RESUMO
Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.
Assuntos
Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Leishmania/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Benzoquinonas/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/química , Leishmania/metabolismo , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Simulação de Acoplamento Molecular , Proteínas de Protozoários/metabolismo , Tripanossomicidas/químicaRESUMO
BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.
Assuntos
Isoquinolinas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Macrófagos/parasitologia , Animais , Leishmania braziliensis/ultraestrutura , Leishmania major/ultraestrutura , Leishmania mexicana/ultraestrutura , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos CBA , Microscopia Eletrônica de Transmissão , Testes de Sensibilidade Parasitária , Fatores de TempoRESUMO
BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.