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1.
Parasitology ; 151(5): 506-513, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38533610

RESUMO

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-ß-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.


Assuntos
Modelos Animais de Doenças , Leishmania mexicana , Leishmaniose Cutânea , Camundongos Endogâmicos BALB C , Animais , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Camundongos , Feminino , Masculino , Leishmania mexicana/efeitos dos fármacos , Tubercidina/farmacologia , Tubercidina/análogos & derivados , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Antiprotozoários/administração & dosagem , Macrófagos Peritoneais/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Leishmania/efeitos dos fármacos
2.
PLoS Negl Trop Dis ; 10(9): e0004972, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27606425

RESUMO

BACKGROUND: Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. CONCLUSIONS/SIGNIFICANCE: This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.


Assuntos
Antiparasitários/uso terapêutico , Resistência a Medicamentos/genética , Retículo Endoplasmático/genética , Leishmania major/genética , Leishmaniose/tratamento farmacológico , Tubercidina/uso terapêutico , Sequência de Aminoácidos , Animais , Linhagem Celular , Leishmania major/efeitos dos fármacos , Estrutura Terciária de Proteína , Fatores de Transcrição/genética
3.
PLoS One ; 7(9): e44282, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22957058

RESUMO

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl(6) and Br(8) substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.


Assuntos
Fosfatos/química , Purina-Núcleosídeo Fosforilase/química , Aciclovir/farmacologia , Adenosina/análogos & derivados , Adenosina/química , Bacillus subtilis/enzimologia , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Ganciclovir/farmacologia , Terapia Genética/métodos , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Neoplasias/genética , Neoplasias/terapia , Pró-Fármacos/química , Estrutura Quaternária de Proteína , Serina/química , Treonina/química , Tubercidina/farmacologia
4.
Parasitol Res ; 104(2): 223-8, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18787843

RESUMO

Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB-NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB-NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.


Assuntos
Antiparasitários/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Tioinosina/análogos & derivados , Tionucleotídeos/uso terapêutico , Tubercidina/uso terapêutico , Animais , Antiparasitários/farmacologia , Antiparasitários/toxicidade , Células Cultivadas , Quimioterapia Combinada , Inibidores Enzimáticos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Schistosoma mansoni/efeitos dos fármacos , Tioinosina/farmacologia , Tioinosina/uso terapêutico , Tionucleotídeos/farmacologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Tubercidina/farmacologia , Tubercidina/toxicidade
5.
Mol Biochem Parasitol ; 90(2): 505-11, 1997 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-9476797

RESUMO

The TOR gene (TOxic nucleoside Resistance gene) was mapped to a 2.3 kb fragment on the amplified DNA from tubercidin resistant Leishmania (TUB). This DNA fragment conferred upon wild type cells resistance to tubercidin, inosine dialdehyde, formycin A and B and allopurinol riboside and a reduced ability to accumulate purine nucleobases and nucleosides. These properties were characteristic of the parental TUB cells which carried the intact amplified DNA and have been hypothesized to be caused by a reduction in the activity of the multiple purine transporters within this organism. The TOR gene was found to be partially homologous to the rodent and human Oct-6/SCIP/Tst-1 gene. It lacked, however, the POU specific domain of this class of transcription factors and contained only the first two helices of the POU homeodomain. This truncated homeodomain was not required to confer resistance upon wild type cells to toxic nucleosides, suggesting that TOR was not a repressor with independent DNA binding capability.


Assuntos
Leishmania mexicana/genética , Peptídeos/genética , Peptídeos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alopurinol/análogos & derivados , Alopurinol/farmacologia , Sequência de Aminoácidos , Animais , Antiprotozoários/farmacologia , Transporte Biológico , Clonagem Molecular , Resistência a Medicamentos , Formicinas/farmacologia , Genes de Protozoários/genética , Humanos , Inosina/análogos & derivados , Inosina/farmacologia , Leishmania mexicana/química , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/metabolismo , Dados de Sequência Molecular , Fator 6 de Transcrição de Octâmero , Peptídeos/química , Purinas/metabolismo , Mapeamento por Restrição , Ribonucleosídeos/farmacologia , Fatores de Transcrição/química , Transformação Genética , Tubercidina/farmacologia
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