with m- and p-tyramine (9 and 10, respectively) is likely due to the restriction of the side-chain conformation. The conformationally defined analogues 22-24 were less active than the conformationally restricted ones, 12-15, although the low-energy half-chair conformation of 2-aminotetralin is defined in 22-24. The reduced activity of 22-24 compared with the activity of 12-15 is probably due to the steric hindrance from the extra bridging atoms in binding to PNMT. The interaction of the p-hydroxyl group of the tyramine moiety may involve hydrogen bonding since the corresponding methyl ethers show a greatly reduced affinity for the active site of PNMT (Ki = 34 and 389 microM for methoxy analogues 28 and 35, compared to Ki = 4.7 and 111 microM for the corresponding phenolic analogues 14 and 23).     

Synthesis and evaluation of 3-substituted analogues of 1,2,3,4-tetrahydroisoquinoline as inhibitors of phenylethanolamine N-methyltransferase

1,2,3,4-Tetrahydroisoquinoline (THIQ) and aryl-substituted derivatives of THIQ are potent inhibitors of the enzyme that catalyzes the formation of epinephrine--phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28). In previous studies, we found that substitution of the 3-position of THIQ with a methyl group resulted in enhanced activity as an inhibitor for 3-methyl-THIQ with respect to THIQ itself. To more fully delineate this region of the PNMT active site, we have synthesized and evaluated other 3-substituted THIQ analogues that vary in both steric and electronic character. Extension of the methyl side chain in 8 by a single methylene unit results in diminished potency for 3-ethyl-THIQ, suggesting that this zone of the active site is spatially compact; furthermore, the region of steric intolerance may be located principally on only "one side" of the 3-position of bound THIQs, since the carbonyl containing (bent) analogues 3-(methoxycarbonyl)-THIQ and 3-(aminocarbonyl)-THIQ are much less capable of forming a strong enzyme-inhibitor dissociable complex compared to straight-chain derivatives possessing a similar steric component. The good activity of 3-(hydroxymethyl)-THIQ as a PNMT inhibitor cannot be explained solely by steric tolerance for this side chain. We believe that an active-site amino acid residue capable of specific (i.e., hydrogen bond) interactions is located in close proximity to the 3-position of bound THIQs and that association of the OH functionality with this active-site residue results in the enhanced in vitro potency of this analogue (Ki = 2.4 microM) compared to that of THIQ (Ki = 10.3 microM). Incorporation of a hydroxymethyl substituent onto the 3-position of the potent PNMT inhibitor 7,8-dichloro-THIQ (SKF 64139, Ki = 0.24 microM) did not result in the same enhancement in inhibitor potency for 17 (Ki = 0.38 microM). This result suggests that simultaneous binding in an optimal orientation of the aromatic halogens, secondary amine, and side-chain hydroxyl functionalities to the PNMT active site is not allowed in this analogue.     

Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier

Summary The neuronal noradrenaline uptake mechanism (uptake₁) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K _m) and the maximal initial transport rate (V _max) were determined. Furthermore, the dissociation constants (K _D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V _max, K _m, and K _D. Neither K _m and V _max nor K _D and V _max were found to be correlated. However, the discrepancy between K _m and K _D expressed as the ratio, K_m/K_D, was negatively correlated with V _max (r = – 0.9315, n = 7, p < 0.01).For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake₁. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K _m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K _D). (2) The discrepancy between K _m and K _D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V _max).The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K _m/K _D and V_max supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to E. Schömig at the above address     

Quantitative structure-activity relationship studies of non-aromatic analogues of ethanolamine as inhibitors of phenylethanolamine N-methyltransferase

The phenylethanolamine N-methyltransferase inhibition potency of non-aromatic ethanolamines is found by regression analysis to be significantly correlated with van der Waals volume, Vw. For the proper inhibition of this enzyme the size of a substituent on beta-carbon of ethanolamine chain should be comparable to the cavity size of enzyme and there should be no other bigger group present on this carbon, which can cause a steric crowding, when the molecule approaches the reactive site of 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.     

Studies on the characterization and inhibition of rat brain phenylethanolamine N-methyltransferase

Summary The existence of phenylethanolamine N-methyltransferase (PNMT) activity in the rat brain and spinal cord was confirmed using both substrate specificity and selective inhibitors of the adrenal enzyme as biochemical tools. The enzyme was not generally localized throughout the central nervous system but was found primarily in the brain stem and spinal cord with lesser amounts occurring in the midbrain. No significant PNMT activity was found in the cerebellum or forebrain. The central enzyme was markedly inhibited both in vitro and in vivo by low concentrations (doses) of SK&F 64139, a potent inhibitor of the adrenal enzyme.