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Leptospirosis is the most common zoonotic disease caused by pathogenic Leptospira, which is classified into three groups according to virulence. Its pathogenic and intermediate species contain leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic strains. In this study, we presented the crystal structure of LSS_11580 (rLRR20) from pathogenic L. santarosai serovar Shermani. X-ray diffraction at a resolution of 1.99 Å revealed a horseshoe-shaped structure containing seven α-helices and five β-sheets. Affinity assays indicated that rLRR20 interacts with E-cadherin on the cell surface. Interestingly, its binds to the extracellular (EC) 1 domain in human epithelial (E)-cadherin, which is responsible for binding to another E-cadherin molecule in neighboring cells. Several charged residues on the concave face of LRR20 were predicted to interact with EC1 domain. In the affinity assays, these charged residues were replaced by alanine, and their affinities to E-cadherin were measured. Three vital residues and mutation variants of LRR20, namely D56A, E59A, and E123A, demonstrated significantly reduced affinity to E-cadherin compared with the control. Besides, we also demonstrated that rLRR20 induced the expression of neutrophil gelatinase-associated lipocalin (NGAL) in HK2 cells. The low ability of the three mutation variants to induce NGAL expression further demonstrates this induction. The present findings indicate that LRR20 from pathogenic Leptospira binds to E-cadherin and interacts with its EC1 domain. In addition, its induction of NGAL expression in HK2 cells is associated with acute kidney injury in human.

This study presents structure and function of the sodium-dependent phosphate transporter, TmPiT-Pi/Na., Inorganic phosphate (Pi) is a fundamental and essential element for nucleotide biosynthesis, energy supply, and cellular signaling in living organisms. Human phosphate transporter (hPiT) dysfunction causes numerous diseases, but the molecular mechanism underlying transporters remains elusive. We report the structure of the sodium-dependent phosphate transporter from Thermotoga maritima (TmPiT) in complex with sodium and phosphate (TmPiT-Na/Pi) at 2.3-angstrom resolution. We reveal that one phosphate and two sodium ions (Pi-2Na) are located at the core of TmPiT and that the third sodium ion (Nafore) is located near the inner membrane boundary. We propose an elevator-like mechanism for sodium and phosphate transport by TmPiT, with the TmPiT-Na/Pi complex adopting an inward occluded conformation. We found that disease-related hPiT variants carry mutations in the corresponding sodium- and phosphate-binding residues identified in TmPiT. Our three-dimensional structure of TmPiT provides a framework for understanding PiT dysfunction and for future structure-based drug design.

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield β-d-glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only α-(1,6)-linked isomaltotriose (RoSBD-isoG3) and isomaltotetraose (RoSBD-isoG4) have been determined at 1.2 and 1.3 A, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD-isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch. Proteins 2014; 82:1079–1085. © 2013 Wiley Periodicals, Inc.

Uridylate kinase (UMPK; EC 2.7.4.22) transfers the γ-phosphate of ATP to UMP, forming UDP. It is allosterically regulated by GTP. Structures of Helicobacter pylori UMPK (HpUMPK) complexed with GTP (HpUMPK-GTP) and with UDP (HpUMPK-UDP) were determined at 1.8 and 2.5 Å resolution, respectively. As expected, HpUMPK-GTP forms a hexamer with six GTP molecules at its centre. Interactions between HpUMPK and GTP are made by the β3 strand of the sheet, loop β3α4 and the α4 helix. In HpUMPK-UDP, the hexameric symmetry typical of UMPKs is absent. Only four of the HpUMPK molecules bind UDP; the other two HpUMPK molecules are in the UDP-free state. The asymmetric hexamer of HpUMPK-UDP, which has an exposed dimer interface, may assist in UDP release. Furthermore, the flexibility of the α2 helix, which interacts with UDP, is found to increase when UDP is absent in HpUMPK-UDP. In HpUMPK-GTP, the α2 helix is too flexible to be observed. This suggests that GTP binding may affect the conformation of the α2 helix, thereby promoting UDP release.

Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in the glycolysis-gluconeogenesis metabolism pathway. The Helicobacter pylori TIM gene (HpTIM) was cloned, and HpTIM was expressed and purified. The enzymatic activity of HpTIM for the substrate GAP was determined (K(m) = 3.46 +/- 0.23 mM and k(cat) = 8.8 x 10(4) min(-1)). The crystal structure of HpTIM was determined by molecular replacement at 2.3 A resolution. The overall structure of HpTIM was (beta/alpha)beta(beta/alpha)(6), which resembles the common TIM barrel fold, (beta/alpha)(8); however, a helix is missing after the second beta-strand. The conformation of loop 6 and binding of phosphate ion suggest that the determined structure of HpTIM was in the "closed" state. A highly conserved Arg-Asp salt bridge in the "DX(D/N)G" motif of most TIMs is absent in HpTIM because the sequence of this motif is "(211)SVDG(214)." To determine the significance of this salt bridge to HpTIM, four mutants, including K183S, K183A, D213Q, and D213A, were constructed and characterized. The results suggest that this conserved salt bridge is not essential for the enzymatic activity of HpTIM; however, it might contribute to the conformational stability of HpTIM.

