Binding of α-hydroxy-β-amino acid inhibitors to methionine aminopeptidase. The performance of two types of scoring functions

The binding mode of a recently described set of -hydroxy--amino acid inhibitors of methionine aminopeptidase type 2 is suggested in the present work. The binding mode is supported by analysis of published structures of transition state analogues co-crystallised with E. coli methionine aminopeptidase and by a comparison of molecular interaction fields calculated using GRID for E. coli and human methionine aminopeptidase. Based on the suggested binding mode two types of scoring functions have been evaluated and compared. These are the commercially available consensus score, CScore, and scoring of the ligands employing energies calculated using the Merck Molecular Force Field (MMFF). Enriched subsets of ligands were obtained when using CScore, but these scores could not be used to assess the relative affinities of individual compounds. Although still not sufficiently accurate for reliable predictive purposes, an improved correlation was obtained between structure and affinity using a combined force field energy including contributions from solvation and conformational energy penalty for binding.     

Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.     

Ionic immobilization, diversification, and release: application to the generation of a library of methionine aminopeptidase inhibitors

Development of an ionic immobilization, diversification, and release method for the generation of methionine aminopeptidase inhibitors is reported. This method involves the immobilization of 5-bromofuran-2-carboxylic acid and 5-bromothiophene-2-carboxylic acid onto PS-BEMP, followed by Suzuki reaction on a resin-bound intermediate and subsequent release to provide products in moderate yields and excellent purities. Compound potencies were evaluated on the Co(II), Mn(II), Ni(II), and Fe(II) forms of Escherichia coli MetAP1. The furoic-acid analogs were found to be Mn(II) selective with IC 50 values in the low micromolar range. Qualitative SAR analysis, supplemented by molecular modeling studies, provides valuable information on structural elements responsible for potency and selectivity.     

Direct determination of methionine sulfoxide in milk proteins by enzyme hydrolysis/high-performance liquid chromatography

A direct and simultaneous HPLC/UV determination of methionine and methionine sulfoxide in enzyme-hydrolyzed milk proteins is described. Protein hydrolysis is accomplished by a three-enzyme (pronase, leucine aminopeptidase, prolidase) 20-h/37 degrees C digestion. A gradient elution reversed-phase HPLC system with UV detection at 214 nm and 280 nm is then used to determine the quantitative releases of methionine sulfoxide, methionine, tyrosine, and tryptophan. The ease of methionine oxidation by a wide variety of oxidants, coupled with the quantitative release of both methionine and its sulfoxide by the three-enzyme hydrolysis, renders the approach valuable for identifying oxidized milk proteins. The relatively simple method proved accurate and precise in its application to commercial milk products, finding methionine sulfoxide levels as high as 74% of the total methionine.     

Which one among Zn(II), Co(II), Mn(II), and Fe(II) is the most efficient ion for the methionine aminopeptidase catalyzed reaction?

The catalytic hydrolysis of a methionyl-peptide substrate by a methionine aminopeptidase active site model cluster was investigated at the DF/B3LYP level of theory, in the gas-phase and in the protein environment. Zn(II), Co(II), Mn(II), and Fe(II) transition metals were examined as the potential catalytic metals of this enzyme involved in protein maturation. Two different mechanisms in which Glu204 was present as protonated or deprotonated residue were considered. The energetic profiles show lower barriers as the protonated glutamate is involved. The rate-determining step of the hydrolysis reaction is always the nucleophilic addition of the hydroxide on substrate carbon, followed by less energetically demanding methionine-peptide C-N bond scission. The lowest activation energy is obtained in the case of zinc dication while the other metals show very high energetic barriers, so that methionine aminopeptidase can be in principle recognized as a dizinc enzyme.     

Investigation of the metal binding site in methionine aminopeptidase by density functional theory

All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co²⁺ions or Zn²⁺ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions. This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E. colimethionine aminopeptidase type 1 (eMetAP-1). Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion.     

