Marcadores de resistencia en Leishmania: Susceptibilidad in vitro a drogas leishmanicidas vs retención de calceina en aislados de pacientes venezolanos con Leishmaniasis Cutánea Difusa

La identificación de parásitos quimio-resistentes con marcadorescelulares fáciles de cuantificar, no ha sido descrita en Leishmania.En este trabajo comparamos la susceptibilidad in vitro a drogasleishmanicidas vs. la retención intracelular de calceina, unamedida indirecta de la actividad de los transportadores MDR1y MRP1, en parásitos aislados de pacientes con LeishmaniasisCutánea Difusa, con fracaso terapéutico a Glucantime. Estos hallazgoslos comparamos con resultados obtenidos en especiesde referencia de la Organización Mundial de la Salud. Encontramosque el ensayo de susceptibilidad a drogas leishmanicidas,es fácil, rápido y reproducible, mas no necesariamente predictivode fenotipo quimio-resistente. Adicionalmente, la detecciónfluorométrica de la retención de calceina permitió diferenciar alos aislados de acuerdo a la funcionalidad de los transportadoresABC. Estos resultados sugieren que de validarse su utilidad, estametodología podría incorporarse al esquema de diagnóstico ytratamiento de la leishmaniasis, como predictiva del éxito de laterapia leishmanicida

Challenges in drug discovery and description targeting Leishmania spp.: enzymes, structural proteins, and transporters

Leishmaniasis is a complex tropical disease caused by the protozoan parasite Leishmania spp. Classical chemotherapy includes pentavalent antimonial; however, pentamidine, amphotericin B, and miltefosine have been used. Chemo-resistance remains a risk for successful treatment; thus, target identification and development of selective drugs remain a priority in controlling this disease. Evidence indicates that 6-phosphogluconate dehydrogenase (6PGDH), β-tubulin protein, and ATP-dependent transporters (ABCs-T) are potential targets to be addressed. The pentose phosphate pathway key enzyme 6PGDH is essential for protecting kinetoplastid parasites from oxidative stress and differs from the mammalian host enzyme (<35% AA sequence identity). An optimized 3D model has been used to select high -affinity compounds toward the enzyme through virtual screening and subsequent evaluation in vivo. In kinetoplasts, tubulins are highly conserved proteins essential for microtubule formation. However, compared to other eukaryotic cells, there is a differential susceptibility of kinetoplastid proteins to antimicrotubular agents, e.g., colchicine resistance. A comparison of experimental models between bovine and Leishmania β-tubulin protein allowed us to identify structural modification products of various amino acid substitutions, which hinder the access of colchicine to the binding pocket of the Leishmania protein. Similar changes are found in the β-tubulin sequence of other kinetoplastids such as Trypanosoma cruzi, T. brucei, and T. evansi. The evaluation of the β-tubulin protein as a therapeutic target and the compounds that selectively interact with it was carried out using in silico approaches. The activities of ABC-Transporters are related to the main causes of drug resistance, and the collected evidence suggests that for the ABC-Transporter blocker glibenclamide, there is a: (1) differential susceptibility of Leishmania spp. vs. macrophages; (2) greater susceptibility of axenic amastigotes vs. promastigotes; and (3) glibenclamide-glucantime synergistic drug interaction in macrophage-infected cells. Herein, we discuss the potential value of designing ABC-Transporter blockers for combination therapy in the treatment of leishmaniasis. The examples mentioned above highlight the importance of the search for new therapeutic targets and pharmacophores for the design of alternative treatments for the disease

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of “resistance” related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined

Leishmanicidal activity of betulin derivatives in Leishmania amazonensis; Effect on plasma and mitochondrial membrane potential, and macrophage nitric oxide and superoxide production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI(50) 45 mu M, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p <0.05) and the mitochondrial membrane potential (p <0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.Peer reviewe

Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis

Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis