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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 hypoxanthine (A) and guanine (B) with Mpn HPRT. Kinetic parameters for Hx and Gua were determined by using the DE81 filter paper assay with [3H]-Hx and [3H]-Gua as the labelled substrates as described in the experimental procedures. Data are from at least three independent measurements and are presented as mean ± standard deviation (SD).
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Figure 2: Substrate saturation curves of hypoxanthine (A) and guanine (B) with Mpn HPRT. Kinetic parameters for Hx and Gua were determined by using the DE81 filter paper assay with [3H]-Hx and [3H]-Gua as the labelled substrates as described in the experimental procedures. Data are from at least three independent measurements and are presented as mean ± standard deviation (SD).

Mentions: The purified Mpn HPRT used both hypoxanthine (Hx) and guanine (Gua) as substrates but not adenine or uracil. With Hx as substrate the reaction was linear with time for up to 25 min and the substrate saturation curve was hyperbolic, which indicated that the enzyme followed Michaelis–Menten kinetics with a Km value of 100.1 ± 6.5 μM and Vmax value of 15.8 ± 0.8 μmol min-1 mg-1 (Figure 2A). However, with Gua as a substrate, the reverse reaction rate was very high and the reaction reached equilibrium in less than 5 min under the same conditions used for Hx. Therefore, the kinetic study with Gua was conducted differently as described in the experimental procedures. Substrate saturation for Gua exhibited a biphasic curve and therefore, data was fitted using the Hill equation. The Vmax value was 2.7 ± 0.1 μmol min-1 mg-1 and S0.5 was 107.6 ± 6.2 μM with a Hill coefficient of 3.5 (Figure 2B), indicating positive cooperativity with Gua binding.


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 hypoxanthine (A) and guanine (B) with Mpn HPRT. Kinetic parameters for Hx and Gua were determined by using the DE81 filter paper assay with [3H]-Hx and [3H]-Gua as the labelled substrates as described in the experimental procedures. Data are from at least three independent measurements and are presented as mean ± standard deviation (SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Substrate saturation curves of hypoxanthine (A) and guanine (B) with Mpn HPRT. Kinetic parameters for Hx and Gua were determined by using the DE81 filter paper assay with [3H]-Hx and [3H]-Gua as the labelled substrates as described in the experimental procedures. Data are from at least three independent measurements and are presented as mean ± standard deviation (SD).
Mentions: The purified Mpn HPRT used both hypoxanthine (Hx) and guanine (Gua) as substrates but not adenine or uracil. With Hx as substrate the reaction was linear with time for up to 25 min and the substrate saturation curve was hyperbolic, which indicated that the enzyme followed Michaelis–Menten kinetics with a Km value of 100.1 ± 6.5 μM and Vmax value of 15.8 ± 0.8 μmol min-1 mg-1 (Figure 2A). However, with Gua as a substrate, the reverse reaction rate was very high and the reaction reached equilibrium in less than 5 min under the same conditions used for Hx. Therefore, the kinetic study with Gua was conducted differently as described in the experimental procedures. Substrate saturation for Gua exhibited a biphasic curve and therefore, data was fitted using the Hill equation. The Vmax value was 2.7 ± 0.1 μmol min-1 mg-1 and S0.5 was 107.6 ± 6.2 μM with a Hill coefficient of 3.5 (Figure 2B), indicating positive cooperativity with Gua binding.

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