New Antibiotic Candidates against<i>Helicobacter pylori</i>

Wang, Shanzhi; Cameron, Scott A.; Clinch, Keith; Evans, Gary B.; Wu, Zhimeng; Schramm, Vern L.; Tyler, Peter C.

doi:10.1021/jacs.5b06110

Cited by 40 publications

(97 citation statements)

References 35 publications

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“…8,52 Derivatives of two distinct chemical scaffolds gave 14 compounds with IC 90 values at or below 16 ng/mL for H. pylori growth (Figure 10). Blocking growth of the bacteria requires entry into the cell and inhibition of the H. pylori MTAN.…”

Section: Bacterial Menaquinone Synthesis: the Futalosine Pathwaymentioning

confidence: 99%

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Immucillins in Infectious Diseases

2017

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The Immucillins are chemically stable analogues that mimic the ribocation and leaving-group features of N-ribosyltransferase transition states. Infectious disease agents often rely on ribosyltransferase chemistry in pathways involving precursor synthesis for nucleic acids, salvage of nucleic acid precursors, or synthetic pathways with nucleoside intermediates. Here, we review three infectious agents and the use of the Immucillins to taget enzymes essential to the parasites. First, DADMe-Immucillin-G is a purine nucleoside phosphorylase (PNP) inhibitor that blocks purine salvage and shows clinical potential for treatment for the malaria parasite Plasmodium falciparum, a purine auxotroph requiring hypoxanthine for purine nucleotide synthesis. Inhibition of the PNPs in the host and in parasite cells leads to apurinic starvation and death. Second, Helicobacter pylori, a causative agent of human ulcers, synthesizes menaquinone, an essential electron transfer agent, in a pathway requiring aminofutalosine nucleoside hydrolysis. Inhibitors of the H. pylori methylthioadenosine nucleosidase (MTAN) are powerful antibiotics for this organism. Synthesis of menaquinone by the aminofutalosine pathway does not occur in most bacteria populating the human gut microbiome. Thus, MTAN inhibitors provide high-specificity antibiotics for H. pylori and are not expected to disrupt the normal gut bacterial flora. Third, Immucillin-A was designed as a transition state analogue of the atypical PNP from Trichomonas vaginalis. In antiviral screens, Immucillin-A was shown to act as a prodrug. It is active against filoviruses and flaviviruses. In virus-infected cells, Immucillin-A is converted to the triphosphate, is incorporated into the viral transcript, and functions as an atypical chain-terminator for RNA-dependent RNA polymerases. Immucillin-A has entered clinical trials for use as an antiviral. We also summarize other Immucillins that have been characterized in successful clinical trials for T-cell lymphoma and gout. The human trials support the potential development of the Immucillins in infectious diseases.

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Section: Bacterial Menaquinone Synthesis: the Futalosine Pathwaymentioning

confidence: 99%

“…5,6 BT-DADMe-Immucillin-A and related analogues are powerful antibiotics for Helicobacter pylori . 7,8 …”

Section: Figurementioning

confidence: 99%

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Immucillins in Infectious Diseases

2017

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“…One such target in this pathway is a homodimeric enzyme, H. pylori 5′-methylthioadenosine nucleosidase (HpMTAN), that hydrolyzes the N-ribosidic bond of 6-amino-6-deoxyfutalosine (Fig. 1A) (2)(3)(4). Additionally, HpMTAN hydrolyzes the N-ribosidic bond of other adenosine-containing metabolites such as S-adenosylhomocysteine (SAH) ( Table S1) and 5′-deoxyadenosine (5-7) and therefore functions as a central metabolic hub.…”

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confidence: 99%

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Banco

Mishra

Ostermann

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple protontransfer events. Of particular interest is the protonation state of residue D198, which possesses a pK a above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wildtype HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.neutron diffraction | enzyme mechanism | proton transfer | nucleosidase | Helicobacter

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“…17,18 Transition-state analogue inhibitors of H. pylori MTAN are more powerful antibiotics against this organism than any of the antibiotics currently approved for H. pylori infections. 19,20 …”

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Continuous Fluorescence Assays for Reactions Involving Adenine

et al. 2016

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5′-Methylthioadenosine phosphorylase (MTAP) and 5′-methylthioadenosine nucleosidase (MTAN) catalyze the phosphorolysis and hydrolysis of 5′-methylthioadenosine (MTA), respectively. Both enzymes have low KM values for their substrates. Kinetic assays for these enzymes are challenging, as the ultraviolet absorbance spectra for reactant MTA and product adenine are similar. We report a new assay using 2-amino-5′-methylthioadenosine (2AMTA) as an alternative substrate for MTAP and MTAN enzymes. Hydrolysis or phosphorolysis of 2AMTA forms 2,6-diaminopurine, a fluorescent and easily quantitated product. We kinetically characterize 2AMTA with human MTAP, bacterial MTANs and use 2,6-diaminopurine as a fluorescent substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used as the substrate to kinetically characterize the dissociation constants for three-transition-state analogue inhibitors of MTAP and MTAN. Kinetic values obtained from continuous fluorescent assays with MTA were in good agreement with previously measured literature values, but gave smaller experimental errors. Chemical synthesis from ribose and 2,6-dichloropurine provided crystalline 2AMTA as the oxalate salt. Chemo-enzymatic synthesis from ribose and 2,6-diaminopurine produced 2-amino-S-adenosylmethionine for hydrolytic conversion to 2AMTA. Interaction of 2AMTA with human MTAP was also characterized by pre-steady-state kinetics and by analysis of the crystal structure in a complex with sulfate as a catalytically inert analogue of phosphate. This assay is suitable for inhibitor screening by detection of fluorescent product, for quantitative analysis of hits by rapid and accurate measurement of inhibition constants in continuous assays, and pre-steady-state kinetic analysis of the target enzymes.

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New Antibiotic Candidates againstHelicobacter pylori

Cited by 40 publications

References 35 publications

Immucillins in Infectious Diseases

Immucillins in Infectious Diseases

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Continuous Fluorescence Assays for Reactions Involving Adenine

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