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

Biological evaluation and structure-activity relationships of imidazole-based compounds as antiprotozoal agents

We discovered a series of azole antifungal compounds as effective antiprotozoal agents. They displayed promising inhibitory activities within the micromolar-submicromolar range against P. falciparum, L. donovani, and T. b. rhodesiense. Moreover, most of such compounds showed excellent nanomolar IC50against T. cruzi, showing also very low cytotoxicity. Discussion of structure-activity relationships and biological data for these compounds are provided against the different parasites. To assess the mechanism of action against T. cruzi we proved that the most potent compounds (3b, 3j-l) inhibited the T. cruzi CYP51. Moreover, the most active derivative 3j dramatically reduced parasitemia in T. cruzi mouse model without acute toxicity

Rapid Chagas Disease Drug Target Discovery Using Directed Evolution in Drug-Sensitive Yeast

Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds' molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here, we use directed evolution and whole-genome sequencing of a drug-sensitive S. cerevisiae strain to identify the yeast ortholog of TcCyp51, lanosterol-14-alpha-demethylase (TcCyp51), as the target of MMV001239, a benzamide compound with activity against Trypanosoma cruzi, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another TcCyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug-protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other antitrypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for TcCyp51 inhibition

Design, synthesis, and biological evaluation of new 1-(Aryl-1H-pyrrolyl)(phenyl)methyl-1H-imidazole derivatives as antiprotozoal agents

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogs possess micromolar activities against T. b. rhodesiense and L. donovani and nanomolar potency against P. falciparum. Most of the analogs displayed the IC50 within the low nanomolar range against T. cruzi, with very high selectivity towards the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, 8b) was assessed by a target-based assays using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits was assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for four consecutive days

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

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

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