Helicobacter pylori cell binding factor 2 (HpCBF2) is an antigenic virulence factor belonging to the SurA-like peptidyl-prolyl cis-trans isomerase family with implications for pathogenicity in the human gastrointestinal tract. HpCBF2 possesses PPIase activity and could act as a periplasmic chaperone to regulate outer membrane protein assembly. Here, we measured the isomerization and chaperone activity of HpCBF2, and determined the crystal structure of HpCBF2 in complex with an inhibitor, indole-2-carboxylic acid (I2CA), at 2.4A resolution. HpCBF2-I2CA forms a homodimer encasing a large central hydrophobic cavity with a basket-like structure, and each monomer contains a PPIase and a chaperone domain. In the HpCBF2-I2CA dimer, the two PPIase domains separate by a distance of 22.8A, while the two chaperone domains arrange in a domain-swap manner. The PPIase domains bound with I2CA ligand face towards the chaperone domains and are shielded by surrounding hydrophobic residues. With the aid of SAXS experiments, we also revealed domain motion between the apo- and I2CA-bound states of HpCBF2. The domain motion in HpCBF2 might be necessary for the isomerization activity of PPIase and the accommodation of the unfolded and partially folded peptides to refold by chaperone domain.

5-Halogeno-6-amino-2′-deoxyuridines were synthesized from 2′-deoxyuridine as potential thymidine phosphorylase (ThdPase) inhibitors. Among the compounds synthesized, 5-bromo-6-amino-2′-deoxyuridine (6) and 5-iodo-6-amino-2′-deoxyuridine (9) were found to inhibit ThdPase activity with IC50 values of 1.3 μM and 6.5 μM, respectively. In vitro cell culture studies showed that compound (6) can significantly enhance the cytotoxic effects of 5-fluoro-2′-deoxyuridine against a human colon cancer HCT-8 cell line.

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12-60hpf) were treated with different dosages of AAP (0-45mM) for different time courses (12-60h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0mM) and 2.25mM AAP-exposure (12-72hpf) groups. In contrast, after higher doses (22.5 and 45mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12h) and dose (22.5mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12-24hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60-72hpf). With an exposure time course less than 24h of 45mM AAP, no embryo survived by the developmental stage of 72hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.

It has been reported that in vitro uridine (Urd) can reverse azidothymidine (AZT) cytotoxicity without decreasing anti-human immunodeficiency virus (HIV) activity. Our studies in mice have shown that daily oral doses of benzylacyclouridine (BAU), an inhibitor of Urd breakdown, also reduces AZT hematologic toxicity, presumably by elevating the plasma concentration of Urd. We now extend these murine studies and report the effect of various doses of exogenous Urd, various doses of BAU, or the combination of BAU and Urd, administered daily, on AZT-induced toxicity. In mice receiving concomitant AZT, daily doses of Urd of 1,000 to 2,000 mg/kg increase peripheral reticulocytes and slightly reduce AZT-induced hematologic toxicity. However, the range of effective doses is narrow, and higher doses of Urd (greater than 3,000 mg/kg/d) significantly enhance hematologic toxicity. At its most effective dose, (2,000 mg/kg/d), Urd produces 28% mortality. In contrast, BAU doses up to 300 mg/kg/d reduced AZT-related hematologic toxicity in a dose-dependent manner without mortality. Higher daily doses of BAU and the combination of BAU with low doses of Urd were not more effective. Studies conducted in mice infected with the Rauscher murine leukemia virus (RLV) indicate that BAU does not impair the antiretroviral effect of AZT when administered at doses that reduce AZT-induced anemia and leukopenia. These findings may be significant for the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Helicobacter pylori PAPT (HpPAPT) has a low sequence identity with other PAPTs and lacks the signature sequence found in other PAPTs. The crystal structure of HpPAPT, determined by multiwavelength anomalous dispersion, revealed an N-terminal β-stranded domain and a C-terminal Rossmann-like domain. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. HpPAPT lacks the gatekeeping loop that facilitates substrate binding in other PAPTs. PAPTs are essential for bacterial cell viability; thus, HpPAPT may be a potential antimicrobial drug target for H. pylori owing to its characteristic PAPT sequence and distinct conformation. Proteins 2007. © 2007 Wiley-Liss, Inc.

A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with Ki values in the range of 10−7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.

