Drug repurposing against arabinosyl transferase (EmbC) of Mycobacterium tuberculosis: Essential dynamics and free energy minima based binding mechanics analysis.

Arabinosyl tranferases (embA, embB, embC) are the key enzymes responsible for biogenesis of arabinan domain of arabinogalactan (AG) and lipoarabinomannan (LAM), two major heteropolysaccharide constituents of the peculiar mycobacterial cell envelope. EmbC is predominantly responsible for LAM synthesis and has been commonly associated with Ethambutol resistance. We have screened the FDA library against EmbC to reposition a drug better than Ethambutol with higher binding affinity to Embc. High throughput virtual screening followed by extra precision docking using Glide gave two best leads i.e. Terlipressin and Amikacin with docking score of -11.39 kcal/mol and -10.71 kcal/mol, respectively. Binding mechanics of the selected drugs was elucidated through long range molecular dynamics simulations (100 ns) using binding free energy rescoring, essential dynamics and free energy minima based approaches, thus revealing the most stable binding modes of Terlipressin with EmbC. Our study establishes the EmbC binding potential of the repurposed drugs Terlipressin and Amikacin.

Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory.

RATIONALE: Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality. OBJECTIVES: To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. METHODS: Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined. MEASUREMENTS AND MAIN RESULTS: A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability. CONCLUSIONS: The GenoType MTBDRsl LPA is a rapid and reliable DST that can be easily incorporated into the diagnostic algorithm. This assay significantly improved diagnostic yield (P < 0.001) while simultaneously decreasing diagnostic delay for reporting second-line DST. The rapid dissemination of second-line DST results will guide initiation of appropriate treatment, thereby reducing transmission and improving treatment outcome.

Expression of the bifunctional suicide gene CDUPRT increases radiosensitization and bystander effect of 5-FC in prostate cancer cells.

PURPOSE: To test the hypothesis that, with 5-fluorocytosine (5-FC) treatment, the co-expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) can lead to greater radiosensitization and bystander effect than CD-expression alone. METHODS AND MATERIALS: R3327-AT cell lines stably expressing CD or CDUPRT were generated. The 5-FC and 5-FU cytotoxicity, and the radiosensitivity with/without 5-FC treatment, of these cells were evaluated under both aerobic and hypoxic conditions. The bystander effect was assessed by apoptosis staining and clonogenic survival. The pharmacokinetics of 5-FU and 5-FC metabolism was monitored in mice bearing CD- or CDUPRT-expressing tumors using 19F MR spectroscopy (MRS). RESULTS: CDUPRT-expressing cells were more sensitive to 5-FC and 5-FU than CD-expressing cells. CDUPRT-expression further enhanced the radiosensitizing effect of 5-FC, relative to that achieved by CD-expression alone. A 25-fold lower dose of 5-FC resulted in the same magnitude of radiosensitization in CDUPRT-expressing cells, relative to that in CD-expressing cells. The 5-FC cytotoxicity in co-cultures of parental cells mixed with 10-20% CDUPRT cells was similar to that in 100% CDUPRT cells. 19F MRS measurements showed that expression of CDUPRT leads to enhanced accumulation of fluorine nucleotide (FNuc), relative to that associated with CD-expression alone. CONCLUSION: Our study suggests that CDUPRT/5-FC strategy may be more effective than CD/5-FC, especially when used in combination with radiation.

Is prochloraz a potent synergist across aquatic species? A study on bacteria, daphnia, algae and higher plants.

Fungicides inhibiting the biosynthesis of ergostrol, such as the triazoles and imidazoles, have been shown to enhance the effect of insecticides on birds, mammals and invertebrates in the terrestrial environment. The synergy is proposed to be due to an effect on P450 monooxygenase enzymes active in pesticide metabolism in these organisms. Fungicides often enter the aquatic environment jointly with other pesticides. It is therefore possible that they could act as synergists also in the aquatic environment. In this study we tested the joint effect of the imidazole fungicide prochloraz together with the herbicides acifluorfen, diquat and terbuthylazine, the fungicide azoxystrobin and the insecticides chlorfenvinphos, dimethoate, and pirimicarb on the bacteria Vibrio fischeri (six binary mixtures), the crustacean Daphnia magna (four binary mixtures), the algae Pseudokirchneriella subcapitata (four binary mixtures) and the floating plant Lemna minor (three binary mixtures). All the binary mixtures were evaluated both in relation to the model of concentration addition (CA) and independent action (IA) using isobolograms. The study showed strong synergy in relation to CA between prochloraz and azoxystrobin, diquat and esfenvalerat on D. magna with sums of toxic units for the 50:50% effect mixture ( summation TU(50:50)) as low as 0.25. The mixture with dimethoate was however antagonistic with summation TU(50:50) of 2.04. Four out of the six mixtures testes on V. fisheri showed synergy in relation to CA, but for three of the mixtures the response could be explained by IA. Only the mixture with diquat showed synergy in relation to both IA and CA with summation TU(50:50) around 0.50. There was no significant synergy for any of the combinations tested on the plant and the algae species in relation to CA and only for diquat in the algae-test in relation to IA. Hence, prochloraz does synergise the effect of some pesticides in the aquatic environment, but not consistently across species. The organism most susceptible to synergy by prochloraz in this study was D. magna. Especially the combination with insecticides such as esfenvalerate, where the concentration needed to immobilize 50% of the daphnia was reduced from >3microgL(-1) to less than 0.5microgL(-1) when prochloraz was added, could be problematic as these concentrations are environmentally realistic. Furthermore, insecticides and ergostrol-biosynthesis-inhibitors (EBI-fungicides) are often applied together, and are therefore likely to co-exist in surface waters, enhancing the problem of the already very potent insecticides.

Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target.

