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Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs.

Sun R, Wang L - BMC Microbiol. (2013)

Bottom Line: Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth.The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition.We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden.

ABSTRACT

Background: Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.

Results: Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml(-1), whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml(-1). In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.

Conclusion: We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.

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Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ-32P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.
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Figure 3: Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ-32P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.

Mentions: TFT is a known substrate of human TK1, and has been used as an antiviral and anticancer drug. We found that TFT strongly inhibited Mpn growth, suggesting that Mpn TK may have a role in growth inhibition caused by TFT. The mechanism of inhibition by TFT and 5-fluorodeoxyuridine (5FdU) was proposed to be linked to inhibition of TS [38]. However, we observed that TFT and 5FdU inhibited both the wild type and the thyA mutant strains to similar extents, suggesting that the mechanism of inhibition is unlikely to be solely by inhibition of TS activity. Therefore, we sought to characterize TFT phosphorylation by Mycoplasma TK and compared this with the human enzymes. Kinetic studies with TFT were performed with purified recombinant human TK1 (a cytosolic enzyme), TK2 (a mitochondrial isoenzyme), and Ureaplasma TK. Because Ureaplasma TK and Mpn TK share 46% sequence identity and they also share 36% respective 32% sequence identity to human TK1 [30,39], and Mpn TK is not available. The phosphorylation of TFT by human TK1, TK2, and Ureaplasma TK followed Michaelis-Menten kinetics and the Km values for the three enzymes were in the same range, while the kcat values varied between the three enzymes (Figure 3). Ureaplasma TK had the highest kcat value and human TK2 had the lowest kcat value (Table 4). However, the overall efficiency was highest with human TK1 and lowest with human TK2 (Table 4).


Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs.

Sun R, Wang L - BMC Microbiol. (2013)

Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ-32P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750255&req=5

Figure 3: Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ-32P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.
Mentions: TFT is a known substrate of human TK1, and has been used as an antiviral and anticancer drug. We found that TFT strongly inhibited Mpn growth, suggesting that Mpn TK may have a role in growth inhibition caused by TFT. The mechanism of inhibition by TFT and 5-fluorodeoxyuridine (5FdU) was proposed to be linked to inhibition of TS [38]. However, we observed that TFT and 5FdU inhibited both the wild type and the thyA mutant strains to similar extents, suggesting that the mechanism of inhibition is unlikely to be solely by inhibition of TS activity. Therefore, we sought to characterize TFT phosphorylation by Mycoplasma TK and compared this with the human enzymes. Kinetic studies with TFT were performed with purified recombinant human TK1 (a cytosolic enzyme), TK2 (a mitochondrial isoenzyme), and Ureaplasma TK. Because Ureaplasma TK and Mpn TK share 46% sequence identity and they also share 36% respective 32% sequence identity to human TK1 [30,39], and Mpn TK is not available. The phosphorylation of TFT by human TK1, TK2, and Ureaplasma TK followed Michaelis-Menten kinetics and the Km values for the three enzymes were in the same range, while the kcat values varied between the three enzymes (Figure 3). Ureaplasma TK had the highest kcat value and human TK2 had the lowest kcat value (Table 4). However, the overall efficiency was highest with human TK1 and lowest with human TK2 (Table 4).

Bottom Line: Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth.The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition.We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden.

ABSTRACT

Background: Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.

Results: Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml(-1), whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml(-1). In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.

Conclusion: We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.

Show MeSH
Related in: MedlinePlus