Miransertib (ARQ 092), an orally-available, selective Akt inhibitor is effective against Leishmania.
ABSTRACT: Leishmaniasis is amongst the most important neglected diseases, afflicting more than 12 million people in 88 countries. There is an urgent need for safe orally bioavailable and cost-effective drugs for the treatment of leishmaniasis. It has recently been shown that Leishmania activates host macrophage serine/threonine kinase Akt, to promote survival of both parasites and infected cells. Here, we sought to evaluate a compound, Miransertib (ARQ 092), an orally bioavailable and selective allosteric Akt inhibitor currently in clinical trials for patients with PI3K/Akt-driven tumors or Proteus syndrome. Miransertib was tested against Leishmania donovani and Leishmania amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Cultured promastigotes were susceptible to Miransertib. In addition, Miransertib was markedly effective against intracellular amastigotes of L. donovani or L. amazonensis-infected macrophages. Miransertib also enhanced mTOR dependent autophagy in Leishmania-infected macrophages, which may represent one mechanism of Miransertib-mediated killing of intracellular Leishmania. Whereas parasite clearance in the spleen of mice infected with L. donovani and treated with Miransertib was comparable to that when treated with miltefosine, Miransertib caused a greater reduction in the parasite load in the liver. In the cutaneous leishmaniasis infection model, lesions were reduced by 40% as compared to mock treated mice. Together, these results provide direct evidence to support the conclusion that Miransertib is an excellent lead compound for the development of a new oral drug therapy for visceral and cutaneous leishmaniasis.
Project description:Paromomycin is an aminoglycoside antibiotic approved in 2006 for the treatment of visceral leishmaniasis caused by Leishmania donovani in Southeast Asia. Although this drug is not approved for the treatment of visceral and cutaneous leishmaniasis in Brazil, it is urgent and necessary to evaluate the potential of this drug as alternative for the treatment against species responsible for these clinical forms of the disease. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse cutaneous leishmaniasis. The diffuse cutaneous form of the disease is difficult to treat and frequent relapses are reported, mainly when the treatment is interrupted. Here, we evaluated paromomycin susceptibility in vitro of a L. amazonensis clinical isolate from a patient with cutaneous leishmaniasis and the reference strain L. amazonensis M2269, as well as its in vivo efficacy in a murine experimental model. Although never exposed to paromomycin, a significant differential susceptibility between these two lines was found. Paromomycin was highly active in vitro against the clinical isolate in both forms of the parasite, while its activity against the reference strain was less active. In vivo studies in mice infected with each one of these lines demonstrated that paromomycin reduces lesion size and parasite burden and a direct correlation between the susceptibility in vitro and the effectiveness of this drug in vivo was found. Our findings indicate that paromomycin efficacy in vivo is dependent on intrinsic susceptibility of the parasite. Beyond that, this study contributes for the evaluation of the potential use of paromomycin in chemotherapy of cutaneous leishmaniasis in Brazil caused by L. amazonensis.
Project description:Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.
Project description:Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.
Project description:Leishmania parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo, and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.
Project description:We conducted a molecular study of parasite sequences from a cohort of cutaneous leishmaniasis patients in Himachal Pradesh, India. Results revealed atypical cutaneous disease caused by Leishmania donovani parasites. L. donovani variants causing cutaneous manifestations in this region are different from those causing visceral leishmaniasis in northeastern India.
Project description:Himachal Pradesh in India is a newer endemic state with co-existence of cutaneous and visceral leishmaniasis. The cutaneous leishmaniasis cases are on an increase in the region and reported to be unusually caused by Leishmania donovani with limited molecular validation. In order to molecularly characterize the causative parasite of the cutaneous disease, parasite specific Internal-Transcribed Spacer 1 (ITS1) PCR RFLP and sequence analysis was performed on skin lesional biopsies from cutaneous leishmaniasis patients. Interestingly, we found the presence of Leptomonas seymouri in 38.5% (22/57) of the patients along with L. donovani detected in all the samples. L. seymouri is a monoxenous insect trypanosomatid, generally incapable of infecting humans. In recent years, the parasite is also reported to co-infect humans with L. donovani in visceral and post kala-azar dermal leishmaniasis (PKDL) cases prevalent in northeastern India. The finding of L. seymouri-L. donovani co-infection in unusual cutaneous cases from Himachal Pradesh is the first ever to our knowledge and imply a newer disease paradigm. There is an urgent need to understand the biology of Leptomonas co-infection with L. donovani and its possible role in visceral and/or dermotropic disease outcome. Importantly, L. seymouri co-infection in cutaneous cases and previously reported visceral and PKDL cases needs to be recognized as a newer phenomenon by the leishmaniasis surveillance program in India.
Project description:Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the ?-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of ?-Gal than the former. A polyvalent ?-Gal conjugate on the immunogenic Q? virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 ?-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-?-Gal antibodies. As expected, ?-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Q?-?-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the infection and proliferation of parasites in the liver and spleen as probed by qPCR. The ?-Gal epitope may therefore be considered as a vaccine candidate to block human cutaneous and visceral leishmaniasis.
Project description:A central question in Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection.
Project description:To investigate the relationship of cutaneous leishmaniasis isolates from Sri Lanka to known species, we performed DNA sequencing and microsatellite analyses. We identified Leishmania donovani as the agent of Sri Lanka cutaneous leishmaniasis and showed that these parasites are closely related to those causing visceral leishmaniasis in the Indian subcontinent.
Project description:We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.