In vivo experiments demonstrate the potent antileishmanial efficacy of repurposed suramin in visceral leishmaniasis.

BACKGROUND: Treatment failure and resistance to the commonly used drugs remains a major obstacle for successful chemotherapy against visceral leishmaniasis (VL). Since the development of novel therapeutics involves exorbitant costs, the effectiveness of the currently available antitrypanosomatid drug suramin has been investigated as an antileishmanial, specifically for VL,in vitro and in animal model experiments. METHODOLOGY/PRINCIPAL: Leishmania donovani promastigotes were treated with suramin and studies were performed to determine the extent and mode of cell mortality, cell cycle arrest and other in vitro parameters. In addition, L. donovani infected BALB/c mice were administered suramin and a host of immunological parameters determined to estimate the antileishmanial potency of the drug. Finally, isothermal titration calorimetry (ITC) and enzymatic assays were used to probe the interaction of the drug with one of its putative targets namely parasitic phosphoglycerate kinase (LmPGK). FINDINGS: The in vitro studies revealed the potential efficacy of suramin against the Leishmania parasite. This observation was further substantiated in the in vivo murine model, which demonstrated that upon suramin administration, the Leishmania infected BALB/c mice were able to reduce the parasitic burden and also generate the host protective immunological responses. ITC and enzyme assays confirmed the binding and consequent inhibition of LmPGK due to the drug. CONCLUSIONS/SIGNIFICANCE: All experiments affirmed the efficacy of suramin against L. donovani infection, which could possibly lead to its inclusion in the repertoire of drugs against VL.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

AIM: We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. METHODS: The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. RESULTS: EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. CONCLUSION: Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Curcumin inhibits prostate cancer by targeting PGK1 in the FOXD3/miR-143 axis.

PURPOSE: Curcumin is a potent antitumor agent. The objective of this study was to explore the interaction between curcumin and PGK1, an oncogene in the FOXD3/miR-143 axis, in prostate cancer therapy. METHODS: MiRNA microarray analysis was used to identify miRNAs upregulated by curcumin treatment. MiR-143 was dramatically upregulated by curcumin. Cells were treated with antimiR-143 in combination to curcumin, followed by examining cell viability and migration. Bioinformatics analysis was used to investigate target genes of miR-143. The interaction between miR-143 and PGK1 was evaluated with dual-luciferase assay. Since FOXD3 is important in the regulation of miR-143, we explored whether curcumin regulated FOXD3 expression. FOXD3 was also ectopically overexpressed to investigate its effects on curcumin's regulation of miR-143. RESULTS: Curcumin treatment significantly upregulated miR-143 and decreased prostate cancer cell proliferation and migration. Those effects were attenuated by anti-miR-143 transfection. Both miR-143 overexpression and curcumin treatment inhibited PGK1 expression and ectopic expression of PGK1 antagonized curcumin's antitumor effects. FOXD3 was upregulated by miR-143. Ectopic expression of FOXD3 synergized with curcumin in upregulating miR-143 expression. CONCLUSION: Curcumin inhibits prostate cancer by upregulating miR-143. PGK1 is downregulated by miR-143, and FOXD3 upregulation is essential for the antitumor effect of curcumin.

Terazosin activates Pgk1 and Hsp90 to promote stress resistance.

Drugs that can protect against organ damage are urgently needed, especially for diseases such as sepsis and brain stroke. We discovered that terazosin (TZ), a widely marketed α1-adrenergic receptor antagonist, alleviated organ damage and improved survival in rodent models of stroke and sepsis. Through combined studies of enzymology and X-ray crystallography, we discovered that TZ binds a new target, phosphoglycerate kinase 1 (Pgk1), and activates its enzymatic activity, probably through 2,4-diamino-6,7-dimethoxyisoquinoline's ability to promote ATP release from Pgk1. Mechanistically, the ATP generated from Pgk1 may enhance the chaperone activity of Hsp90, an ATPase known to associate with Pgk1. Upon activation, Hsp90 promotes multistress resistance. Our studies demonstrate that TZ has a new protein target, Pgk1, and reveal its corresponding biological effect. As a clinical drug, TZ may be quickly translated into treatments for diseases including stroke and sepsis.

Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs.

Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine L-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both D- and L-configuration. Human PGK demonstrated catalytic efficiencies in the 10(4)-10(5)M(-1)s(-1) range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in D- or L-configuration. In contrast, it was poorly active with natural pyrimidine D-nucleotides (less than 10(3)M(-1)s(-1)). Pyrimidine L-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their D-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, L-2'-deoxycytosine and L-2'-deoxythymidine.

Mapping oxidative DNA damage at nucleotide level.

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the il vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50mM H2O2, or purified genomic DNA was treated with 5 mM H2O2, 100 microM Ascorbate, and 50 microM, 100 microM, or 100 microM of Cu(II), Fe(II), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H2O2-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H2O2.

Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The C-terminal end is an integral part of the N-terminal domain. The consequences of the deletion of fifteen and three C-terminal amino acids on the conformational state and on the guanidine hydrochloride-induced and thermal unfolding of PGK were investigated by using near- and far-UV CD, tryptophan fluorescence, 1-anilinonaphthalene-8-sulfonic acid binding, accessibility to chemical modification, and differential scanning calorimetry. The results of these studies indicate that the conformations of both domains and of the interdomain region were altered by these deletions. In the absence of the 15-amino acid C-terminal peptide [delta(401-415)], the N-terminal domain exhibits several characteristics of a molten globule state, whereas the C-terminal domain retains native-like, although distinctly different, tertiary structure. Deletion of three C-terminal amino acids [delta(413-415)] also globally affects PGK conformation, although to a much lesser extent. Both C-terminal deletions resulted in a significant decrease in protein stability, as demonstrated by their increased susceptibility to guanidine-induced and thermal denaturation. These results suggest that the formation of a native tertiary fold of PGK requires the presence of a complete polypeptide chain.

Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo.

Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.

Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation.

The function of arginine 65, a conserved residue located at the periphery of the active site cleft in yeast 3-phosphoglycerate kinase (PGK), has been investigated by site-directed mutagenesis. Mutant enzymes with glutamine, serine and alanine at position 65 all have very similar kinetic properties. The maximum velocities, determined in the absence of sulfate anion, are approximately 100% higher than the Vmax of wild-type PGK. The Km values are increased 2- to 3-fold for ATP and 5- to 6-fold for 3-phosphoglycerate (3PG). These results demonstrate that arginine 65 is not essential for catalysis. In contrast to wild-type enzyme, the mutants are not activated by sulfate ions. In addition, steady-state kinetic experiments indicate that the mutants are no longer activated by high concentrations of either 3PG or ATP. The dissociation constants for anions were determined by spectral titrations of the R65Q mutant labeled with a chromophoric probe. The Kd for 3PG is increased 6-fold, as compared to wild-type PGK, whereas the Kd for ATP is essentially unchanged. The Kd for sulfate is decreased less than 2-fold. The suppression of substrate- and sulfate-dependent activation suggests that arginine 65 participates in the regulatory mechanism responsible for activation of the enzyme.