Identification of clinically approved small molecules that inhibit growth and affect transcript levels of developmentally regulated genes in the African trypanosome

Abstract

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. We screened a library of 1,585 U.S. or foreign-approved drugs and identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wished to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites lack the immune evasion mechanisms prevalent in bloodstream forms, making them vulnerable to the host immune system. To identify drugs that increase transcript levels of an invariant, insect-stage specific surface protein called procyclin, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction or stabilization of the procyclin transcript. Using these bloodstream reporter strains in combination with automated flow cytometry, we identified eflornithine, spironolactone, and phenothiazine as small molecules that increase abundance of procyclin transcript. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. While we failed to identify compounds that increase levels of procyclin protein on the cell surface, this study is proof of principle that these fluorescent reporter parasites represent a useful tool for future small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions.

Antileishmanial Chemotherapy through Clemastine Fumarate Mediated Inhibition of the Leishmania Inositol Phosphorylceramide Synthase

“…As a licensed medicine, clemastine fumarate has proven pharmacokinetic exposure and the high volume of distribution (9.5 ± 3.8 L kg –1 ) could be contributing to the enhanced perfusion of parasite infected cells. As such, it is perhaps not surprising that clemastine fumarate has been identified in other repurposing studies for NTDs, − including those looking for new antileishmanial applications . However, significant antiparasitic activity has not been reported nor has a putative mode-of-action been identified.…”

Chemical inhibition of bromodomain proteins in insect stage African trypanosomes perturbs silencing of the Variant Surface Glycoprotein repertoire and results in widespread changes in the transcriptome

“…Specifically, the EP genes that are downregulated and the BARP genes that are upregulated during the transition to the epimastigote stage do not show significantly altered transcript levels following I-BET151 treatment. We verified that procyclin expression was not altered in procyclic parasites following I-BET151 treatment using a previously validated EP1/GFP reporter parasite line 30 (Supplemental Figure 1). We observed no difference in EP1/GFP expression in I-BET151 treated parasites (Supplemental Figure 2).…”

“…In parasites, bromodomain inhibitors have been shown to bind to bromodomain proteins in T. brucei, T. cruzi, P. falciparum, and L. dovani 23,27,[41][42][43][44] . Inhibition of bromodomain proteins in parasites has been shown to affect differentiation processes in multiple parasite systems 23,45,46 , and therapeutic strategies targeting chromatin interacting proteins have also been proposed and/or demonstrated for trypanosomiasis 23,30 , Chagas disease [47][48][49] , schistosomiasis 50,51 , toxoplasmosis 52,53 , leishmaniasis 54 , and malaria 55,56 .…”

“…Parasites were treated with 20µM I-BET151 (Sigma-Aldrich). EP1/GFP procyclic reporter parasites were generated as described in 30 . EP1/GFP bloodstream parasites 30 and Single Marker (SM) parasites 77 (used as controls for verification of the EP1/GFP reporter construct) were grown in HMI9 supplemented with 10% FCS and 10% Serum Plus at 37 ºC and 5.0% CO 2 .…”

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

“…The authors of that study showed that clemastine treatment reduces Plasmodium tubulin levels and inhibits replication. Clemastine also inhibits the kinetoplastid parasites Leishmania (amastigotes), Trypanosoma cruzi (amastigotes), and Trypanosoma brucei (bloodstream forms) with EC 50 values of 180 nM, 400 nM, and 3.7 µM, respectively [ 38 , 39 , 40 ]. The Leishmania study proposes inositol phosphoryl-ceramide synthase as the target: clemastine inhibits purified IPCS enzyme activity, with an IC 50 of 2.90 μM; parasites with an IPCS gene knockout have reduced drug sensitivity, and clemastine-treated amastigotes show altered lipid profiles [ 38 ].…”