Human PNMT Research Articles

Structure-Based Drug Design of Bisubstrate Inhibitors of Phenylethanolamine N-Methyltransferase Possessing Low Nanomolar Affinity at Both Substrate Binding Domains1.

The enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) catalyzes the final step in the biosynthesis of epinephrine and is a potential drug target, primarily for the control of hypertension. Unfortunately, many potent PNMT inhibitors also possess significant affinity for the a2-adrenoceptor, which complicates the interpretation of their pharmacology. A bisubstrate analogue approach offers the potential for development of highly selective inhibitors of PNMT. This paper documents the design, synthesis, and evaluation of such analogues, several of which were found to possess human PNMT (hPNMT) inhibitory potency <5 nM versus AdoMet. Site-directed mutagenesis studies were consistent with bisubstrate binding. Two of these compounds (19 and 29) were co-crystallized with hPNMT and the resulting structures revealed both compounds bound as predicted, simultaneously occupying both substrate binding domains. This bisubstrate inhibitor approach has resulted in one of the most potent (20) and selective (vs the a2-adrenoceptor) inhibitors of hPNMT yet reported.

Abstract 2897: ERBB2 amplicon passenger genes: A novel class of breast cancer antigens

Abstract DNA copy number amplification at the ERBB2 locus on 17q12-21 is a frequent genomic event, present in 10-15% of breast tumors. This gene-dense amplicon contains several genes that are aberrantly overexpressed in ERBB2+ breast cancer. We hypothesized that a subset of amplicon genes with minimal expression in normal tissue could serve as candidates for shared tumor-specific antigens. We eliminated from consideration other amplicon genes with high expression in specific tissues or broad expression in normal tissues due to the potential for autoimmune toxicity. Immunohistochemistry of a breast tumor microarray showed expression of MIEN, GRB7, PGAP3 proteins specifically in ERBB2+ tumor cells but not in stroma or in ERBB2 non-amplified breast tumor samples. We tested several peptide epitopes with predicted high affinity for HLA-A2 (&amp;lt;50 nM) and confirmed that these could stabilize MHC expression on T2 cells. In vitro sensitization of normal donor lymphocytes with the predicted epitopes led to production of IFN-γ. Moreover, Dendritic cells pulsed with lysate from SKBR3 (ERBB2 amplified) but not MCF7 breast cancer cells could sensitize autologous lymphocytes to epitopes from amplicon proteins. This suggests that the natural abundance of the amplicon proteins in ERBB2 positive tumors is sufficient to provide cross-priming. To test whether the amplicon proteins could serve as tumor-rejection antigens we used the murine TM15 breast cancer cell line which has amplification of a syntenic region surrounding the murine Her2/neu gene. Quantitative PCR showed 50-fold overexpression of the amplicon gene pnmt in TM15 cells. Mice vaccinated with recombinant murine or human PNMT protein, but not ovalbumin, rejected challenge with TM15 tumor without any overt toxicity and developed memory against delayed re-challenge.These data indicate that normal host genes which are in immediate proximity to the oncogene ERBB2 are abnormally expressed at high levels in ERBB2+ breast tumor cells. Although the molecular mechanism leading to aberrant expression is unknown, analysis of several breast cancer microarray databases as well as TCGA data also show a very high correlation between ERBB2 overexpression and high expression of these passenger genes so this seems to be a general phenomenon. T lymphocytes normally have tolerance to non-mutated host proteins however tolerance can be reversed with proper sensitization conditions. Antigen overexpression in tumor cells might allow effector T cells to surpass an activation threshold while preserving tolerance for normal tissue. DNA copy number amplification of specific oncogenes is a frequent genomic event in cancer so the general strategy of targeting passenger genes on amplicons may have broader use in cancer immunotherapy. Citation Format: Gregory E. Plautz, Arun Modi, Li-Xin Wang. ERBB2 amplicon passenger genes: A novel class of breast cancer antigens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2897. doi:10.1158/1538-7445.AM2014-2897

