Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Transition state analogues of Plasmodium falciparum and human orotate phosphoribosyltransferases.

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nM. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5'-phosphate bound to OPRTs with Kd values near 40 nM. Analogues designed with a C5-pyrimidine carbon-carbon bond to ribocation mimics gave Kd values in the range of 80-500 nM. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5'-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.

Methylthioadenosine deaminase in an alternative quorum sensing pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.

2,2,2-Trifluoroethyl 6-thio-ß-D-glucopyranoside as a selective tag for cysteines in proteins.

A synthetic route to a trifluoromethyl and thiol containing glucose derivative (2,2,2-trifluoroethyl 6-thio-ß-D-glucopyranoside) is presented, which is based on microwave-assisted Fischer glycosylation under increased pressure. This water-soluble, neutral thiol-compound can be used to selectively introduce a fluorine probe for (19)F NMR spectroscopy on cysteines in proteins. It can be attached under mild conditions in an aqueous environment without the risk of denaturing the protein. This tag has been applied to determine the redox-state of two cysteine residues in a bacterial transcription activator. Qualitative information about the solvent accessibility can be obtained from F-19 solvent PREs.

Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics.

Several acyclic hydroxy-methylthio-amines with 3-5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and Escherichia coli and Neisseria meningitidis MTANs and gave K(i) values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon-carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts.

A fluorescent probe for GM1 gangliosidosis related ß-galactosidase: N-(dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol.

N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol, a strong competitive inhibitor of ß-galactosidase, enhances residual ß-galactosidase activities in fibroblasts and serves as lead en route to diagnostic compounds for tracking the fate of mutant ß-gal as well as aberrant GM1 gangliosides by live cell imaging.

1-Deoxy-D-galactonojirimycins with dansyl capped N-substituents as ß-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines.

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-D-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-D-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-D-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-D-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.

1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines.

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of d-glucosidases and could also be shown to distinctly improve, at sub-inhibitory concentrations, the activity of beta-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.

7-(5-Methyl-sulfanyl-ß-d-erythrofuran-osyl)-7H-pyrrolo-[2,3-d]pyrimidin-4-amine monohydrate (MT-tubercidin·H(2)O).

The title compound, C(12)H(16)N(4)O(3)S·H(2)O, which has potential as a possible anti-malarial drug, was studied when small deviations in melting points, for two differently aged preparations, were observed. The unexpected existence of a water mol-ecule of crystallization is considered to be the cause of this variation. The 7H-pyrrolo-[2,3-d]pyrimidine unit is very slightly puckered with a total puckering amplitude of 0.035 (2) Å; its mean plane makes an angle of 88.40 (12)° with the mean plane through the ribofuranosyl unit. In the crystal, the mol-ecules are bound by strong O-H⋯N and N-H⋯O hydrogen bonds, utilizing all available protons and linking mainly through the water of crystallization.

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1.

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Third-generation immucillins: syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase.

ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP), developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases. Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin analogues (8-38) in order to identify simplified alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated that the flexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity. SerMe-ImmH (10, K(d) = 5.2 pM) was shown to be orally available in mice with a long biological residence time on blood PNP.

2-Benzyl-myo-inositol monohydrate.

The title structure, C(13)H(18)O(6)·H(2)O, contains two independent 2-benzyl-myo-inositol and water mol-ecules. In the crystal, the mol-ecules are strongly hydrogen bonded into an infinite two dimensional network utilizing all OH protons.

Synthesis of 5'-methylthio coformycins: specific inhibitors for malarial adenosine deaminase.

Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2'-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5'-methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5'-methylthioadenosine. 5'-Methylthiocoformycin and 5'-methylthio-2'-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.