4-aminopyridyl-based lead compounds targeting CYP51 prevent spontaneous parasite relapse in a chronic model and improve cardiac pathology in an acute model of Trypanosoma cruzi infection.

BackgroundChagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice.Methodology/principal findingsBoth acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart.Conclusions/significanceThe positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life

Mapping of host-parasite-microbiome interactions reveals metabolic determinants of tropism and tolerance in Chagas disease

Chagas disease (CD) is a parasitic disease caused by Trypanosoma cruzi protozoa, presenting with cardiomyopathy, megaesophagus, and/or megacolon. To determine the mechanisms of gastrointestinal (GI) CD tissue tropism, we systematically characterized the spatial localization of infection-induced metabolic and microbiome alterations, in a mouse model of CD. Notably, the impact of the transition between acute and persistent infection differed between tissue sites, with sustained large-scale effects of infection in the esophagus and large intestine, providing a potential mechanism for the tropism of CD within the GI tract. Infection affected acylcarnitine metabolism; carnitine supplementation prevented acute-stage CD mortality without affecting parasite burden by mitigating infection-induced metabolic disturbances and reducing cardiac strain. Overall, results identified a previously-unknown mechanism of disease tolerance in CD, with potential for new therapeutic regimen development. More broadly, results highlight the potential of spatially resolved metabolomics to provide insight into disease pathogenesis and infectious disease drug development.Open Access fees paid for in whole or in part by the University of Oklahoma Libraries. This work was supported by start-up funds from the University of Oklahoma to L.-I.M. and the National Institute of Allergy and Infectious Diseases of the NIH under award number R21AI148886 to L.-I.M. Initial tissue collection was supported by a postdoctoral fellowship to L.-I.M. from the Canadian Institutes of Health Research (award number 338511; www.cihr-irsc.gc.ca/). Microbial community analysis was supported, in part, by an NIH grant (award number NIH 2R01-GM089886 to K.S). Immunological characterization was performed on instrumentation from the OU Protein Production and Characterization Core facility, supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the NIH under grant number P20GM103640. Histology samples were processed by the University of Oklahoma Health Sciences Center, Stephenson Cancer Center Tissue Pathology Shared Resource, supported by the National Cancer Institute Cancer Center Support Grant P30CA225520 and COBRE P20GM103639. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.Ye

Metabolite Profiling Of Experimental Cutaneous Leishmaniasis Lesions Demonstrates Significant Perturbations In Tissue Glycerophosphocholines

Each year 700,000 to 1.2 million new cases of cutaneous leishmaniasis (CL) are reported and yet CL remains one of thirteen diseases classified as neglected tropical diseases (NTDs). Leishmania major is one of several different species of that same genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can lead to new drug targets. In this study, samples were collected from mice infected in the ear and footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including select phosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through the host metabolism and may lead to new curative measures against infection with Leishmania

Quantitative analysis of <i>ex vivo</i> bioluminescence in the internal organs of chronically-infected mice.

<p>Each animal is represented by an individual data point. Treatment groups are labeled along the x-axis by the gender and drug name. (<b>A)</b> Cumulative signal from all the organs in each individual animal. (<b>B</b>) Signal from the GI tract alone. The majority of the bioluminescent parasites are associated with the GI tract.</p

Cumulative <i>in vivo</i> effect of 9 and 12.

<p>No alteration in the liver (ALT, AST, BIL, ALB, ALP) or kidney (BUN, CRE) markers were observed in the course of mice treated with <b>9</b> or <b>12</b> at 25 mg/kg, for 28 days, b.i.d. Treatment groups are labeled along the x-axis by the gender/drug combination.</p

The effects of 4-aminopyridyl lead compounds on mice with acute <i>T</i>. <i>cruzi</i> infection.

<p>(<b>A</b>) Parasitemia levels in Swiss male mice infected with <i>T</i>. <i>cruzi</i> Y strain (10⁴ inoculum) and treated for 28 days with <b>9</b> or <b>12</b> (25 mg/Kg) are undetectable. (<b>B</b>) Survival curve shows partial protection and delayed death of mice treated with <b>9</b> and <b>12</b> (25 mg/Kg) in the acute phase of the infection. (<b>C-F</b>) Biochemical analysis of serum from infected mice treated with <b>9</b> or <b>12</b> (25 mg/Kg) was comparable to serum from unifected controls and shows normal levels of liver enzymes—alanine aminotransferase (ALT;C) and aspartate aminotransferase (AST;D)–as well as the renal function markers—creatinine (CRE; E) and urea (F). dpi—days post infection. *Statistically significant by <i>t</i> test, p≤0.05.</p

Effect of 4-aminopyridyl compounds in chronic infection.

<p>Parasite levels measured by bioluminescence detection in BALB/c males infected with <i>T</i>. <i>cruzi</i> CL-luc strain. (<b>A</b>) Average levels from all groups from 102 to 197 dpi. Individual values for each mouse in the groups represented separately on 123 dpi (<b>B</b>), 151 dpi (<b>C</b>), 179 dpi (<b>D</b>) and 197 dpi (<b>E</b>).</p

Histopathological analysis of heart tissue.

<p>H&E staining of heart tissue from acute models of <i>T</i>. <i>cruzi</i> infection. (<b>A-D</b>) BALB/c female mice infected with <i>T</i>. <i>cruzi</i> CL-luc. (<b>F-I</b>) Lethal infection of Swiss male mice infected with <i>T</i>. <i>cruzi</i> Y strain. In <b>F</b>, the parasite nests are labeled with the asterisks. Bar = 100μM. (<b>E, J</b>) Inflammation quantified using FIJI software.[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006132#pntd.0006132.ref044" target="_blank">44</a>]. * Statistically significant by <i>t</i> test, p≤0.05.</p

Post-treatment evolution of parasitemia in acutely infected mice monitored by bioluminescence imaging of live animals.

<p>Stochastic profile of <i>T</i>. <i>cruzi</i> infection is shown. Abbreviations are M—male, F—female, Bz—benznidazole, Pos—posaconazole, Sol—solutol, <b>9</b> and <b>12</b> –lead compounds.</p

Development of parasitemia in acutely infected mice after the 28-day course of treatment with 9 or 12.

<p>The photon count values are displayed for each individual animal up to 81 dpi, showing progressive parasite relapse. Post-treatment evolution of parasetimea in live animals is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006132#pntd.0006132.g006" target="_blank">Fig 6</a>.</p