Gary L. Grunewald

Getting the Adrenaline Going: Crystal Structure of the Adrenaline-Synthesizing Enzyme PNMT

BACKGROUND Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. RESULTS We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. CONCLUSIONS An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Relative stereochemistry and solution conformation of the novel paclitaxel-like antimitotic agent epothilone A

Abstract A nearly complete set of 1 H 1 H coupling constants and NOEs were determined for the novel paclitaxel-like antimitotic agent epothilone A from phase sensitive DFQ COSY and NOESY experiments in 75% DMSO- d 6 /D 2 O. This data was employed as distance constraints for molecular modeling studies. Molecular dynamic simulations were carried out on a complete series of diastereoisomers, and the results show only two relative stereochemistries to be in agreement with the NOE data.

Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors.

CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.

A liquid chromatographic assay for phenylethanolamine N-methyltransferase

Abstract A new assay technique for phenylethanolamine N -methyltransferase (PNMT) is described. The assay involves the separation of the PNMT substrate (e.g., norepinephrine) from the N -methylated product (e.g., epinephrine) using high-performance liquid chromatography on a cation-exchange resin. The detection and quantitation of these catecholamines are accomplished using an electrochemical detector. This assay technique has sensitivity and precision comparable to the radiochemical assays. In addition, it has the added advantage of specific product identification and quantitation.

The oxetane conformational lock of paclitaxel: Structural analysis of D-secopaclitaxel

Analysis of the 1H NMR data of paclitaxel in comparison with its oxetane ring-opened analogue D-secopaclitaxel suggests that the oxetane moiety (D-ring) of paclitaxel serves as a conformational lock for the diterpene moiety and the C13 side chain.

A reinvestigation of the taxol content of himalayan taxus wallichiana zucc. and a revision of the structure of brevifoliol.

Abstract When taken at the appropriate time and place, biorenewable leaves of the abundant Himalayan yew, Taxus wallichiana Zucc., contain significant quantities (0.045–0.130%) of taxol and other useful taxanes including 10-deacetylbaccatin III and brevifoliol. Spectroscopic reexamination of brevifoliol indicates that its structure must be revised to 11 . This makes brevifoliol and recently reported taxchinin A ( 12 ) close molecular relatives.

Effects of a 3-alkyl-, 4-hydroxy- and/or 8-aromatic-substituent on the phenylethanolamine N-methyltransferase inhibitor potency and α2-adrenoceptor affinity of 2,3,4,5-tetrahydro-1H-2-benzazepines

2,3,4,5-Tetrahydro-1H-2-benzazepine (THBA; 1) is nearly 100-fold more selective an inhibitor of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) versus the alpha2-adrenoceptor than is 1,2,3,4-tetrahydroisoquinoline (THIQ; 2) (1: PNMT K(i)= 3.3 microM, alpha2-adrenoceptor K(i) = 11 microM, selectivity [alpha2 K(i)/PNMT K(i)] = 3.3; 2: PNMT K(i) = 9.7 microM, alpha2 K(i) = 0.35 microM, selectivity=0.036;). Since the PNMT inhibitory activity and selectivity of THIQ were enhanced by the introduction of a hydrophilic electron-withdrawing 7-substituent and a 3-alkyl-substituent, a similar study was conducted on THBA. 8-Nitro-THBA (3) was found to be as potent an inhibitor of PNMT as its THIQ analogue (21) and to be more selective due to its reduced alpha2-adrenoceptor affinity (3: PNMT K(i) = 0.39 microM, alpha2 K(i) = 66 microM, selectivity = 170; 21: PNMT K(i) = 0.41 microM, alpha2 K(i) = 4.3 microM, selectivity = 10). Introduction of a 3-alkyl substituent on the THBA nucleus decreased both the alpha2-adrenoceptor affinity and the PNMT inhibitory activity, suggesting an area of steric bulk intolerance at both sites. 4-Hydroxy-THBA (15), which can be considered a conformationally-restricted analogue of 3-hydroxymethyl-THIQ (30), exhibited poorer PNMT inhibitory activity and less selectivity than 30 (15: PNMT K(i) = 58 microM, alpha2 K(i) = 100 microM, selectivity = 1.7; 30: PNMT K(i) = 1.1 microM, alpha2 K(i) = 6.6 microM, selectivity = 6.0). While the addition of an 8-nitro group to 15 increased the selectivity of 16 as compared to its THIQ analogue (31), it was not as potent at PNMT nor as selective as 8-nitro-THBA (3) (16, PNMT K(i) = 5.3 microM, alpha2 K(i) = 680 microM, selectivity = 130; 31: PNMT K(i) = 0.29 microM, alpha2 K(i) = 19 microM, selectivity = 66). Compound 3 is the most selective (PNMT/alpha2) and one of the more potent at PNMT compounds yet reported in the benzazepine series, and should have sufficient lipophilicity to penetrate the blood-brain barrier (CLogP = 1.8).

