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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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Schematic overview of M. pneumoniae nucleotide biosynthesis. Hx, hypoxanthine; Gua, guanine; Ura, uracil; Thy, thymine; dT, thymidine; dA, deoxyadenosine; dC deoxycytidine; dG, deoxyguanosine; PRPP, phosphoribosyl pyrophosphate; NMP, nucleoside monophosphate; NDP, nucleoside diphosphate, NTP, nucleoside triphosphate; dNDP, deoxynucleoside diphosphate; dNTP, deoxynucleoside triphosphate; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; 5FdU-MP, 5-fluorodeoxyuridine monophosphate; 5FdU-TP, 5-fluorodeoxyuridine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdC-TP, gemcitabine triphosphate; 6-TG, 6-thioguanine; 6-TG-TP, 6-thioguanine triphosphate. Enzymes: hpt, hypoxanthine guanine phosphoribosyl transferase (MPN672); apt, adenine phosphoribosyl transferase (MPN395); upp, uracil phosphoribosyl transferase (MPN033); deoA, thymidine phosphorylase (MPN064); tdk, thymidine kinase (MPN044); thyA, thymidylate synthase (MPN320); tmk, thymidylate kinase (MPN006); adk, adenylate kinase (MPN185); gmk, guanylate kinase (MPN246); cmk, cytidylate kinase (MPN476); nrdE/nrdF, ribonucleotide reductase (MPN322 and MPN324); pyrH, uridylate kinase (MPN632); deoxyadenosine kinase (MPN386). I = inhibition.
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Figure 4: Schematic overview of M. pneumoniae nucleotide biosynthesis. Hx, hypoxanthine; Gua, guanine; Ura, uracil; Thy, thymine; dT, thymidine; dA, deoxyadenosine; dC deoxycytidine; dG, deoxyguanosine; PRPP, phosphoribosyl pyrophosphate; NMP, nucleoside monophosphate; NDP, nucleoside diphosphate, NTP, nucleoside triphosphate; dNDP, deoxynucleoside diphosphate; dNTP, deoxynucleoside triphosphate; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; 5FdU-MP, 5-fluorodeoxyuridine monophosphate; 5FdU-TP, 5-fluorodeoxyuridine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdC-TP, gemcitabine triphosphate; 6-TG, 6-thioguanine; 6-TG-TP, 6-thioguanine triphosphate. Enzymes: hpt, hypoxanthine guanine phosphoribosyl transferase (MPN672); apt, adenine phosphoribosyl transferase (MPN395); upp, uracil phosphoribosyl transferase (MPN033); deoA, thymidine phosphorylase (MPN064); tdk, thymidine kinase (MPN044); thyA, thymidylate synthase (MPN320); tmk, thymidylate kinase (MPN006); adk, adenylate kinase (MPN185); gmk, guanylate kinase (MPN246); cmk, cytidylate kinase (MPN476); nrdE/nrdF, ribonucleotide reductase (MPN322 and MPN324); pyrH, uridylate kinase (MPN632); deoxyadenosine kinase (MPN386). I = inhibition.

Mentions: Mycoplasmas differ from their hosts in the biosynthesis of precursors for DNA and RNA because they cannot synthesize purine and pyrimidine bases de novo. Therefore, they rely totally on the salvage pathway for nucleotide biosynthesis (depicted in Figure 4). Purine bases such as Hx, Gua, and Ade are recycled by HPRT and adenine phosphoribosyl transferase, whereas the pyrimidine base, uracil is salvaged by uracil phosphoribosyl transferase [31,32]. The salvage of deoxynucleosides is catalyzed by deoxynucleoside kinases, including TK and deoxyadenosine/deoxyguanosine kinase [29]. The deoxynucleoside monophosphates are further phosphorylated to their corresponding triphosphates and are used as precursors for DNA synthesis. The ribonucleoside monophosphates are further phosphorylated to their triphosphate forms, and are then incorporated into RNA, or the diphosphate forms can be reduced by ribonucleotide reductase to produce precursors for DNA synthesis (Figure 4). Of 17 genes involved in nucleotide biosynthesis, 15 are essential [33,34]. Therefore, it has been suggested that this pathway may be a therapeutic target for future development of antibiotics [42].


