Research Article: Development of a Fluorescence-based Trypanosoma cruzi CYP51 Inhibition Assay for Effective Compound Triaging in Drug Discovery Programmes for Chagas Disease

Date Published: September 22, 2015

Publisher: Public Library of Science

Author(s): Jennifer Riley, Stephen Brand, Michael Voice, Ivan Caballero, David Calvo, Kevin D. Read, Michael P. Pollastri.

Abstract: 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.

Partial Text: 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.



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