Furanosyl analogs of BAU (5-benzylacyclouridine) and BBAU (5-benzyloxybenzylacyclouridine), two potent inhibitors of uridine phosphorylase, were synthesized and evaluated as potential cancer chemotherapeutic agents. The analogs included ribosides, 2,2′-anhydro nucleosides, arabinosides and deoxyribosides. The anhydrouridine intermediates were potent inhibitors of uridine phosphorylase and good potentiators of FdUrd activity in human tumor cells in culture.

Eighty-seven pyrimidine base and nucleoside analogs were evaluated as inhibitors of uridine phosphorylase (UrdPase) and thymidine phosphorylase (dThdPase). These findings, together with an extensive literature review, have allowed construction of structure-activity relationships for the binding of ligands to UrdPase and dThdPase and provide a basis for the rational design of new inhibitors of these enzymes. Additionally, 2,6-pyridinediol and 6-benzyl-2-thiouracil have been identified as being potent inhibitors of UrdPase and dThdPase respectively.

Hydroxymethyl analogs of 5-benzylacyclouridine (BAU) and 5-benzyloxybenzylacyclouridine (BBAU) were synthesized by the condensation of appropriately blocked 2-(chloromethyl)glycerols with substituted 2, 4-dimethoxypyrimidines. The HM derivatives were found to be potent inhibitors of the enzyme uridine phosphorylase and to potentiate significantly the growth-inhibiting action of FdUrd in cell culture.

Survival studies were performed in mice bearing Sarcoma 180 ascites tumor treated with 6-thio and 6-seleno analogs of guanine and guanosine. The selenium-containing analogs were somewhat superior to the sulfur-containing compounds in antitumor activity and therapeutic index. The formation of 6-SeGMP from 6-seleno-guanine (6-SeG) was demonstrated in Sarcoma 180 ascites cells. Guanine, 6-thioguanine (6-TG) and 6-SeG show comparable substrate activity whereas 8-azaguanine is a much poorer substrate for hypoxanthine-guanine phosphoribosyl transferase from Sarcoma 180 cells. Both 6-TG and 6-SeG are good substrates for purine nucleoside phosphorylase from Sarcoma 180 cells. Chemically and enzymatically synthesized 6-SeGMP behaved as a competitive inhibitor ( K i 1 × 10 −4 M) of erythrocytic and Sarcoma 180 guanylate kinases. Weak substrate activity was demonstrated in the presence of large amounts of erythrocytic guanylate kinase.

The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli. Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-[(2'-hydroxy-1'-hydroxymethyl)methyl]urac il) with a Ki value of 0.15 microM. The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources [Niedzwicki et al., Biochem. Pharmac. 31, 1857 (1982) and Naguib et al., manuscript in preparation] indicating that the structure of the active site of uridine phosphorylase from E. coli may resemble that of the mammalian enzyme.

A new class of nucleoside analogs, the pyridimine acyclonucleosides, are competitive inhibitors of uridine phosphorylase but have no effect on thymicline phosphorylase, uridine kinase or thymidine kinase. The most potent of the series is acyclothymidine [5-methyl-1-(2′-hydroxyethoxymethyl)uracil] with a Ki value of 3 μM. Ki values of less than 30 μM were estimated for other analogs substituted at the 5-position of the pyrimicline ring. Extracts of xenografts of six human tumors were assayed for tissue levels of uricline phosphorylase and thymicline phosphorylase and for inhibition of 5-fluoro-2′-deoxyuridine (FUdR) phosphorolytic activity by acyclouridine [1-(2′-hydroxyethoxymethyl) uracil]. FUdR cleavage was inhibited most in those tissues in which the ratio of thymidine phosphorylase to uricline phosphorylase was low. Potential usage of these uricline phosphorylase inhibitors with the chemotherapeutic agent FUdR is discussed.

Various pyrimidine acyclonucleosides (l-(2'-hydroxyethoxymethyl)uracils) are specific inhibitors of uridine phosphorylase [Niedzwicki et al., Biochem. Pharmac. 30 , 2097 (1981)]. 5-Benzyluracils have also been shown to inhibit this enzyme [Baker and Kelley. J. med. Chem. 13 , 461 (1970); Woodman et al., Biochem. Pharmac . 29 ,1059 (1980)]. We have synthesized the acyclonucleoside analogs of 5-benzyluracil (BU) and 5-benzyloxybenzyluracil (BBU). These compounds, 5-benzyl-1-(2'-hydroxy-ethoxymethyl) uracil (BAU) and 5-( m -benzyloxybenzyl)-1-(2'-hydroxyethoxymethyl)uracil (BBAU), are potent inhibitors of uridine phosphorylase. K i values of 98 and 32 nM were estimated for BAU and BBAU respectively. These compounds are better inhibitors of uridine phosphorylase than BU ( K i = 1575 nM), BBU ( K i = 270 nM), and all other compounds previously tested, and they have no effect on thymidine phosphorylase, uridine-cytidine kinase, or thymidine kinase. Potential chemotherapeutic applications of BAU and BBAU are discussed.