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

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

Schematic overview of M. pneumoniae nucleotide biosynthesis. Hx, hypoxanthine; Gua, guanine; Ura, uracil; Thy, thymine; dT, thymidine; dA, deoxyadenosine; dC deoxycytidine; dG, deoxyguanosine; PRPP, phosphoribosyl pyrophosphate; NMP, nucleoside monophosphate; NDP, nucleoside diphosphate, NTP, nucleoside triphosphate; dNDP, deoxynucleoside diphosphate; dNTP, deoxynucleoside triphosphate; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; 5FdU-MP, 5-fluorodeoxyuridine monophosphate; 5FdU-TP, 5-fluorodeoxyuridine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdC-TP, gemcitabine triphosphate; 6-TG, 6-thioguanine; 6-TG-TP, 6-thioguanine triphosphate. Enzymes: hpt, hypoxanthine guanine phosphoribosyl transferase (MPN672); apt, adenine phosphoribosyl transferase (MPN395); upp, uracil phosphoribosyl transferase (MPN033); deoA, thymidine phosphorylase (MPN064); tdk, thymidine kinase (MPN044); thyA, thymidylate synthase (MPN320); tmk, thymidylate kinase (MPN006); adk, adenylate kinase (MPN185); gmk, guanylate kinase (MPN246); cmk, cytidylate kinase (MPN476); nrdE/nrdF, ribonucleotide reductase (MPN322 and MPN324); pyrH, uridylate kinase (MPN632); deoxyadenosine kinase (MPN386). I = inhibition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Schematic overview of M. pneumoniae nucleotide biosynthesis. Hx, hypoxanthine; Gua, guanine; Ura, uracil; Thy, thymine; dT, thymidine; dA, deoxyadenosine; dC deoxycytidine; dG, deoxyguanosine; PRPP, phosphoribosyl pyrophosphate; NMP, nucleoside monophosphate; NDP, nucleoside diphosphate, NTP, nucleoside triphosphate; dNDP, deoxynucleoside diphosphate; dNTP, deoxynucleoside triphosphate; TFT, trifluorothymidine; TFT-MP, trifluorothymidine monophosphate; TFT-TP, trifluorothymidine triphosphate; 5FdU-MP, 5-fluorodeoxyuridine monophosphate; 5FdU-TP, 5-fluorodeoxyuridine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdC-TP, gemcitabine triphosphate; 6-TG, 6-thioguanine; 6-TG-TP, 6-thioguanine triphosphate. Enzymes: hpt, hypoxanthine guanine phosphoribosyl transferase (MPN672); apt, adenine phosphoribosyl transferase (MPN395); upp, uracil phosphoribosyl transferase (MPN033); deoA, thymidine phosphorylase (MPN064); tdk, thymidine kinase (MPN044); thyA, thymidylate synthase (MPN320); tmk, thymidylate kinase (MPN006); adk, adenylate kinase (MPN185); gmk, guanylate kinase (MPN246); cmk, cytidylate kinase (MPN476); nrdE/nrdF, ribonucleotide reductase (MPN322 and MPN324); pyrH, uridylate kinase (MPN632); deoxyadenosine kinase (MPN386). I = inhibition.
Mentions: Mycoplasmas differ from their hosts in the biosynthesis of precursors for DNA and RNA because they cannot synthesize purine and pyrimidine bases de novo. Therefore, they rely totally on the salvage pathway for nucleotide biosynthesis (depicted in Figure 4). Purine bases such as Hx, Gua, and Ade are recycled by HPRT and adenine phosphoribosyl transferase, whereas the pyrimidine base, uracil is salvaged by uracil phosphoribosyl transferase [31,32]. The salvage of deoxynucleosides is catalyzed by deoxynucleoside kinases, including TK and deoxyadenosine/deoxyguanosine kinase [29]. The deoxynucleoside monophosphates are further phosphorylated to their corresponding triphosphates and are used as precursors for DNA synthesis. The ribonucleoside monophosphates are further phosphorylated to their triphosphate forms, and are then incorporated into RNA, or the diphosphate forms can be reduced by ribonucleotide reductase to produce precursors for DNA synthesis (Figure 4). Of 17 genes involved in nucleotide biosynthesis, 15 are essential [33,34]. Therefore, it has been suggested that this pathway may be a therapeutic target for future development of antibiotics [42].

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