Identification of Trypanocidal Activity for Known Clinical Compounds Using a New Trypanosoma cruzi Hit-Discovery Screening Cascade

Abstract

Chagas disease is a significant health problem in Latin America and the available treatments have significant issues in terms of toxicity and efficacy. There is thus an urgent need to develop new treatments either via a repurposing strategy or through the development of new chemical entities. A key first step is the identification of compounds with anti-Trypanosoma cruzi activity from compound libraries. Here we describe a hit discovery screening cascade designed to specifically identify hits that have the appropriate anti-parasitic properties to warrant further development. The cascade consists of a primary imaging-based assay followed by newly developed and appropriately scaled secondary assays to predict the cidality and rate-of-kill of the compounds. Finally, we incorporated a cytochrome P450 CYP51 biochemical assay to remove compounds that owe their phenotypic response to inhibition of this enzyme. We report the use of the cascade in profiling two small libraries containing clinically tested compounds and identify Clemastine, Azelastine, Ifenprodil, Ziprasidone and Clofibrate as molecules having appropriate profiles. Analysis of clinical derived pharmacokinetic and toxicity data indicates that none of these are appropriate for repurposing but they may represent suitable start points for further optimisation for the treatment of Chagas disease.

Identification of clinically approved small molecules that inhibit growth and promote surface remodeling in the African trypanosome

“…We binned each drug into four categories: highly inhibitory, inhibitory, slightly inhibitory, and not inhibitory ( Table, S3 Table). Several other drugs, including nitrofurazone, aminacrine, clemastine fumarate, bepridil hydrochloride, amiodarone hydrochloride, prochlorperazine dimaleate, protriptyline hydrochloride, fluoxetine hydrochloride, apomorphine hydrochloride, and proadifen hydrochloride have been shown to inhibit growth in T. cruzi (33,(36)(37)(38)(39)(40)(41)(42)(43)(44). Finally, cyproheptadine hydrochloride was shown to inhibit growth in T. evansi (S2 Table) (45).…”

“…We chose three drugs from our most highly inhibitory category: flunarizine hydrochloride, aprepitant, and clemastine fumarate. Clemastine fumarate is an antihistaminic drug that has been shown to inhibit growth in T. cruzi (37). Aprepitant is an antiemetic and flunarizine hydrochloride is a vasodilator.…”

Discovery and Optimization of 5-Amino-1,2,3-triazole-4-carboxamide Series against Trypanosoma cruzi

“…22 Compounds were initially tested at a single concentration (15 μM) over 3 days. The potency of suitable hits ( n = 495), defined as those which exhibited >80% inhibition of parasite growth and <30% inhibition of growth of VERO cells, was then determined by 10-point dose response (potency range 0.0025–50 μM).…”

“…22 The “rate of kill” of T. cruzi intracellular parasites was also measured and compared to nifurtimox ( 2 ) (which has a very rapid onset (0–24 h) of parasite killing) and posaconazole ( 9 ) (which has a slower lag (24–48 h) before killing occurs (Figure 3). The rate of kill of ATC series compounds 11 , 20 , 48 , 58 , 59 (amides), and 37 (oxadiazole) was slower than nifurtimox.…”

Drug repositioning approaches to parasitic diseases: a medicinal chemistry perspective

“…The first approach is based on the identification of parasitic protein orthologs that correspond to validated targets in other organisms, allowing the use of target-focused compound collections and series of analogs; the strategy also offers opportunities for medicinal chemistry work on structurePage 7 of 44 A c c e p t e d M a n u s c r i p t 7 based scaffold optimization [14]. However, the latter approach provides the advantages of examining drug-induced effects on cells, evaluating drug uptake issues and identifying synergic events in complex biological networks [15,16]. Owing to the small number of fully validated targets in NTDs, most of the repurposed compounds have arisen from phenotypic campaigns rather than target-based strategies [17].…”