Anales RANF

P.06 ALFAXOLONE-INDUCED P2X4R ALLOSTERIC AGONISM CONFIRMED BY MOLECULAR DYNAMIC CALCULATIONS Juan. P. Huidobro-Toro 1,2 , Verónica Latapiat 1 , Felipe Montenegro 3 and Nelson P. Barrera 3 1 Universidad de Santiago de Chile, Santiago, Estación Central, Chile; 2 Centro de NanoCiencias y Nanotecnología, CEDENNA, Santiago, Estación Central, Chile; 3 Pontificia Universidad Católica de Chile, Santiago, Chile. The P2X4R is modulated allosterically by multiple ligands including trace metals, steroids and ivermectin an antiparasitic agent. While trace metals such as Cu(II) or Zn(II) have sites in the extracellular part of the P2XR domain, steroids and ivermectin interact with P2XR sites in the transmembrane domain. Neurosteroids based on pregnanolone derivatives, are brain synthesized that influence animal behavior by interacting with membrane receptor sites. Alfaxolone (A) is a synthetic steroid derivative formerly used as an anesthetic agent. 1-10 µM A applications to oocytes or HEK cells transfected with rP2X4R, caused ATP-gated currents potentiation; however, at 30 µM, A evidenced per se gating currents sensitive to suramin blockade. This finding allowed to propose that A is a P2X4R allosteric agonist. We now examined the molecular mechanism of the allosteric agonism, hypothesizing that this steroid must somehow open the receptor pore in the absence of ATP. Bioinformatic tools such steroid docking analysis, rP2X4R modeling and molecular dynamic simulations, and hole analysis, were used to generate rP2X4R model in the apo and holo states in the absence and in the presence or A. Molecular dynamics in the different rP2X4R states revealed allosteric-induced stability. Pore and lateral fenestration measurements of the different rP2X4R states showed that A can induce a larger pore opening in the absence of ATP, as expected and consistent with an allosteric agonist. Altogether, the present findings are compatible with A binding to a membrane localized allosteric site eliciting rP2X4R conformational changes compatible with the notion of allosteric agonism. Future studies will allow the design of novel allosteric agonists or human P2X4R antagonists with proved beneficial clinicals as analgesic drugs. Funded by Newton Picarte DPI Conicyt 20140080 grant, FONDECYT 117-0842; additional funds were provided by CEDENNA, FB 0807 grant.