Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51.

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), is endemic in Latin America and emerging in North America and Europe through human migration. It is a severe global health problem with 8–10 million people infected and an estimated 12,000 deaths annually. Current treatment options are poorly efficacious and have severe side effects. New drugs are therefore urgently required. Two of these potential new drugs, posaconazole and ravuconazole, both targeting an enzyme in T. cruzi called CYP51, have recently failed in clinical development. Therefore, in light of these recent clinical failures and in order to better balance the overall risk in the drug discovery portfolio for Chagas disease, it has become prudent to assess whether new chemical start points for drug discovery programmes have a mode of action predominantly driven by T. cruzi CYP51 inhibition. In this paper we report a fluorescence based assay to determine whether compounds inhibit T. cruzi CYP51. This provides a high throughput screen to help prioritise medicinal chemistry resource on those T. cruzi active new chemical series that do not have a mode of action predominantly driven by CYP51 inhibition.

Competing interests: DC and IC are employed by GlaxoSmithKline. This does not alter their adherence to all PLOS NTDs policies on sharing data and materials. They have declared that no competing interests exist. MV is director of CYPEX Ltd. This does not alter his adherence to all PLOS NTDs policies on sharing data and materials. However, he cannot make the plasmid (or E.coli containing the plasmid) freely available as the ompA part is patented and would be in breach of his licence. He has no competing interests although it should be noted that the T. cruzi CYP51 enzyme developed in this paper will be made available for sale commercially through CYPEX. KR, JR and SB have declared that no competing interests exist.

Funding: Funding under Wellcome Trust strategic award WT092340 and WT100476 supported this work for JR, SB and KR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Riley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Introduction

Chagas disease is a tropical parasitic disease caused by the flagellate eukaryotic (protozoan) parasite Trypanosoma cruzi (T. cruzi), endemic in Latin America and now emerging in North America and Europe through human migration. It is becoming a severe global health problem with approximately 8–10 million people infected, an estimated 12,000 deaths per year, and placing 100 million people at risk. Transmission to humans and other mammals is predominantly by an insect vector, the blood-sucking "kissing bugs" of the subfamily Triatominae (family Reduviidae) [1]. Transmission has also been reported to occur through contaminated food, blood transfusions and from mother to child.

Clinical Chagas disease can be classified into two distinct phases, acute and chronic. In the acute phase, lasting a few weeks, parasites begin to multiply in the organs and tissues. Symptoms are usually mild and non-specific with patients rarely being diagnosed. However, life-threatening myocarditis or meningoencephalitis can occur during the acute phase with a death rate for people in this phase of about ten percent. Ten to fifty percent of infected survivors develop chronic Chagas disease. People in the chronic phase can be asymptomatic for many years, with parasites generally undetectable in the blood. However, the disease causes organ and tissue damage, particularly potentially lethal cardiopathy and megacolon or megaoesophagus, caused by the sequential induction of inflammatory response to the parasite. Nitroheterocyclic compounds, benznidazole and nifurtimox, developed in the 1960’s [2], are currently the only two drugs used for the treatment of Chagas disease. Both have low efficacy in the chronic stage and, with prolonged dosing regimens, both drugs have significant side effects including skin irritation, neurotoxicity, and digestive system disorders [3]. Newer, safer and more efficacious treatments are therefore in desperate need.

Inhibition of sterol 14α-demethylase (CYP51) has been considered a viable target against T. cruzi for over 30 years [2,4,5,6,7,8]. Found in a broad variety of organisms including animals, plants, fungi and protozoa, this enzyme plays an essential role in the sterol biosynthetic pathway, catalysing the oxidative removal of the 14α-methyl group from sterol precursors such as lanosterol or eburicol [9]. The products of the pathway, cholesterol in humans or ergosterol in fungi, are required for the integrity of the eukaryotic cell membrane. These sterols are required for membrane function in T. cruzi. Inhibition of CYP51 activity is lethal as the T. cruzi parasites are unable to scavenge and utilise host cholesterol [10]. The CYP51 gene is known to be expressed in all stages of the parasite life cycle and indeed it has also been shown to be up-regulated in multiplying forms [9]. As with other members of the Cytochrome family, CYP51 is a haem containing protein located on the membrane of the endoplasmic reticulum that relies upon electron transfer by NADPH reductase for activation [11].

Azole inhibitors, which interfere with sterol biosynthesis, essential in eukaryotic cells, have already been used with success in humans in the treatment of fungal infections. Several of these drugs have been considered as possible treatments for Chagas disease [12]. Ketoconazole, fluconazole, itraconazole, ravuconazole and posaconazole are known to inhibit CYP51 in vitro, competitively binding to the haem within CYP51 and occupying the active site preventing any substrate from binding. Although ketoconazole and itraconazole have not demonstrated significant curative activity in humans with chronic Chagas disease [6], other azoles, with greater potency and improved pharmacokinetic properties, which have been shown to have potent activity against T. cruzi, including posaconazole [13,14] and ravuconazole (Fig 1), are in clinical development with the Drugs for Neglected Diseases initiative (DNDi).

To prevent enrichment of candidates against a single target, and thus reduce risk in the overall drug discovery portfolio for Chagas disease, it has therefore become necessary to evaluate and prioritise medicinal chemistry resource on new chemical series active against T. cruzi but with such activity not likely driven via T. cruzi CYP51 inhibition. Recent findings from clinical trials with posaconazole [15] and ravuconazole [16] has indicated re-emergence of parasitaemia in two thirds of patients once dosing has been completed, thus reinforcing the need to strengthen the overall drug discovery portfolio for Chagas disease with new chemical lead series not working via this mechanism of action.

Evaluating compounds as potential inhibitors of T. cruzi CYP51 has previously been demonstrated measuring the apparent dissociation constants (Kd) by spectral titration [4,17,18] utilising the shift of the haem iron soret band in response to binding [18]. One of the drawbacks to this methodology is that micromolar protein concentrations are required for screening causing potential interference with the optical properties and/or solubility of test compounds [4]. There are many potential reasons why affinity estimates measured by binding may not correlate with functional inhibition. These include allosteric sites, non or uncompetitive modes of inhibition or slow kinetics [19].

Inhibition of endogenous substrate lanosterol, eburicol and obtusifoliol has also been used as an in vitro tool using recombinant expressed human CYP51 enzyme [20]. In particular, measuring effect on CYP51 driven metabolism of lanosterol to follicular fluid meiosis activating sterol (FF-MAS) in the presence of test substances [21] is well established (Fig 2). However, FF-MAS detection requires mass spectrometry limiting the number of compounds that can be tested and consequently limiting the value of such an assay for triaging large numbers of phenotypic screening T. cruzi hits toward identifying modes of action away from CYP51.

Metabolism of fluorogenic probe substrate to a product, detectable by fluorescence is well established with recombinantly expressed cytochrome P450 enzymes (CYP’s) for the purpose of assessing possible drug-drug interactions [22]. Measuring CYP inhibition by this method provides a high throughput screening approach, avoiding time consuming analysis by mass spectrometry and minimising use of expensive substrates. The O-dealkylation of Vivid substrate benzyloxymethylocyanocoumarin (BOMCC) to fluorescent product cyanohydroxycoumarin (CHC) is commonly used to evaluate CYP3A4 activity in recombinantly expressed membrane preparations (Fig 2). Valuably, O-dealkylation activity in the presence of recombinantly expressed T. cruzi CYP51 was observed. This has enabled the creation of a fast, high-throughput, 96 and 384 well microtitre method to assess the inhibitory potential of compounds against T. cruzi CYP51, which is described in this paper.