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Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin.

Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H, Rottenberg ME - PLoS Negl Trop Dis (2009)

Bottom Line: Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment.Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites.Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/principal findings: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/significance: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

No MeSH data available.


Related in: MedlinePlus

Sequencing of candidate genes.A) The PCR DNA amplification from T.b. brucei pleomorphic AnTat1.1 or monomorphic Lister 427 parasites is shown. Primers recognizing the 5′ and 3′ UTR and the 5′ and 3′ ends of the ORF of the TbAT1 gene were used. B) A single base insertion at position 61 and a single base deletion at position 631, causing the synthesis of a non-functional truncated gene product in clones of the resistant pleomorphic strain but not in the parental or in the genome annotated strain (Lister 427). C) TbAT1−/− and parental parasites were incubated with different doses of cordycepin for 70 h, when WST-1 reagent added. Two h after incubation, parasite viability was measured. The fraction of parasites in comparison to untreated controls is depicted.
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pntd-0000495-g010: Sequencing of candidate genes.A) The PCR DNA amplification from T.b. brucei pleomorphic AnTat1.1 or monomorphic Lister 427 parasites is shown. Primers recognizing the 5′ and 3′ UTR and the 5′ and 3′ ends of the ORF of the TbAT1 gene were used. B) A single base insertion at position 61 and a single base deletion at position 631, causing the synthesis of a non-functional truncated gene product in clones of the resistant pleomorphic strain but not in the parental or in the genome annotated strain (Lister 427). C) TbAT1−/− and parental parasites were incubated with different doses of cordycepin for 70 h, when WST-1 reagent added. Two h after incubation, parasite viability was measured. The fraction of parasites in comparison to untreated controls is depicted.

Mentions: While several polymorphisms were found, no deleterious mutations were found in the genes sequenced except for TbAT1. TbAT1 is a nucleoside adenosine transporter present in the genome of T. brucei as a 1392 bp single copy gene where both alleles have been found to be almost identical. We found two mutations in the cordycepin-resistant AnTat1.1 strain TbAT1 gene that were not present in the respective parental DNA. There was a single base insertion at position 61 and a single base deletion at position 631, that could cause synthesis of a non-functional truncated gene product were found (Figure 10B). Five out of 12 sequenced colonies presented the one base insertion while the other seven carried the deletion, both groups with a read coverage of 15×, which confirmed the mutations. We assume that these two sequences detected correspond to the two alleles of TbAT1 carrying different mutations but rendering the same non-functional transporter protein.


Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin.

Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H, Rottenberg ME - PLoS Negl Trop Dis (2009)

Sequencing of candidate genes.A) The PCR DNA amplification from T.b. brucei pleomorphic AnTat1.1 or monomorphic Lister 427 parasites is shown. Primers recognizing the 5′ and 3′ UTR and the 5′ and 3′ ends of the ORF of the TbAT1 gene were used. B) A single base insertion at position 61 and a single base deletion at position 631, causing the synthesis of a non-functional truncated gene product in clones of the resistant pleomorphic strain but not in the parental or in the genome annotated strain (Lister 427). C) TbAT1−/− and parental parasites were incubated with different doses of cordycepin for 70 h, when WST-1 reagent added. Two h after incubation, parasite viability was measured. The fraction of parasites in comparison to untreated controls is depicted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713411&req=5

pntd-0000495-g010: Sequencing of candidate genes.A) The PCR DNA amplification from T.b. brucei pleomorphic AnTat1.1 or monomorphic Lister 427 parasites is shown. Primers recognizing the 5′ and 3′ UTR and the 5′ and 3′ ends of the ORF of the TbAT1 gene were used. B) A single base insertion at position 61 and a single base deletion at position 631, causing the synthesis of a non-functional truncated gene product in clones of the resistant pleomorphic strain but not in the parental or in the genome annotated strain (Lister 427). C) TbAT1−/− and parental parasites were incubated with different doses of cordycepin for 70 h, when WST-1 reagent added. Two h after incubation, parasite viability was measured. The fraction of parasites in comparison to untreated controls is depicted.
Mentions: While several polymorphisms were found, no deleterious mutations were found in the genes sequenced except for TbAT1. TbAT1 is a nucleoside adenosine transporter present in the genome of T. brucei as a 1392 bp single copy gene where both alleles have been found to be almost identical. We found two mutations in the cordycepin-resistant AnTat1.1 strain TbAT1 gene that were not present in the respective parental DNA. There was a single base insertion at position 61 and a single base deletion at position 631, that could cause synthesis of a non-functional truncated gene product were found (Figure 10B). Five out of 12 sequenced colonies presented the one base insertion while the other seven carried the deletion, both groups with a read coverage of 15×, which confirmed the mutations. We assume that these two sequences detected correspond to the two alleles of TbAT1 carrying different mutations but rendering the same non-functional transporter protein.

Bottom Line: Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment.Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites.Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/principal findings: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/significance: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

No MeSH data available.


Related in: MedlinePlus