Short-course combination treatment for experimental chronic Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.

Discovery of pyrazolopyrrolidinones as potent, broad-spectrum inhibitors of Leishmania infection.

Introduction: Leishmaniasis is a parasitic disease that affects more than 1 million people worldwide annually, predominantly in resource-limited settings. The challenge in compound development is to exhibit potent activity against the intracellular stage of the parasite (the stage present in the mammalian host) without harming the infected host cells. We have identified a compound series (pyrazolopyrrolidinones) active against the intracellular parasites of Leishmania donovani and L. major; the causative agents of visceral and cutaneous leishmaniasis in the Old World, respectively. Methods: In this study, we performed medicinal chemistry on a newly discovered antileishmanial chemotype, with over 100 analogs tested. Studies included assessments of antileishmanial potency, toxicity towards host cells, and in vitro ADME screening of key drug properties. Results and discussion: Members of the series showed high potency against the deadliest form, visceral leishmaniasis (approximate EC50 ≥ 0.01 µM without harming the host macrophage up to 10.0 µM). In comparison, the most efficient monotherapy treatment for visceral leishmaniasis is amphotericin B, which presents similar activity in the same assay (EC50 = 0.2 µM) while being cytotoxic to the host cell at 5.0 µM. Continued development of this compound series with the Discovery Partnership with Academia (DPAc) program at the GlaxoSmithKline Diseases of the Developing World (GSK DDW) laboratories found that the compounds passed all of GSK's criteria to be defined as a potential lead drug series for leishmaniasis. Conclusion: Here, we describe preliminary structure-activity relationships for antileishmanial pyrazolopyrrolidinones, and our progress towards the identification of candidates for future in vivo assays in models of visceral and cutaneous leishmaniasis.

Novel Diarylthioether Compounds as Agents for the Treatment of Chagas Disease.

Herein, we describe the hit optimization of a novel diarylthioether chemical class found to be active against Trypanosoma cruzi; the parasite responsible for Chagas disease. The hit compound was discovered through a whole-cell phenotypic screen and as such, the mechanism of action for this chemical class is unknown. Our investigations led to clear structure-activity relationships and the discovery of several analogues with high in vitro potency. Furthermore, we observed excellent activity during acute in vivo efficacy studies in mice infected with transgenic T. cruzi. These diarylthioether compounds represent a promising new chemotype for Chagas disease drug discovery and merit further development to increase oral exposure without increasing toxicity.

Identification and development of a series of disubstituted piperazines for the treatment of Chagas disease.

Approximately 6-7 million people around the world are estimated to be infected with Trypanosoma cruzi, the causative agent of Chagas disease. The current treatments are inadequate and therefore new medical interventions are urgently needed. In this paper we describe the identification of a series of disubstituted piperazines which shows good potency against the target parasite but is hampered by poor metabolic stability. We outline the strategies used to mitigate this issue such as lowering logD, bioisosteric replacements of the metabolically labile piperazine ring and use of plate-based arrays for quick diversity scoping. We discuss the success of these strategies within the context of this series and highlight the challenges faced in phenotypic programs when attempting to improve the pharmacokinetic profile of compounds whilst maintaining potency against the desired target.

Rate-of-Kill (RoK) assays to triage large compound sets for Chagas disease drug discovery: Application to GSK Chagas Box.

Chagas disease (CD) is a human disease caused by Trypanosoma cruzi. Whilst endemic in Latin America, the disease is spread around the world due to migration flows, being estimated that 8 million people are infected worldwide and over 10,000 people die yearly of complications linked to CD. Current chemotherapeutics is restricted to only two drugs, i.e. benznidazole (BNZ) and nifurtimox (NIF), both being nitroaromatic compounds sharing mechanism of action and exerting suboptimal efficacy and serious adverse effects. Recent clinical trials conducted to reposition antifungal azoles have turned out disappointing due to poor efficacy outcomes despite their promising preclinical profile. This apparent lack of translation from bench models to the clinic raises the question of whether we are using the right in vitro tools for compound selection. We propose that speed of action and cidality, rather than potency, are properties that can differentiate those compounds with better prospect of success to show efficacy in animal models of CD. Here we investigate the use of in vitro assays looking at the kinetics of parasite kill as a valuable surrogate to tell apart slow- (i.e. azoles targeting CYP51) and fast-acting (i.e. nitroaromatic) compounds. Data analysis and experimental design have been optimised to make it amenable for high-throughput compound profiling. Automated data reduction of experimental kinetic points to tabulated curve descriptors in conjunction with PCA, k-means and hierarchical clustering provide drug discoverers with a roadmap to guide navigation from hit qualification of a screening campaign to compound optimisation programs and assessment of combo therapy potential. As an example, we have studied compounds belonging to the GSK Chagas Box stemmed from the HTS campaign run against the full GSK 1.8 million compounds collection [1].

