Direct molecular haplotyping of multiple polymorphisms within exon 4 of the human catechol-O-methyltransferase gene by liquid chromatography–electrospray ionization time-of-flight mass spectrometry

The applicability of ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS) for the haplotyping of five SNPs (rs769223, rs4818, rs4986871, rs8192488, rs4680) located within exon 4 of the human catechol-O-methyltransferase (COMT, EC 2.1.1.6) gene is demonstrated. Two differently sized products of polymerase chain reaction—a 71-bp amplicon partially covering the sequence of a 124-bp amplicon—were used to determine unequivocally the allelic states of the single nucleotide polymorphisms linked on both chromosomes. The two amplicons were co-loaded onto the chromatographic column and simultaneously analyzed within a single gradient run. Using the described strategy, 101 individuals representing an Austrian population sample were typed. The obtained haplotype frequencies will serve as reference values in future association studies to examine the impact of the COMT gene on neuropsychiatric disorders. Additionally, two newly discovered polymorphic sites within the sequence of the COMT gene are described (a synonymous C>T mutation at the third position of the amino acid codon 99 in the soluble COMT protein or 149 in the membrane-bound COMT protein; a non-synonymous G>A substitution at the second position of the amino acid codon 95 in the soluble COMT protein or 145 in the membrane-bound-COMT protein).     

A Ready‐to‐Use Electrochemical Kit Design for the Diagnosis of Single Nucleotide Polymorphisms

In this study, for the first time a model electrochemical kit was constructed for the detection of a functional polymorphism in catechol‐O‐methyl transferase (COMT) gene which is important for diagnosis of neuropsychiatric disorders as Alzheimer disease. The disposable pencil graphite electrode (PGE) is designed as a “kit” and the probe DNA covered PGE can detect single nucleotide polymorphisms (SNPs) from real samples based on the guanine oxidation signal even after 5 months of kit preparation (150 days durability).The detection limit (S/N=3) of the biosensor was calculated as 1.18 pmol of synthetic target sequence and 6.09×10⁵ molecules of real samples in 30 min detection time.     

The effect of temperature on the analysis of metanephrine for catechol-O-methyltransferase activity assay by HPLC with electrochemical detection

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.     

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.     

6,7-二氧代-4-芳胺香豆素的设计、合成及分子对接研究

茶酚-氧位-甲基转移(COMT)酶抑制剂在治疗帕金森病中起到重要作用.通过对现有COMT酶抑制剂托卡朋和恩托卡朋结构与活性关系分析,推断含有儿茶酚结构的香豆素类化合物可能具有潜在的COMT酶抑制活性,因此设计合成了一类新型的6,7-二氧代-4-芳胺香豆素,通过理论计算,研究了此类化合物对COMT酶抑制活性.结果表明,设计的10种6,7-二氧代-4-芳胺香豆素与COMT酶的对接效果均较好,其中具有甲氧基乙基保护的儿茶酚结构化合物6,7-二[2-(甲氧基)乙氧]-4-(苯胺)香豆素(6b4)和6,7-二[2-(甲氧基)乙氧]-4-[(3-乙炔基)苯胺]香豆素(6b5)与COMT酶的对接效果尤为显著.     

Computation of the binding affinities of catechol‐O‐methyltransferase inhibitors: Multisubstate relative free energy calculations

Alchemical free energy simulations are amongst the most accurate techniques for the computation of the free energy changes associated with noncovalent protein–ligand interactions. A procedure is presented to estimate the relative binding free energies of several ligands to the same protein target where multiple, low‐energy configurational substates might coexist, as opposed to one unique structure. The contributions of all individual substates were estimated, explicitly, with the free energy perturbation method, and combined in a rigorous fashion to compute the overall relative binding free energies and dissociation constants. It is shown that, unless the most stable bound forms are known a priori, inaccurate results may be obtained if the contributions of multiple substates are ignored. The method was applied to study the complex formed between human catechol‐O‐methyltransferase and BIA 9‐1067, a newly developed tight‐binding inhibitor that is currently under clinical evaluation for the therapy of Parkinson's disease. Our results reveal an exceptionally high‐binding affinity (K_d in subpicomolar range) and provide insightful clues on the interactions and mechanism of inhibition. The inhibitor is, itself, a slowly reacting substrate of the target enzyme and is released from the complex in the form of O‐methylated product. By comparing the experimental catalytic rate (k_cat) and the estimated dissociation rate (k_off) constants of the enzyme‐inhibitor complex, one can conclude that the observed inhibition potency (K_i) is primarily dependent on the catalytic rate constant of the inhibitor's O‐methylation, rather than the rate constant of dissociation of the complex. © 2012 Wiley Periodicals, Inc.     

A semiempirical study on inhibition of catechol O-methyltransferase by substituted catechols

Catechol and endogenous catechol derivatives are readily methylated by catechol O-methyltransferase (COMT). In contrast, many catechol derivatives possessing electronegative substituents are potent COMT inhibitors. The X-ray structure of the active site of COMT suggests that the methylation involves a lysine as a general base. The lysine can activate one of the catecholic hydroxyl groups for a nucleophilic attack on the active methyl group of the coenzyme S-adenosyl-l-methionine (AdoMet). We studied the effect of dinitrosubstitution of the catecholic ring at the semiempirical PM3 level on the methylation reaction catalysed by COMT. The electronegative nitro groups make the ionized catechol hydroxyls less nucleophilic than the corresponding hydroxyl groups of the non-substituted catechol. As a consequence, dinitrocatechol is not methylated but is instead a potent COMT inhibitor. The implications of this mechanism to the design of COMT inhibitors are discussed.     

Molecular dynamics simulations of the enzyme Catechol-O-Methyltransferase: methodological issues1

Results from extensive 70 ns all-atom molecular dynamics simulations of catechol-O-methyltransferase (COMT) enzyme are reported. The simulations were performed with explicit TIP3P water and Mg^2?+ ions. Four different crystal structures of COMT, with and without different ligands, were used. These simulations are among the most extensive of their kind and as such served as a stability test for such simulations. On the methodological side we found that the initial energy minimization procedure may be a crucial step: particular hydrogen bonds may break, and this can initiate an irreversible loss of protein structure that becomes observable in longer time scales of the order of tens of nanoseconds. This has important implications for both molecular dynamics and quantum mechanics–molecular mechanics simulations.