Perspectives and challenges in developing small molecules targeting purine nucleoside phosphorylase.

As a cytosolic enzyme involved in the purine salvage pathway metabolism, purine nucleoside phosphorylase (PNP) plays an important role in a variety of cellular functions but also in immune system, including cell growth, apoptosis and cancer development and progression. Based on its T-cell targeting profile, PNP is a potential target for the treatment of some malignant T-cell proliferative cancers including lymphoma and leukemia, and some specific immunological diseases. Numerous small-molecule PNP inhibitors have been developed so far. However, only Peldesine, Forodesine and Ulodesine have entered clinical trials and exhibited some potential for the treatment of T-cell leukemia and gout. The most recent direction in PNP inhibitor development has been focused on PNP small-molecule inhibitors with better potency, selectivity, and pharmacokinetic property. In this perspective, considering the structure, biological functions, and disease relevance of PNP, we highlight the recent research progress in PNP small-molecule inhibitor development and discuss prospective strategies for designing additional PNP therapeutic agents.

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.

Transition state analogue of MTAP extends lifespan of APCMin/+ mice.

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5'-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5'-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-L-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP-/- cancer cell lines.

Binding thermodynamics and interaction patterns of human purine nucleoside phosphorylase-inhibitor complexes from extensive free energy calculations.

Human purine nucleoside phosphorylase (hPNP) plays a significant role in the catabolism of deoxyguanosine. The trimeric protein is an important target in the treatment of T-cell cancers and autoimmune disorders. Experimental studies on the inhibition of the hPNP observe that the first ligand bound to one of three subunits effectively inhibits the protein, while the binding of more ligands to the subsequent sites shows negative cooperativities. In this work, we performed extensive end-point and alchemical free energy calculations to determine the binding thermodynamics of the trimeric protein-ligand system. 13 Immucillin inhibitors with experimental results are under calculation. Two widely accepted charge schemes for small molecules including AM1-BCC and RESP are adopted for ligands. The results of RESP are in better agreement with the experimental reference. Further investigations of the interaction networks in the protein-ligand complexes reveal that several residues play significant roles in stabilizing the complex structure. The most commonly observed ones include PHE200, GLU201, MET219, and ASN243. The conformations of the protein in different protein-ligand complexes are observed to be similar. We expect these insights to aid the development of potent drugs targeting hPNP.

A resistant mutant of Plasmodium falciparum purine nucleoside phosphorylase uses wild-type neighbors to maintain parasite survival.

Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'-Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe-ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression, and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of PfPNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant PfPNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed PfPNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid PfPNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid PfPNP gave near-native Km values for substrate, but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately ninefold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits is responsible for altered properties of the hybrid protein. Thus, gene amplification of PfPNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.

Plasmodium falciparum purine nucleoside phosphorylase as a model in the search for new inhibitors by high throughput screening.

Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: KM 17 µM, kcat 1.2 s-1, VMax 2.2 U/mg and kcat/KM 7 × 10-4; for MESG they were: KM 131 µM, kcat 2.4 s-1, VMax 4.4 U/mg and kcat/KM 1.8 × 10-4. The thermodynamic parameters for the substrate Phosphate were: ΔG - 5.8 cal mol-1, ΔH - 6.5 cal mol-1 and ΔS - 2.25 cal mol-1/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.

Purine nucleoside phosphorylase inhibition ameliorates age-associated lower urinary tract dysfunctions.

In the aging population, lower urinary tract (LUT) dysfunction is common and often leads to storage and voiding difficulties classified into overlapping symptom syndromes. Despite prevalence and consequences of these syndromes, LUT disorders continue to be undertreated simply because there are few therapeutic options. LUT function and structure were assessed in aged (>25 months) male and female Fischer 344 rats randomized to oral treatment with a purine nucleoside phosphorylase (PNPase inhibitor) 8-aminoguanine (8-AG) or vehicle for 6 weeks. The bladders of aged rats exhibited multiple abnormalities: tactile insensitivity, vascular remodeling, reduced collagen-fiber tortuosity, increased bladder stiffness, abnormal smooth muscle morphology, swelling of mitochondria, and increases in urodamaging purine metabolites. Treatment of aged rats with 8-AG restored all evaluated histological, ultrastructural, and physiological abnormalities toward that of a younger state. 8-AG is an effective treatment that ameliorates key age-related structural and physiologic bladder abnormalities. Because PNPase inhibition blocks metabolism of inosine to hypoxanthine and guanosine to guanine, likely uroprotective effects of 8-AG are mediated by increased bladder levels of uroprotective inosine and guanosine and reductions in urodamaging hypoxanthine and xanthine. These findings demonstrate that 8-AG has translational potential for treating age-associated LUT dysfunctions and resultant syndromes in humans.

