Re-evaluating pretomanid analogues for Chagas disease: Hit-to-lead studies reveal both in vitro and in vivo trypanocidal efficacy
Graphical abstract
Introduction
Chagas disease (CD) or American trypanosomiasis is a neglected tropical disease that afflicts approximately 7 million people worldwide and claims at least 10,000 lives every year [1]. Once endemic almost exclusively to poor or rural areas of Latin America, CD has now spread into many high-income countries in Europe, North America, and the Western Pacific [2,3]. The causative parasite, Trypanosoma cruzi (T. cruzi), is transmitted primarily via the faeces of a blood-sucking triatomine insect known as the ‘kissing bug’, which commonly infests substandard housing; many animals also function as disease reservoirs [[1], [2], [3]]. Following infection, a one- to two-month acute illness develops, which is usually mild or asymptomatic, although it can be fatal in 5% of diagnosed cases due to the high parasitic burden. The host immune system then takes over, rendering even parasite detection extremely difficult [2,4]. Eventually, sometimes decades later, a chronic disease state can manifest in digestive or cardiac disorders, which may lead to malnutrition, progressive heart failure and sudden death [1,3]. However, in 2015, it was estimated that more than 80% of individuals affected by CD globally did not have access to diagnosis and specific treatment [5].
There is currently no vaccine for CD and just two drugs are available for therapy, benznidazole (1) and nifurtimox (2) (Fig. 1), both of which can induce serious adverse effects during dosing periods of 60–90 days [2,6,7]. Recent clinical trial failures with two azole drugs, posaconazole (3) and E1224 (5, a prodrug of ravuconazole 4), have emptied the late-stage candidate pipeline, providing fresh impetus for the development of new assessment tools with superior predictivity [4,[8], [9], [10], [11], [12]]. One encouraging result is that the repositioned antimicrobial agent fexinidazole (6), a safe and effective newly approved drug for late-stage human African trypanosomiasis (HAT) [13], has shown curative activity potentially better than 1 in acute stage CD mouse models and equivalent to 1 in the chronic stage models, as revealed by highly sensitive bioluminescence imaging [14]. Although a phase II proof of concept clinical study of 6 was interrupted because of safety and tolerability issues at higher doses and longer treatment times, good efficacy against CD was observed at more tolerable doses; therefore, a second clinical trial has been initiated [15,16]. A close analogue of the HAT clinical candidate SCYX-7158 (7), oxaborole SCYX-6759 (8), also exhibited strong in vivo activity against CD but was not curative in the chronic model [17]. Amongst many novel lead molecules evaluated in mice [[18], [19], [20], [21]], the most promising was proteasome inhibitor GNF6702 (9), which delivered an 88% cure rate for treating chronic infection when dosed twice daily for 20 days [22]. Nevertheless, a five day schedule using higher doses of 9 did not achieve any cures in the acute model [23], and a new article by Rao et al. [24] that compares 9 with a more recent analogue (for HAT) still provides no indication whether this pan-kinetoplastid drug candidate has progressed beyond preclinical toxicology. Hence, there remains a compelling need to discover new medicines for CD.
In CD research, successful target-based drug discovery has been hindered by the lack of well-validated targets [25]. Hence, phenotypic screening of various compound collections, seeking to exploit existing chemical matter, is still considered to be the most useful and cost-effective strategy to identify new leads or starting points [25,26]. Pretomanid (PA-824, 10) is an orally active drug that was recently approved by the FDA to treat highly challenging cases of tuberculosis [27]. We have previously recounted that phenotypic screening (instigated by the Drugs for Neglected Diseases initiative, DNDi [28]) of bicyclic nitroimidazole derivatives generated in our quest for an improved backup to 10 (with the TB Alliance [29]) led to the novel discovery of their potent antileishmanial activity [30,31]. Collaborative lead optimisation efforts with DNDi in the very promising 7-substituted 2-nitroimidazooxazine class eventually culminated in the development of clinical candidate DNDI-0690 (11) [31], having comparable efficacy but greater safety than the original preclinical nominee, DNDI-VL-2098 (12) [30]. As part of the early investigations (2010), a 900-member subset of the pretomanid analogue library above was counter-screened against intracellular Y strain T. cruzi amastigotes, using an image-based assay [8] (with 7 data points and 3-fold dilutions) run at the Institut Pasteur Korea (IPK). Following initial profiling of the best screening hits, we conducted some limited hit expansion work alongside our primary studies directed at leishmaniasis. Testing of these new compounds against T. cruzi pinpointed several that displayed submicromolar activity, some of which have been further assessed (e.g., for solubility, microsomal stability, T. cruzi CYP51 inhibition and maximal parasite killing in a 48 h exposure assay). We now report the findings from our accumulated efforts to identify a suitable nitroimidazooxazine-based lead candidate for CD.
Section snippets
Initial examination of library screening hits
The structures and in vitro biological assay data for selected IPK screening hits (based mainly on % inhibition data) are summarised in Table 1. The IC50 and IC90 data against intracellular T. cruzi (transfected Tulahuen strain TcVI) or L6 host cells (rat skeletal myoblasts) were derived from replicate assays performed at Murdoch University [32], using fresh sample stock. Most of the hits provided submicromolar IC50 and IC90 values and displayed very low cytotoxicity, resulting in excellent
Conclusions
To summarise, in response to a dire need for new medications to treat CD, an early-stage chemical library of antitubercular bicyclic nitroimidazole derivatives was screened against T. cruzi, and a selection of 12 hits was profiled in vitro. Mouse pharmacokinetic data generated on five on these directed our initial focus toward the evaluation of 17 in a CD mouse model, albeit, promising efficacy in the acute phase was overturned after immunosuppression with cyclophosphamide, implying the need
General information
Elemental analyses were performed by the Campbell Microanalytical Laboratory, University of Otago, Dunedin, New Zealand. Melting points were determined using an Electrothermal IA9100 melting point apparatus and are as read. NMR spectra were measured on a Bruker Avance 400 spectrometer at 400 MHz for 1H and 100 MHz for 13C and were referenced to Me4Si or solvent resonances. Chemical shifts and coupling constants were recorded in units of ppm and hertz, respectively. High-resolution electrospray
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank the Drugs for Neglected Diseases initiative for financial support through a collaborative research agreement. For this project, DNDi received financial support from the following donors: Department for International Development (DFID), UK; Federal Ministry of Education and Research (BMBF), through KfW, Germany; Directorate-General for International Cooperation (DGIS), The Netherlands; Ministry of Foreign and European Affairs (MAEE), France; Swiss Agency for Development and
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- 1
Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
- 2
Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Lower Hutt 5010, New Zealand.
- 3
PathWest Laboratory Medicine, Locked Bag 2009, Nedlands, WA 6009, Australia.