Rubén Martín-Escolano

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of New Arylamine Mannich Base-Type Derivatives

Chagas disease is a neglected tropical disease with 6-7 million people infected worldwide, and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity, and a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model results show that compounds 3, 4, 7, and 10 induced a remarkable decrease in parasitemia levels in acute phase and the parasitemia reactivation following immunosuppression, and curative rates were higher than with benznidazole. These high antiparasitic activities encourage us to propose these compounds as promising molecules for developing an easy to synthesize anti-Chagas agent.

An in vitro iron superoxide dismutase inhibitor decreases the parasitemia levels of Trypanosoma cruzi in BALB/c mouse model during acute phase

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by 1H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.

Library of Seleno-Compounds as Novel Agents against Leishmania Species

ABSTRACT The in vitro leishmanicidal activities of a series of 48 recently synthesized selenium derivatives against Leishmania infantum and Leishmania braziliensis parasites were tested using promastigotes and intracellular amastigote forms. The cytotoxicity of the tested compounds for J774.2 macrophage cells was also measured in order to establish their selectivity. Six of the tested compounds (compounds 8, 10, 11, 15, 45, and 48) showed selectivity indexes higher than those of the reference drug, meglumine antimonate (Glucantime), for both Leishmania species; in the case of L. braziliensis, compound 20 was also remarkably selective. Moreover, data on infection rates and amastigote numbers per macrophage showed that compounds 8, 10, 11, 15, 45, and 48 were the most active against both Leishmania species studied. The observed changes in the excretion product profile of parasites treated with these six compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe superoxide dismutase (Fe-SOD) in the two parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials, and straightforward synthesis make these compounds appropriate molecules for the development of affordable antileishmanicidal agents.

Synthesis and in vitro leishmanicidal activity of novel [1,2,3]triazolo[1,5-a]pyridine salts

Leishmaniasis remains a significant worldwide problem; it is of great interest to develop new drugs to fight this disease. Recently we described some [1,2,3]triazolo[1,5-a]pyridine compounds with significant leishmanicidal activity. The importance of water solubility in drug action made us realise that we could transform non charged triazolopyridines into charged analogues that could increase the degree of water solubility. With this objective we report here the synthesis of novel [1,2,3]triazolo[1,5-a]pyridinium salts 2–7 from triazolopyridines 1, and the study of their in vitro leishmanicidal activity. The activity was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, using promastigote and intracellular amastigote forms. The cytotoxicity of the tested compounds on J774.2 macrophage cells was also measured. Five of the tested compounds (2b, 4a, 4c, 6, 7d) showed selectivity indexes higher than those of the reference drug Glucantime for the three Leishmania species. Moreover, the data on infection rate and on amastigotes showed that these compounds are the most active against the three Leishmania species. The changes in the excretion product profiles of parasites treated with the compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe-SOD in the three parasite species considered whereas their impact on human CuZn-SOD was low.

Tetradentate polyamines as efficient metallodrugs for Chagas disease treatment in murine model.

A series of tetraamine-based compounds was prepared, and their trypanocidal effects against Trypanosoma cruzi and cytotoxicity were determined through the determination of IC50 values. In vivo assays were performed in mice, where parasitaemia levels were quantified by fresh blood examination and the assignment of a cure was determined by polymerase chain reaction and reactivation of blood parasitaemia levels after immunosuppression. The mechanisms of action were elucidated at metabolic and ultra-structural levels, by 1H NMR, Fe-SOD inhibition and TEM studies. The high-selectivity indexes observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. Compound 6 induced a remarkable decrease in the reactivation of parasitaemia after immunosuppression and curative rates of 33%. The experiments allowed us to select compound 6 as a promising candidate for treating Chagas disease, but a further high-level study should be considered to obtain an improved efficiency.

Second Generation of Mannich Base-Type Derivatives with in Vivo Activity against Trypanosoma cruzi

Chagas disease is a potentially life-threatening and neglected tropical disease caused by Trypanosoma cruzi. One of the most important challenges related to Chagas disease is the search for new, safe, effective, and affordable drugs since the current therapeutic arsenal is inadequate and insufficient. Here, we report a simple and cost-effective synthesis and the biological evaluation of the second generation of Mannich base-type derivatives. Compounds 7, 9, and 10 showed improved in vitro efficiency and lower toxicity than benznidazole, in addition to no genotoxicity; thus, they were applied in in vivo assays to assess their activity in both acute and chronic phases of the disease. Compound 10 presented a similar profile to benznidazole from the parasitological perspective but also yielded encouraging data, as no toxicity was observed. Moreover, compound 9 showed lower parasitaemia and higher curative rates than benznidazole, also with lower toxicity in both acute and chronic phases. Therefore, further studies should be considered to optimize compound 9 to promote its further preclinical evaluation.

Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability

Over a quarter million of protein phosphorylation sites have been identified so far, although the effects of site-specific phosphorylation on protein function and stability, as well as their possible impact in the phenotypic manifestation in genetic diseases are vastly unknown. We investigated here the effects of phosphorylating S82 in human NADP(H):quinone oxidoreductase 1, a representative example of disease-associated flavoprotein in which protein stability is coupled to the intracellular flavin levels. Additionally, the cancer-associated P187S polymorphism causes inactivation and destabilization of the enzyme. By using extensive in vitro and in silico characterization of phosphomimetic S82D mutations, we showed that S82D locally affected the flavin binding site of the wild-type (WT) and P187S proteins thus altering flavin binding affinity, conformational stability and aggregation propensity. Consequently, the phosphomimetic S82D may destabilize the WT protein intracellularly by promoting the formation of the degradation-prone apo-protein. Noteworthy, WT and P187S proteins respond differently to the phosphomimetic mutation in terms of intracellular stability, further supporting differences in molecular recognition of these two variants by the proteasomal degradation pathway. We propose that phosphorylation could have critical consequences on stability and function of human flavoproteins, important for our understanding of genotype-phenotype relationships in their related genetic diseases.

Synthesis and Biological in vitro and in vivo Evaluation of 2-(5-Nitroindazol-1-yl)ethylamines and Related Compounds as Potential Therapeutic Alternatives for Chagas Disease

Chagas disease, a neglected tropical disease caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially life‐threatening illness that affects 5–8 million people in Latin America, and more than 10 million people worldwide. It is characterized by an acute phase, which is partly resolved by the immune system, but then develops as a chronic disease without an effective treatment. There is an urgent need for new antiprotozoal agents, as the current standard therapeutic options based on benznidazole and nifurtimox are characterized by limited efficacy, toxicity, and frequent failures in treatment. In vitro and in vivo assays were used to identify some new low‐cost 5‐nitroindazoles as a potential antichagasic therapeutic alternative. Compound 16 (3‐benzyloxy‐5‐nitro‐1‐vinyl‐1H‐indazole) showed improved efficiency and lower toxicity than benznidazole in both in vitro and in vivo experiments, and its trypanocidal activity seems to be related to its effect at the mitochondrial level. Therefore, compound 16 is a promising candidate for the development of a new anti‐Chagas agent, and further preclinical evaluation should be considered.

In vitro antileishmanial activity and iron superoxide dismutase inhibition of arylamine Mannich base derivatives

Leishmaniasis is one of the worlds most neglected diseases, and it has a worldwide prevalence of 12 million. There are no effective human vaccines for its prevention, and treatment is hampered by outdated drugs. Therefore, research aiming at the development of new therapeutic tools to fight leishmaniasis remains a crucial goal today. With this purpose in mind, we present 20 arylaminoketone derivatives with a very interesting in vitro and in vivo efficacy against Trypanosoma cruzi that have now been studied against promastigote and amastigote forms of Leishmania infantum, Leishmania donovani and Leishmania braziliensis strains. Six out of the 20 Mannich base-type derivatives showed Selectivity Index between 39 and 2337 times higher in the amastigote form than the reference drug glucantime. These six derivatives affected the parasite infectivity rates; the result was lower parasite infectivity rates than glucantime tested at an IC25 dose. In addition, these derivatives were substantially more active against the three Leishmania species tested than glucantime. The mechanism of action of these compounds has been studied, showing a greater alteration in glucose catabolism and leading to greater levels of iron superoxide dismutase inhibition. These molecules could be potential candidates for leishmaniasis chemotherapy.

Synthesis and In Vitro Antiparasitic Activity of Novel Arylamine Mannich Base-Type Derivatives against Trypanosoma cruzi and Leishmania spp.

Chagas disease and Leishmaniasis are trypanosomatid diseases considered as neglected tropical diseases by the WHO. [...]