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Effect of GSK-343, an inhibitor of histone methyltransferase EZH2, on the parasite Schistosoma mansoni

Grant number: 18/23466-9
Support type:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): March 11, 2019
Effective date (End): March 10, 2020
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Sergio Verjovski Almeida
Grantee:Adriana Silva Andrade Pereira
Supervisor abroad: Conor Caffrey
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Local de pesquisa : University of California, San Diego (UC San Diego), United States  
Associated to the scholarship:16/10046-6 - Effect of GSK343, an inhibitor of the histone methyltransferase EZH2, on the parasite Schistosoma mansoni, BP.PD

Abstract

Schistosomiasis is a chronic and debilitating disease caused by a trematode of the genus Schistosoma and is one of the most prevalent and neglected diseases of tropical and subtropical regions, affecting more than 190 million people in 78 countries. Current schistosomiasis treatment is based on the use of praziquantel (PZQ), which is effective against all Schistosoma species infecting humans. The possibility of emergence of PZQ-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. My preliminary data to the present proposal (now published) investigated the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control. Specifically, different developmental forms (schistosomula, and adult male and female worms) of Schistosoma mansoni were incubated in vitro with different concentrations of a selective inhibitor of human EZH2, namely GKS34. For the preparation of histone acid extracts, fifty adult pairs or approximately 10,000 schistosomula were used. From each sample, 10 ¼g of extracts was loaded on 15 % SDS-polyacrylamide gels, and after electrophoretic protein separation, western blot were performed. RNA-seq and proteomics were applied to identify the metabolic pathways affected by GSK343. Western blotting showed a decrease in the H3K27me3 histone mark in GSK343-treated adult worms and schistosomula. Also, adult worms were less motile with a 40% reduction in egg laying compared to controls (0.1 % DMSO). GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis, egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. RNA-Seq analysis of iEZH2 treated schistosomula pointed to altered expression of genes related to cell adhesion, membrane constituent, transmembrane transport, drug binding and in the gynecophoral canal. Our results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Based on the foregoing data, and as part of a Research Internships Abroad period, I we now propose to test the hypothesis that GSK343 disrupts the balance between important histone marks of activation and inhibition of transcription. To address these questions, I will employ ChIP-Seq with anti-H3K27me3, anti-H3K27ac and anti-H3K4me3 antibodies, in adult worms treated with GSK343, and by silencing of SmEZH2 (Smp_078900), accompanied by measurement of oviposition in treated females as well as measurement with RT-qPCR of the expression level of genes in the pathways of DNA replication and ncRNAs metabolism. Here we discuss the available results and explain which experiments will be done, also justifying why such experiments should be carried out in the laboratory of Prof. Conor R. Caffrey at the University of California San Diego, CA, USA.