Inhibition of neuronal uptake of 3H-biogenic amines into rat cerebral cortex by partially and fully saturated derivatives of imipramine and desipramine: The importance of the aromatic ring in adrenergic amines—part 3

Abstract In order to assess the importance of the aromatic rings in inhibition of neuronal amine uptake produced by tricyclic antidepressant drugs, derivatives of imipramine (IMI) and dcsipramine (DMI) were prepared in which either one (IMIH, DMIH) or both (IMIH2, DMIH2) of the aromatic rings were fully reduced to cyclohexane rings. The relative abilities of these compounds to inhibit the uptake of l-[ 3 H ]- norepinephrine (NE), [ 3 H]-dopamine (DA) and [ 3 H]-5-hydroxytryptamine (5-HT) into chopped tissue of rat cerebral cortex were determined. Reduction of one or both aromatic rings did not alter significantly the inhibition of uptake of [ 3 H]-DA or [ 3 H]5-HT produced by either IMI or DMI ( IC 50 values 25–80 μM). However, saturation of one or both rings abolished the selectivitv of DMI for inhibition of NE uptake ( IC 50 0.12 μM). decreasing potency 150-fold ( IC 50 18.3 μM) and 250-fold ( IC 50 29.4 μM) respectively. The effect of aromatic ring reduction on the IMI-induced inhibition of NE uptake was much less pronounced. The results suggest that hydrophobic rather than π-electron attractive forces are involved in the interaction of DMI or IMI with DA or 5-HT uptake sites. However, the loss in selectivity for inhibition of NE uptake upon reduction of DMI may reflect loss of π-electron interactions in the binding of DMI to the NE uptake site, or may reflect increased sensitivity to spatial disposition of the hydrophobic binding areas of the drug relative to that found at the DA or 5-HT amine uptake sites.

Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity.

Substrate specificity is critically important for enzyme catalysis. In the adrenaline-synthesizing enzyme PNMT (phenylethanolamine N-methyltransferase), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of six human PNMT complexes, including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline. Determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT-noradrenaline complex with the previously determined PNMT-p-octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that p-octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptability of the PNMT substrate binding site in accepting multi-fused ring systems, such as substituted norbornene, as well as noradrenochrome, the oxidation product of noradrenaline. These results explain why only a subset of ligands recognized by PNMT are methylated by the enzyme; bulky substituents dictate the binding orientation of the ligand and can thereby place the acceptor amine too far from the donor methyl group for methylation to occur. We also show how the critical Glu(185) catalytic residue can be replaced by aspartic acid with a loss of only 10-fold in catalytic efficiency. This is because protein backbone movements place the Asp(185) carboxylate almost coincident with the carboxylate of Glu(185). Conversely, replacement of Glu(185) by glutamine reduces catalytic efficiency almost 300-fold, not only because of the loss of charge, but also because the variant residue does not adopt the same conformation as Glu(185).

A comparative molecular field analysis derived model of the binding of taxol® analogues to microtubules☆

Abstract We investigated a series of Taxol ® ( 1 , paclitaxel) analogues using a 3-D QSAR approach (CoMFA). Published and unpublished data for more than 50 compounds have been included in the analysis and the model has been used to predict the activity of other analogues. The model accurately describes known SARs and also has good predictive power.