The Mycobacterium tuberculosis pyrR gene (Rv1379) encodes a protein that regulates the expression of pyrimidine-nucleotide biosynthesis (pyr) genes in a UMP-dependent manner. Because pyrimidine biosynthesis is an essential step in the progression of TB, the gene product pyrR is an attractive antitubercular drug target. The 1.9 A native structure of Mtb pyrR determined by the TB Structural Genomics Consortium facilities in trigonal space group P3(1)21 is reported, with unit-cell parameters a = 66.64, c = 154.72 A at 120 K and two molecules in the asymmetric unit. The three-dimensional structure and residual uracil phosphoribosyltransferase activity point to a common PRTase ancestor for pyrR. However, while PRPP- and UMP-binding sites have been retained in Mtb pyrR, a distinct dimer interaction among subunits creates a deep positively charged cleft capable of binding pyr mRNA. In silico screening of pyrimidine-nucleoside analogs has revealed a number of potential lead compounds that, if bound to Mtb pyrR, could facilitate transcriptional attenuation, particularly cyclopentenyl nucleosides.

Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP.

Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl alpha-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95 degrees C than at 37 degrees C, but the functional half-life was short at 95 degrees C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20-fold without much effect on K(m) for the substrates. The concentration of GTP required for half-maximal activation was about 80 microM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase from S. shibatae is an allosteric enzyme which is activated by a purine nucleotide and inhibited by a pyrimidine nucleotide as seen for several enzymes in the pyrimidine nucleotide biosynthetic pathway of Escherichia coli, but not observed before for any phosphoribosyltransferase.

Probing the active site of Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase using covalent modification of cysteine residues.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of Tritrichomonas foetus was inactivated by the thiol reagents iodoacetate and 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). Iodoacetate inactivates the enzyme in a time-dependent and concentration-dependent manner that follows pseudo-first-order kinetics. However, the observation that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in the catalytic activity of the enzyme. Nbs2 caused 50% inactivation rapidly, which was followed by gradual modifications of an additional three cysteine residues leading to complete enzyme inactivation. Analysis of the inactivation using the method developed by Tsou (1962) revealed that modification of two cysteine residues by Nbs2 is sufficient to impair the HGXPRTase activity. Tryptic digestion of HGXPRTase labeled with iodo[2-14C]acetic acid, followed by fractionation of the digest by HPLC and sequence analysis of the labeled peptides allowed the identification of Cys71, Cys129, Cys132, and Cys148 as the reactive cysteine residues. GMP and 5-phosphoribosyl-1-diphosphate provided complete protection against HGXPRTase inactivation by iodoacetate and against carboxymethylation of Cys129, Cys132, and Cys148, Cys71 was not protected by either substrate against iodoacetate, but its carboxymethylation caused no loss in enzyme activity either. There was also no substrate protection of Cys71 against Nbs2, which, however, caused 50% inactivation of the enzyme. Replacing the thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery of the enzyme activity, which indicates that a steric hindrance at the active site was introduced by Nbs but removed by cyanide. Thus, our results demonstrate that although the reactive cysteine residues in HGXPRTase are not directly involved in the catalytic activity, modification of cysteine residues 129, 132, and 148 by iodoacetate or Nbs2 hinders substrate binding which can, in turn, protect the cysteine residues from modifications. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brought about by substrate binding.

Selective inhibition of spontaneous pulmonary metastasis of Lewis lung carcinoma by 5'-deoxy-5-fluorouridine.

5'-deoxy-5-fluorouridine (5'-FUdR) is a cytostatic that is biotransformed to 5-fluorouracil (5-FUra) by pyrimidine nucleoside phosphorylase (PyNPase), the expression of which is up-regulated by tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interferon gamma (IFN gamma). In Lewis lung carcinoma (LLC) cell cultures, these inflammatory cytokines up-regulated the expression of type-IV collagenase, metastatic factor, as well as PyNPase and consequently enhanced the antiproliferative activity of 5'-FUdR. However, the activity of 5-FUra was not enhanced. It appears that 5'-FUdR selectively kills highly metastatic cells which are exposed to these intrinsic cytokines in tumor tissues, because of their high PyNPase activity. In fact, 5'-FUdR inhibited the spontaneous metastasis of LLC from the s.c. inoculation site to the lung. When 5'-FUdR was given during the process of metastasis it greatly reduced the number of tumor nodules in the lung even at doses 46 times lower than those inhibiting the primary tumor growth. In addition, 5'-FUdR, but not 5-FUra, lowered type-IV collagenase levels in the tumors at the low dose showing only anti-metastatic activity. On the other hand, 5-FUra showed anti-metastatic activity at doses similar to or only several times lower than those inhibiting the primary tumor growth.

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans.

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeast Candida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg++ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. 'Ping pong' mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (Km = 70.5 and 186 microM for uracil and PRPP, respectively) as compared with those of E. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.

Effect of non-steroidal anti-inflammatory drugs (NSAIDS) on glycosyltransferase activity from human osteoarthritic cartilage.

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the activity of glycosyltransferases required for the synthesis of the polysaccharide chains of proteoglycans, was studied in human osteoarthritic cartilage in vitro. Using exogenous acceptors, salicylate and indomethacin suppressed the activity of glucuronyl- and xylosyltransferases in a concentration-dependent manner, but had little effect on N-acetylgalactosaminyl- and galactosyltransferases. When used at a concentration derived from the values found in the synovial fluid, salicylate, indomethacin and chloroquine significantly suppressed the activity of glucuronyl- and xylosyltransferases, while tiaprofenic acid, paracetamol (acetaminophen), floctafenine, ketoprofen, ibuprofen and tenoxicam had no effect on the enzymes. An alteration of some glycosyltransferases could explain the reported suppressive effect of some NSAIDs on cartilage proteoglycan synthesis.