A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium’s recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori — two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. Key points • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathione-β-synthase domain

Purine metabolism plays a pivotal role in bacterial life cycle, however, regulation of the de novo and purine salvage pathways have not been extensively detailed in mycobacteria. By gene knockout, biochemical and structural analyses, we identified Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) guaB1 gene product as a novel type of guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine salvage pathway and contains cystathione β-synthase (CBS) domains with atypical orientation in the octamer. CBS domains share a much larger interacting area with a conserved catalytic domain in comparison with the only known CBS containing protozoan GMPR and closely related inosine monophosphate dehydrogenase structures. Our results revealed essential effect of pH on allosteric regulation of Msm GMPR activity and oligomerization with adenine and guanosine nucleotides binding to CBS domains.Bioinformatic analysis indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

MO631XANTHINE OXIDASE INHIBITOR AMELIORATES HIGH GLUCOSE-INDUCED OXIDATIVE STRESS BY ACTIVATING AMPK VIA THE ACTIVATION OF PURINE SALVAGE PATHWAY IN GLOMERULAR ENDOTHELIAL CELLS

Background and Aim Oxidative stress plays a crucial role in the pathogenesis of diabetic nephropathy (DN). Xanthine oxidase (XO) contribute to reactive oxygen species (ROS) production, and XO inhibitor, febuxostat has been reported to the protection of kidney diseases. However, the mechanism of renoprotective effects for febuxostat remained unclear. We investigated the renoprotective mechanism associated with purine salvage pathway of febuxostat against DN. Method Glomerular endothelial cells (GEnCs) exposed to high glucose (HG) were treated with or without febuxostat for 72 hours, and then the changes of purine salvage pathway and the phosphorylation of 5' AMP-activated protein kinase (AMPK) and its related signaling pathway were evaluated. Results Cell survival was significantly decreased in HG-treated GEnCs, and febuxostat treatment enhanced cell survival in a dose-dependent manner. The expressions of xanthine/hypoxanthine, and the levels of xanthine oxidoreductase were significantly increased in HG-treated GEnCs, and these findings were attenuated by febuxostat. The AMP/ATP ratio was inhibited in in HG-treated GEnCs and enhanced by febuxostat treatment. Febuxostat treatment enhanced phosphorylation of AMPK, peroxisome proliferator-activated receptor (PPAR)-α, PPAR-gamma coactivator (PGC)-1α, and dephosphorylation of the Forkhead box O (FoxO)3a in HG-treated GEnCs. Febuxostat treatment also suppressed NADPH oxidase expressions and their catalytic subunits and oxidative stress in HG-treated GEnCs. AMPK inhibition using small interfering RNA blunted the antioxidative effects of febuxostat in HG-treated GEnCs. Conclusions Febuxostat attenuated HG-induced oxidative stress through the activation of purine salvage pathway and AMPK–PGC-1α–NADPH oxidase signaling.

4E Interacting Protein as a Potential Novel Drug Target for Nucleoside Analogues in Trypanosoma Brucei

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

Similar solutions to a common challenge: Regulation of genes encoding Ralstonia solanacearum xanthine dehydrogenase

The stringent response involves accumulation of (p)ppGpp, and it ensures that survival is prioritized. Production of (p)ppGpp requires purine synthesis, and upregulation of an operon that encodes the purine salvage enzyme xanthine dehydrogenase (Xdh) has been observed during stringent response in some bacterial species, where direct binding of ppGpp to a TetR-family transcription factor is responsible for increased xdh gene expression. We show here that the plant pathogen Ralstonia solanacearum has a regulatory system in which the LysR-family transcription factor XanR controls expression of the xan operon; this operon encodes Xdh as well as other enzymes involved in purine salvage, which favor accumulation of xanthine. XanR bound upstream of the xan operon, a binding that was attenuated on addition of either ppGpp or cyclic di-guanosine monophosphate (c-di-GMP). Using a reporter in which enhanced green fluorescent protein (EGFP) is expressed under control of a modified xan promoter, XanR was shown to repress EGFP production. Our data suggest that R. solanacearum features a regulatory mechanism in which expression of genes encoding purine salvage enzymes is controlled by a transcription factor that belongs to a different protein family, yet performs similar regulatory functions.

Intrathecal baclofen therapy for Lesch-Nyhan disease: illustrative case

BACKGROUNDLesch-Nyhan disease (LND) is a very rare metabolic disorder involving the purine salvage pathway. LND manifests hyperuricemia, self-mutilation, cognitive impairment, and movement disorders such as spasticity and dystonia, whose control is difficult pharmaceutically.OBSERVATIONSIntrathecal baclofen (ITB) therapy was received by a 22-year-old male for generalized dystonia. His paroxysmal abnormal dystonic posturing reduced after surgery, making the task of caregivers easier despite the unchanged assignment on the dystonia scale during a follow-up period of 4 years.LESSONSITB may be a safe and feasible option for dystonic symptoms and difficulty with nursing care in patients with LND.

Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5′-deoxy-5′-iodoadenosine (IADO) and 5′-deoxy-5′-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.