The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.

Chagas Disease Drug Discovery: Multiparametric Lead Optimization against Trypanosoma cruzi in Acylaminobenzothiazole Series.

Acylaminobenzothiazole hits were identified as potential inhibitors of Trypanosoma cruzi replication, a parasite responsible for Chagas disease. We selected compound 1 for lead optimization, aiming to improve in parallel its anti-T. cruzi activity (IC50 = 0.63 µM) and its human metabolic stability (human clearance = 9.57 mL/min/g). A total of 39 analogues of 1 were synthesized and tested in vitro. We established a multiparametric structure-activity relationship, allowing optimization of antiparasite activity, physicochemical parameters, and ADME properties. We identified compound 50 as an advanced lead with an improved anti-T. cruzi activity in vitro (IC50 = 0.079 µM) and an enhanced metabolic stability (human clearance = 0.41 mL/min/g) and opportunity for the oral route of administration. After tolerability assessment, 50 demonstrated a promising in vivo efficacy.

Novel chemical starting points for drug discovery in leishmaniasis and Chagas disease.

Visceral leishmaniasis (VL) and Chagas disease (CD) are caused by kinetoplastid parasites that affect millions of people worldwide and impart a heavy burden against human health. Due to the partial efficacy and toxicity-related limitations of the existing treatments, there is an urgent need to develop novel therapies with superior efficacy and safety profiles to successfully treat these diseases. Herein we report the application of whole-cell phenotypic assays to screen a set of 150,000 compounds against Leishmania donovani, a causative agent of VL, and Trypanosoma cruzi, the causative agent of CD, with the objective of finding new starting points to develop novel drugs to effectively treat and control these diseases. The screening campaign, conducted with the purpose of global open access, identified twelve novel chemotypes with low to sub-micromolar activity against T. cruzi and/or L. donovani. We disclose these hit structures and associated activity with the goal to contribute to the drug discovery community by providing unique chemical tools to probe kinetoplastid biology and as hit-to-lead candidates for drug discovery.

Quinoxaline derivatives as potential antitrypanosomal and antileishmanial agents.

Continuous efforts have been made to discover new drugs for the treatment of Chagas' disease, human African trypanosomiasis, and leishmaniasis. We have previously reported the synthesis and antileishmanial and antitrypanosomal (Y strain) properties of 2,3-disubstituted quinoxalines. Considering their promising antiparasitic potential, the present study was conducted to expand our search and take advantage of high-throughput assays to investigate the effects of quinoxaline derivatives against Leishmania donovani, Trypanosoma brucei, and Trypanosoma cruzi (Tulahuen strain). These compounds were active against the kinetoplastid parasites that were evaluated. The 2-chloro-3-methylsulfoxylsulfonyl and 2-chloro-3-methylsulfinyl quinoxalines were the most potent, and some of these derivatives were even more active than the reference drugs. Although the 2,3-diaryl-substituted quinoxalines were not active against all of the parasites, they were active against T. brucei and intracellular amastigotes of T. cruzi, without interfering with mammalian cell viability. These compounds presented encouraging results that will guide our future studies on in vivo bioassays towards the mode of action.

Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas' disease drug discovery.

Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments.

Hit-to-Lead Optimization of a Novel Class of Potent, Broad-Spectrum Trypanosomacides.

The parasitic trypanosomes Trypanosoma brucei and T. cruzi are responsible for significant human suffering in the form of human African trypanosomiasis (HAT) and Chagas disease. Drugs currently available to treat these neglected diseases leave much to be desired. Herein we report optimization of a novel class of N-(2-(2-phenylthiazol-4-yl)ethyl)amides, carbamates, and ureas, which rapidly, selectively, and potently kill both species of trypanosome. The mode of action of these compounds is unknown but does not involve CYP51 inhibition. They do, however, exhibit clear structure-activity relationships, consistent across both trypanosome species. Favorable physicochemical parameters place the best compounds in CNS drug-like chemical space but, as a class, they exhibit poor metabolic stability. One of the best compounds (64a) cleared all signs of T. cruzi infection in mice when CYP metabolism was inhibited, with sterile cure achieved in one mouse. This family of compounds thus shows significant promise for trypanosomiasis drug discovery.

New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.

Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

BACKGROUND: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. CONCLUSIONS/SIGNIFICANCE: We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells.

Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.