Amino analogs of BAU (5-benzylacyclouridine) and BBAU (5-benzyloxybenzylacyclouridine) and their 2′-hydroxymethyl derivatives were synthesized for evaluation as inhibitors of uridine phosphorylase and hence potential cancer chemotherapeutic agents. Both aminomethyl analogs were found to be potent inhibitors of this enzyme and good potentiators of the anti-tumor action of FUdR.

Dimethyl DNS-chol (DDNS), a dansylated analog of acetylcholine, binds with specific sites in Schistosoma mansoni. This treatment provides fluorescent labeling of well-defined areas within the organism. It is believed that these regions of fluorescence reflect areas receiving cholinergic innervation; these regions might include both nervous and non-nervous (effector) tissue, and the DDNS binding sites might include both prejunctional amd postjunctional acetylcholine binding sites. Hycanthone (Hyc), an antischistosomal drug, blocks this labeling by DDNS. Atropine and other known antagonists of acetylcholine also prevent DDNS fluorescent labeling. Measurements of somatic motor activity demonstrate that Hyc and DDNS partially block the paralytic effects of the cholinomimetic drug carbamylcholine on S. mansoni. These data further support the hypothesis that Hyc is an anticholinergic agent, and show that DDNS may be useful as a histochemical label in studies involving structures receiving cholinergic innervation.

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). The LD10 (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. x 5) schedule was 30 mg . kg-1 . day-1. When the animals were pretreated with 0.1 mg . kg-1 . day-1 of dCF, the LD10 dose was reduced to 10 mg . kg-1 . day-1 x 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the LD10 dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 mumoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 mumoles/g of cells when 8-azaAdo was used in combination with dCF (1 microgram/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.

Kinetic parameters for the phosphorolytic activity of uridine phosphorylase (UrdPase) from human and mouse livers have been determined. The values of these parameters are: K P i = 279.0 ± 66.0 μ M, K Urd = 242.0 ± 63.0 μ M and V max = 3940 ± 175 pmol/min/mg, and K P i = 76.0 ± 7.0 μ M, K Urd = 143.0 ± 9.0 μ M and V max = 293.0 ± 5.0 pmol/min/mg, for human and mouse livers respectively. Benzylacyclouridines, the specific inhibitors of UrdPase, and seventeen newly synthesized derivatives, modified at the pyrimidine ring, the benzyl moiety or the acycio tail, have been tested for their potency to inhibit UrdPase and thymidine phosphorylase (dThdPase) from both human and mouse livers. None inhibited dThdPase. In contrast, all of the compounds tested inhibited UrdPase. Competitive inhibition was observed in all cases. Several of the new compounds were superior in their inhibition of UrdPase to the parent compounds. The inhibitory potencies of these compounds with UrdPase from human liver roughly paralleled those obtained with UrdPase from mouse liver. The most potent of these compounds was AM-BBAU (aminomethyl-BBAU or 5-(3'-benzyloxybenzyl)-1-[(1'-aminomethyl-2'-hydroxy-ethoxy)methyl]uracil) with a K i value of 18 nM with UrdPase from mouse liver. Structure-activity relationships of the binding of these inhibitors of UrdPase are discussed.

6-Methylmercaptopurine ribonucleoside (MeMPR) is the most potent inhibitor known of PRPP amidotransferase, the first step in purine biosynthesis de novo. We have examined other 6-methylmercapto- and 6-methylselenopurine ribonucleosides for their ability to act as feedback inhibitors of this pathway as well as for their ability to form analog nucleotides. Methylmercaptopurine ribonucleoside (MeMPR), methylselenopurine ribonucleoside (MeSePR), and methylmercaptopyrazolo pyrimidine ribonucleoside (MeMPPR) were potent inhibitors (90 per cent), methylselenopyrazolo pyrimidine ribonucleoside (MeSePPR) was less potent (75 per cent), and methylselenoguanosine (MeSeGR) did not significantly inhibit this pathway. Large amounts of the corresponding 5'-monophosphate nucleosides were formed from MeMPR and MeMPPR, lesser amounts from MeSePPR and MeSePR and none from MeSeGR. The ability of 6-selenoguanine, its ribonucleoside and the α- and β-anomers of the deoxyribonucleoside were also examined as possible inhibitors of purine synthesis de novo. Further investigation is needed to determine if these compounds have significant activity as antineoplastic agents, but in any case they may lead to structural modifications which will produce active compounds.

Treatment of adenosine, 2-aminoadenosine, adenine arabinoside, formycin, 1-Me-AMP, cyclo-AMP, and 1-Me-cyclo-AMP with H2Se in aqueous pyridine gave the corresponding seleno compounds with good yield.