In vitro and in vivo evaluation of the antimalarial potential of Epirubicin

Author(s)

Letícia Tiburcio Ferreira

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-02-22

Examining board members

Fabio Trindade Maranhão Costa; Danilo Ciccone Miguel; Anna Caroline Campos Aguiar

• Advisor: Fabio Trindade Maranhão Costa

Abstract

Caused by protozoan of the genus "Plasmodium", malaria is one of the most important parasitic diseases in history, reaching more than 200 million new cases every year. Besides significant global advances in disease control, the presence of parasites strains less susceptible to the drugs available is recurrently observed for different "Plasmodium" species. Facing the challenges met by the pharmaceutical industry towards drug development, drug repositioning rises as as a new strategy with reduced risks on the search for antimalarial drugs. In silico chemogenomic approaches previously conducted have identified, based on ligand and target similarity, Epirubicin as a candidate for repositioning as an antimalarial drug. Epirubicin, originally developed to treat cancer, is an anthracyclin approved by the FDA. The present study aimed to validate Epirubicin’s antimalarial activity. Experimental validation started with in vitro fluorimetric assays (SybrGreen) using chloroquine-sensitive (3D7) and multi-resistant (W2 and Dd2) "P. falciparum" strains. Epirubicin demonstrated antiplasmodial activity "in vitro" in nanomolar range (EC50 = 110.7, 68.9 and 99.5 nM, respectively). Cell viability assays in mammalian cell lines (MTT reduction) showed that Epirubicin presents low toxicity (COS-7 and CHO cell lines, CC50 = 5.48 and 4.45µM, respectively), with selectivity indexes of 49.4 and 40.1. Schizont maturation assays identified Epirubicin’s potential to inhibit "P. vivax" isolates with as much potency as chloroquine. We also evaluated the drug’s potential to block parasite transmission using a modified P. berghei line (luciferase expression under specific ookinete promoter). Epirubicin inhibited around 80% of ookinete formation at 1 µM (EC50 = 0.39 µM). Also,mosquito feeding assays in "P. vivax" infected blood by a membrane mimicking skin corroborated Epirubicin’s transmission blocking potential by demonstrating that A. aquasalis mosquitoes had significantly less oocysts found in their midgut after taking a blood meal on an Epirubicin-treated sample. In addition, schizonticidal activity of Epirubicin was evaluated "in vivo" in C57BL/6JUnib mice infected with "P. yoelii" 17XNL, treated with 6 mg/kg of Epirubicin for 4 days. This regimen inhibited approximately 90% of mice parasitemia until the last day of treatment, keeping this inhibition above 60% until the sixth day post-infection. Finally, using synthetic biology, particularly a library screening of heterozygous mutant yeast strains, other possible molecular targets and pathways affected by Epirubicin were introduced beyond the one predicted at first, a gyrase in the apicoplast. Therefore, the results obtained not only show that Epirubicin presents relevant antiplasmodial activity against several parasite strains - including chemorresistant ones, but also validates the drug repurposing strategy as a consistent and suitable option in drug discovery (AU)

• FAPESP's process: 17/02031-1 - Evaluation of the antimalarial potential of Epirubicin (4'-epidoxorubicin)
• Grantee: Letícia Tiburcio Ferreira
• Support Opportunities: Scholarships in Brazil - Master