The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

Centre for Nanobiology and Structural Biology Academy of Sciences of the Czech Republic 373 33 Nové Hrady Czech Republic

Department of Neurosurgery Medical University of Graz A 8010 Graz Austria

Gottfried Schatz Research Centre Medical University of Graz A 8010 Graz Austria

Gottfried Schatz Research Centre Medical University of Graz A 8010 Graz Austria Institute of Biophysics JKU Life Science Centre Johannes Kepler University Linz A 4020 Linz Austria

Institute of Biophysics JKU Life Science Centre Johannes Kepler University Linz A 4020 Linz Austria

Institute of Chemistry University of Graz Heinrichstraße 28 A 8010 Graz Austria

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