Small molecules under development for psoriasis: on the road to the individualized therapies.

Psoriasis is an incurable cutaneous illness characterized by the presence of well-delimited reddish plaques and silvery-white dry scales. So far, there is a limited understanding of its pathogenesis, though recent discoveries on the immunological, genetic and molecular aspects of this disease have significantly contributed to the identification of new targets and the development of novel drugs. Despite these advances, many patients are still dissatisfied, so to improve patient satisfaction, reliability, and clinical outcomes, the individualization of the treatments for this disease becomes a necessity. This review summarizes recent findings related to psoriasis pathogenesis and describes new small molecules and targets recently identified as promising for treatments. Additionally, the current status, challenges and the future directions for achieving individualized therapy for this disease and the need for more collaborative studies are discussed. The individualization of treatments for psoriasis, rather than a goal, is analyzed as a process where a dynamic integration between the needs and characteristics of the patients, the pharmacological progress, and the clinical decisions takes place.

Transition State Analogues Enhanced by Fragment-Based Structural Analysis: Bacterial Methylthioadenosine Nucleosidases.

Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Fused Heterocyclic Systems with an s-Triazine Ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines.

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.

Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase.

Bacterial 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5'-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.

Inhibiting PNP for the therapy of hyperuricemia in Lesch-Nyhan disease: Preliminary in vitro studies with analogues of immucillin-G.

Lesch-Nyhan disease (LND) is a rare X-linked genetic disorder, with complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, uric acid (UA), hypoxanthine and xanthine accumulation, and a devastating neurologic syndrome. UA excess, causing renal failure, is commonly decreased by xanthine oxidoreductase (XOR) inhibitors, such as allopurinol, yielding a xanthine and hypoxanthine increase. Xanthine accumulation may result in renal stones, while hypoxanthine excess seems involved in the neurological disorder. Inhibition of purine nucleoside phosphorylase (PNP) represents a different strategy for lowering urate. PNP catalyzes the cleavage of purine ribo- and d-ribo-nucleosides into ribose/deoxyribose phosphate and free bases, starting catabolism to uric acid. Clinical trials demonstrated that PNP inhibitors, initially developed as anticancer drugs, lowered UA in some gouty patients, in association or not with allopurinol. The present study tested the reliability of an analogue of immucillin-G (C1a), a PNP inhibitor, as a therapy for urate, hypoxanthine, and xanthine excess in LND patients by blocking hypoxanthine production upstream. The therapeutic aim is to limit the administration of XOR inhibitors to LND patients by supplying the PNP inhibitor in low doses, avoiding d-nucleoside toxicity. We report studies conducted in primary cultures of skin fibroblasts from controls and LND patients grown in the presence of the PNP inhibitor. Cell viability, oxypurine release in culture medium, and endocellular nucleotide pattern have been monitored in different growth conditions (inhibitor concentration, time, added inosine). Our results demonstrate effective PNP inhibition by low inhibitor concentration, with reduced hypoxanthine release, and no appreciable toxicity in control or patient cells, suggesting a new therapeutic strategy for LND hyperuricemia.

Hydroxyl-related differences for three dietary flavonoids as inhibitors of human purine nucleoside phosphorylase.

In this work, the hydroxyl-related differences of binding properties and inhibitory activities of dietary flavonoids, namely chrysin, baicalein and apigenin against purine nucleoside phosphorylase (PNP) were investigated. It was found that the hydroxylation on position C4' of chrysin (→apigenin) mildly decreased the binding affinities for PNP, whereas on the position C6 of chrysin (→baicalein) significantly increased binding affinities. Comparatively, the hydroxylation on position C4' and C6 greatly improved their PNP inhibitory effects. The IC50 values of apigenin and baicalein were 6.09 × 10-5 M and 8.94 × 10-5 M, respectively, which is significantly lower than that of chrysin (2.13 × 10-4 M). Results from molecular modeling revealed that there are two binding sites, i.e. active site (major) and tryptophan site (minor) on PNP, and the binding of these flavonoids might induce a serious conformational destabilization of PNP as a result of altering the micro-environment and morphology by flavonoids.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes Pi binding, while a three-site model is needed to characterize FA binding, irrespective of Pi presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with Pi and FA shows, however, that Pi binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that Pi moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. DATABASES: The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). ENZYMES: Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463.