An (R)-specific N-methyltransferase involved in human morphine biosynthesis

The biosynthesis of morphine, a stereochemically complex alkaloid, has been shown to occur in plants and animals. A search in the human genome for methyltransferases capable of catalyzing the N-methylation of benzylisoquinoline alkaloids, as biosynthetic precursors of morphine, yielded two enzymes, PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49). Introduction of an N-terminal poly-histidine tag enabled purification of both proteins by immobilized metal affinity chromatography. Recombinant PNMT and NMT were characterized for their catalytic activity towards four benzylisoquinolines: tetrahydropapaveroline (THP), 6-O-methyl-THP, 4′-O-methyl-THP and norreticuline. Human PNMT accepted none of the offered alkaloids and was only active with its established substrate, phenylethanolamine. The second enzyme, human NMT, converted all four benzylisoquinolines, however, with a strict preference for (R)-configured morphine precursors. Determination of kinetic parameters of NMT for the four (R)-configured benzylisoquinoline alkaloids by LC–MS/MS revealed (R)-norreticuline to be the best substrate with an even higher catalytic activity as compared to the previously reported natural substrate tryptamine. In addition, isolation of the morphine precursor salutaridine from urine of mice injected (i.p.) with (R)-THP provides new evidence that the initial steps of morphine biosynthesis in mammals occur stereochemically and sequentially differently than in plants and suggests an involvement of the herein characterized (R)-specific NMT.

Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT.

Human phenylethanolamine N-methyltransferase pharmacogenomics: gene re-sequencing and functional genomics.

Phenylethanolamine N-methyltransferase (PNMT, EC2.1.1.28) catalyzes the N-methylation of norepinephrine to form epinephrine. As a step toward understanding the possible contribution of inheritance to individual variation in PNMT-catalyzed epinephrine formation, we 're-sequenced' the entire human PNMT gene, including the three exons, the introns and approximately 1 kb of the 5'-flanking region (5'-FR), using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Within the 3.5 kb re-sequenced, 18 single nucleotide polymorphisms (SNPs) were observed, including four non-synonymous coding SNPs (cSNPs) that resulted in the following alterations in encoded amino acid sequence: Asn9Ser, Thr98Ala, Arg112Cys and Ala175Thr. When constructs for the non-synonymous cSNPs were transiently expressed in COS-1 cells, the Ala98 allozyme displayed significantly lower levels of both activity and immunoreactive protein (p < 0.002) than did the wild-type (WT) enzyme due, at least in part, to accelerated protein degradation by a proteasome-mediated process. Luciferase reporter gene constructs were also created for the six common PNMT 5'-FR haplotypes observed. Significant differences were observed among haplotypes in their ability to drive transcription. These observations raise the possibility of inherited variation in the ability to form epinephrine from norepinephrine as a result of variant PNMT polymorphisms and haplotypes.

Crystallization of PNMT, the adrenaline-synthesizing enzyme, is critically dependent on a high protein concentration.

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 A. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 A.

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 Å. 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.

Two single nucleotide polymorphisms in the promoter region of the human phenylethanolamine N-methyltransferase PNMT gene.

Two single nucleotide polymorphisms in the promoter region of the human phenylethanolamine N-methyltransferase PNMT gene.

The 5′-flanking region of the human dopamine β-hydroxylase gene promotes neuron subtype-specific gene expression in the central nervous system of transgenic mice

Dopamine β-hydroxylase (DBH, EC 1.14.17.1) catalyze the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeriic gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631–1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5′-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.

Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase

Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672–7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5′-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the 5′-flanking region of the mouse PNMT gene.

Structure of human phenylethanolamine n-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672-7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5? terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.

Molecular cloning of cDNA and chromosomal assignment of the gene for human phenylethanolamine N-methyltransferase, the enzyme for epinephrine biosynthesis.

Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the synthesis of epinephrine from norepinephrine, the last step of catecholamine biosynthesis. To isolate a cDNA clone for human PNMT, we first isolated a cDNA clone for bovine adrenal medulla PNMT using mixed oligodeoxyribonucleotide probes whose synthesis was based on the partial amino acid sequence of tryptic peptides from the bovine enzyme. By screening a bovine adrenal medulla cDNA library, a cDNA clone with an insert of about 200 base pairs (bp) was isolated. This clone consisted of 84 bp of carboxyl-terminal coding region, which contained amino acid sequences corresponding to two tryptic peptides, and about 100 bp of 3'-untranslated region. Using this cDNA fragment as the probe, we screened a human pheochromocytoma cDNA library and isolated a cDNA clone with an insert of about 1.0 kilobase pairs, which contained the complete coding region of the enzyme. Northern blot analysis of human pheochromocytoma poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1,000 nucleotides, suggesting that this clone is a full-length cDNA. Determination of the nucleotide sequence revealed that human PNMT consists of 282-amino acid residues with a predicted molecular weight of 30,853, including initial methionine. The amino acid sequence of the human PNMT was highly homologous (88%) to that of the bovine enzyme. Chromosomal assignment of the gene for human PNMT was carried out using mouse-human somatic cell hybrids. The PNMT gene was assigned to chromosome 17.