Discovery of 4H-thieno[3,2-b]pyrrole derivatives as potential anticancer agents

In this study, we describe the discovery of the 4H-thieno[3,2-b]pyrrole derivatives as an useful scaffold to obtain potent lead compounds for the treatment of colon cancer. We first started with the 4H-thieno[3,2-b]pyrrole derivatives which come from compound libraries screening, and then optimized their structures based on the cellular activities and pharmacophore models. The inhibition rate of cell growth assay demonstrated that this series compounds showed better inhibitory activities against colon cancer cells than other tested tumor cells. Moreover, the target of the most active compound 8i was explored by target fishing strategy and validated by molecular docking and biological activity analysis. The results of apoptosis and flow cytometry demonstrated that compound 8i induces cell apoptosis probably by inhibiting activity of methionine aminopeptidase 2, therefore compound 8i may be a potent inhibitor to methionine aminopeptidase 2.     

General molecular mechanics method for transition metal carboxylates and its application to the multiple coordination modes in mono- and dinuclear Mn(II) complexes

A general molecular mechanics method is presented for modeling the symmetric bidentate, asymmetric bidentate, and bridging modes of metal-carboxylates with a single parameter set by using a double-minimum M-O-C angle-bending potential. The method is implemented within the Molecular Operating Environment (MOE) with parameters based on the Merck molecular force field although, with suitable modifications, other MM packages and force fields could easily be used. Parameters for high-spin d (5) manganese(II) bound to carboxylate and water plus amine, pyridyl, imidazolyl, and pyrazolyl donors are developed based on 26 mononuclear and 29 dinuclear crystallographically characterized complexes. The average rmsd for Mn-L distances is 0.08 A, which is comparable to the experimental uncertainty required to cover multiple binding modes, and the average rmsd in heavy atom positions is around 0.5 A. In all cases, whatever binding mode is reported is also computed to be a stable local minimum. In addition, the structure-based parametrization implicitly captures the energetics and gives the same relative energies of symmetric and asymmetric coordination modes as density functional theory calculations in model and "real" complexes. Molecular dynamics simulations show that carboxylate rotation is favored over "flipping" while a stochastic search algorithm is described for randomly searching conformational space. The model reproduces Mn-Mn distances in dinuclear systems especially accurately, and this feature is employed to illustrate how MM calculations on models for the dimanganese active site of methionine aminopeptidase can help determine some of the details which may be missing from the experimental structure.     

A chemical and genetic approach to the mode of action of fumagillin

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/beta-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.     

Synthesis and bioactivity of a novel series of 3, 6-disubstituted 1, 2, 4-triazolo [3, 4-b]-1, 3, 4-thiadiazoles

<正>A novel series of 3,6-disubstituted 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles were synthesized by the condensation of 4-amino-5- [2-(4-chlorophenoxymethylbenzimidazole)-1-methylene]-3-mercapto-1,2,4-triazole with various(un)substituted aromatic acids in the presence of phosphorous oxychloride.These compounds were investigated for their inhibitory activity to E.coli methionine aminopeptidase(EcMetAP1).Some of the tested compounds showed significant inhibitory activity.     

4, 6-二芳基-2-氨基嘧啶类衍生物的合成与生物活性

合成并表征了5种4,6-二芳基-2-氨基嘧啶类化合物。 测试了它们对大肠肝菌甲硫酰胺肽酶(EcMetAP)的抑制作用及对CXCR4受体的拮抗作用。 发现5种化合物均对EcMetAP酶活有抑制作用,除化合物2外均对CXCR4受体有拮抗作用。 利用FieldTemplater和FieldAlign软件对化合物1～5的上述活性构效关系进行了分析,初步认为化合物的嘧啶环3位N原子及4位取代苯环上若引入给电子基团,可增强这类化合物的EcMetAP酶抑制活性;在嘧啶环2位引入负电性较强的基团取代,改造2个苯环和嘧啶环的4、5、6位Ｃ原子的结构可增强其CXCR4受体拮抗活性。     

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells

BACKGROUND: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESUTLS: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.     

Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M?+?H?+?O]⁺), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M?+?H?+?O]⁺ product is observed at a much greater abundance than the proton transfer product (viz., [M?+?H]⁺). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to ‘label’ methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

Metalloform-selective inhibitors of escherichia coli methionine aminopeptidase and X-ray structure of a Mn(II)-form enzyme complexed with an inhibitor

Methionine aminopeptidase (MetAP) enzymes require a divalent metal ion such as Mn(II), Fe(II), Co(II), Ni(II), or Zn(II) for its removal of the N-terminal methionine from newly synthesized proteins, but it is not certain which of these ions is most important in vivo. Metalloform-selective MetAP inhibitors could be valuable for defining which metals are physiologically relevant for MetAP activation and could serve as leads for development of new therapeutic agents. We have screened a library of 43 736 small drug-like molecules against Escherichia coli MetAP and identified two groups of potent and highly metalloform-selective inhibitors of the Co(II)-form, and of the Mn(II)-form, of this enzyme. Compound 1 is 790-fold more selective for the Co(II)-form, while compound 4 is over 640-fold more potent toward the Mn(II)-form. The X-ray structure of a di-Mn(II) form of E. coli MetAP complexed with the Mn(II)-form-selective compound 4 was obtained, and it shows that the inhibitor interacts with both Mn(II) ions through the two oxygen atoms of its free carboxylate group. The preferential coordination of the hard (oxygen) donors to Mn(II) may contribute to its superb selectivity toward the Mn(II)-form.     

Solution-phase parallel synthesis of hexahydro-1H-isoindolone libraries via tactical combination of Cu-catalyzed three-component coupling and Diels-Alder reactions

Parallel solution-phase synthesis of combinatorial libraries of hexahydro-1 H-isoindolones exploiting a novel "tactical combination" of Cu-catalyzed three-component coupling and Diels-Alder reactions was accomplished. Three distinct libraries consisting of 24 members (library I), 60 members (library II), and 32 members (library III) were constructed. Variation of three substituents on the isoindolone scaffold in library I was exclusively achieved by the choice of the building blocks. In the syntheses of libraries II and III, sublibraries of isoindolone scaffolds were prepared initially in a one-pot/two-step process and were further diversified via Pd-catalyzed Suzuki cross-coupling reaction with boronic acids at two different diversification points. The Lipinski profiles and calculated ADME properties of the compounds are also reported.     

Thyrocalcitonin. II. Structure of alpha-thyrocalcitonin

The highly active peptide hormones isolated from pig thyroids – i.e. α-thyrocalcitonin and its sulfoxide, containing 32 amino acid residues – were subjected to sequence studies using chemical and enzymatic methods. On the basis of these studies the following structure was derived for α-thyrocalcitonin: In the α-thyrocalcitonin sulfoxide (designated formerly as β-thyrocalcitonin) the methionine residue is replaced by methionine sulfoxide.     

Enantioselective synthesis of non-natural aromatic alpha-amino acids

We present two complementary methods for the stereoselective synthesis of non-natural alpha-amino acids with aromatic or heteroaromatic side chains. One approach is based on the chemical transformation of methionine, whereas the other applies the stereoselective Myers alkylation of glycine. The resulting product types differ in the linker length between glycine and the aromatic substituent. Since methionine and pseudoephedrine are available in both absolute configurations, R- or S-configured enantiopure amino acids with either C(2) or C(3) linkers can be obtained on gram scales. In each case the key step of the synthesis is hydroboration of the unsaturated building blocks 9 and 17, followed by palladium-catalyzed Suzuki cross-coupling with aryl halides. Attention must in certain cases be paid to the stereochemical integrity when basic Suzuki conditions are applied. Our initial difficulties are reported as well as the final "racemization-proof" procedures. The protecting groups chosen for the alpha-amino acids should be compatible with solid-phase peptide synthesis. This was confirmed by the successful synthesis of a series of tripeptides.     

Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-l-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-l-methionine sulfoxide and the product Fmoc-l-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-l-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-l-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis–Menten constant, K _m, and the maximum velocity, v _max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

Direct determination of free methionine in soy-based infant formula

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.