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum.

Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine nucleoside phosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased PfPNP gene copy number and protein expression. At increased drug pressure, additional PfPNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant PfPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PfPNP. Crystal structures of native and mutated PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2136 clonal selection). Transition-state analog inhibitors against PfPNP are slow to induce resistance and may have promise in malaria therapy.

8-Aminoguanine Induces Diuresis, Natriuresis, and Glucosuria by Inhibiting Purine Nucleoside Phosphorylase and Reduces Potassium Excretion by Inhibiting Rac1.

Background 8-Aminoguanosine and 8-aminoguanine are K+-sparing natriuretics that increase glucose excretion. Most effects of 8-aminoguanosine are due to its metabolism to 8-aminoguanine. However, the mechanism by which 8-aminoguanine affects renal function is unknown and is the focus of this investigation. Methods and Results Because 8-aminoguanine has structural similarities with inhibitors of the epithelial sodium channel (ENaC), Na+/H+ exchangers, and adenosine A1 receptors, we examined the effects of 8-aminoguanine on EN aC activity in mouse collecting duct cells, on intracellular pH of human proximal tubular epithelial cells, on responses to a selective A1-receptor agonist in vivo, and on renal excretory function in A1-receptor knockout rats. These experiments showed that 8-aminoguanine did not block EN aC, Na+/H+ exchangers, or A1 receptors. Because Rac1 enhances activity of mineralocorticoid receptors and some guanosine analogues inhibit Rac1, we examined the effects of 8-aminoguanine on Rac1 activity in mouse collecting duct cells. Rac1 activity was significantly inhibited by 8-aminoguanine. Because in vitro 8-aminoguanine is a purine nucleoside phosphorylase ( PNP ase) inhibitor, we examined the effects of a natriuretic dose of 8-aminoguanine on urinary excretion of PNP ase substrates and products. 8-Aminoguanine increased and decreased, respectively, urinary excretion of PNP ase substrates and products. Next we compared in rats the renal effects of intravenous doses of 9-deazaguanine ( PNP ase inhibitor) versus 8-aminoguanine. 8-Aminoguanine and 9-deazaguanine induced similar increases in urinary Na+ and glucose excretion, yet only 8-aminoguanine reduced K+ excretion. Nsc23766 (Rac1 inhibitor) mimicked the effects of 8-aminoguanine on K+ excretion. Conclusions 8-Aminoguanine increases Na+ and glucose excretion by blocking PNP ase and decreases K+ excretion by inhibiting Rac1.

Immucillins in Infectious Diseases.

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.

In Vitro Immunomodulatory Activity of a Transition-State Analog Inhibitor of Human Purine Nucleoside Phosphorylase in Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) is the most common clinical form of American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis. CL is associated with a strong Th1 immune response. This exacerbated inflammatory response is correlated with severity of disease and delays the healing time of the ulcer. The fourth-generation immucillin derivative (DI4G), a potent inhibitor of purine nucleoside phosphorylase, has been proposed as a promising agent in the treatment of diseases associated with T cell activation. Herein, we evaluated the in vitro immunomodulatory activity of DI4G in cells of patients presenting with CL. Peripheral blood mononuclear cells (PBMC) from CL patients were stimulated with soluble leishmania antigen (SLA), in the presence or absence of DI4G, and IFN-γ, TNF, CXCL9, and CXCL10 levels were determined by ELISA. Lymphocyte proliferation in the presence or absence of DI4G was also evaluated, using flow cytometry. DI4G was able to decrease (p < 0.05) IFN-γ production but did not change the TNF, CXCL9, and CXCL10 levels. DI4G decreased (p < 0.05) the lymphoproliferative response mediated by CD8+ T cells, but not that by CD4+ T cells. DI4G is able to attenuate the exaggerated immune response in CL, exhibiting immunomodulatory activity in IFN-γ production and in CD8+ T cell proliferation.