Identification of putative targets of Eg-FKBP-rapalogs complex in Echinococcus sp

CUMINO, ANDREA C.; NICOLAO, MARÍA CELESTE; CLARA ALBANI; GUILLERMO M. DENEGRI

Colonia del Sacramento

Congreso; XXIII Congreso Mundial de Hidatidología; 2009

The macrolides FK506 and rapalogs bind with high affinity to FKB proteins, creating a drug-protein complex that subsequently inactivates target proteins as calcineurin and TOR (target of rapamycin), respectively. TOR is a phosphatidylinositol-3-kinase that controls cell growth, autophagia and stress responses. Our experiments have showed that FK506, rapamycin, and everolimus possess dose-dependent protoscolicidal activity. Also, we have identified an Eg-fkb1 gene that encodes Eg-FKBP, an archetypal protein with all residues implicated in the binding of pharmacological ligands, and in interactions with possible targets. As rapamycin sensitivity is the major criterion used to detect TOR kinase, we identified and analyzed in silico critical residues of putative homologs in Echinococcus genome. BLAST searches revealed two homolog genes in the E. multilocularis genome. These genes predict ORFs included in Em-Contig_0011413 and Em-Contig_0011414 with 44% and 38% of identity compared with human ortholog gene (P42345) and 41% and 30% of identity with TOR of B. malayi (XM_001901052), respectively. These sequences have the conserved highly FRB domain corresponding to FKBP-rapalog binding site with key residues for physiological function, catalytic and FATC domains. Em-Contig_0011413 includes the 50% of coding region of a TOR-like protein, with at least 32 exons that represent the C-terminus end with the catalytic and FATC domains. On the other hand, as rapamycin is the best positive control used to induce autophagy in diverse cellular models, we analyzed if this process could be present in Echinococcus. We showed that treatment with rapamycin and everolimus induce an increase of acridine orange signal in protoscoleces compared to the control group, observable by fluorometric determinations and confocal images, because revealed lysosome-like vesicules, ultrastructural characteristics of the autophagy. These preliminary results will let us continue subsequent studies that may improve the outline for further identification of downstream target proteins, a promising target for chemotherapy of cystic echinococcosis.