As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.
African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and targetbased approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.
The antileishmanial activity of a series of bis-pyridinium derivatives that are analogues of pentamidine have been investigated, and all compounds assayed were found to display activity against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major, with 50% effective concentrations (EC 50 s) lower than 1 M in most cases. The majority of compounds showed similar behavior in both Leishmania species, being slightly more active against L. major amastigotes. However, compound VGP-106 {1,1=-(biphenyl-4,4=-diylmethylene)bis[4-(4-bromo-N-methylanilino)pyridinium] dibromide} exhibited significantly higher activity against L. donovani amastigotes (EC 50 , 0.86 ؎ 0.46 M) with a lower toxicity in THP-1 cells (EC 50 , 206.54 ؎ 9.89 M). As such, VGP-106 was chosen as a representative compound to further elucidate the mode of action of this family of inhibitors in promastigote forms of L. donovani. We have determined that uptake of VGP-106 in Leishmania is a temperature-independent process, suggesting that the compound crosses the parasite membrane by diffusion. Transmission electron microscopy analysis showed a severe mitochondrial swelling in parasites treated with compound VGP-106, which induces hyperpolarization of the mitochondrial membrane potential and a significant decrease of intracellular free ATP levels due to the inhibition of ATP synthesis. Additionally, we have confirmed that VGP-106 induces mitochondrial ROS production and an increase in intracellular Ca 2؉ levels. All these molecular events can activate the apoptotic process in Leishmania; however, propidium iodide assays gave no indication of DNA fragmentation. These results underline the potency of compound VGP-106, which may represent a new avenue for the development of novel antileishmanial compounds.
Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.
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