A New Selective Pharmacological Enhancer of the Orai1 Ca²⁺ Channel Reveals Roles for Orai1 in Smooth and Skeletal Muscle Functions

Abstract

Store-operated calcium (Ca 2+ ) entry is an important homeostatic mechanism in cells, whereby the release of Ca 2+ from intracellular endoplasmic reticulum stores triggers the activation of a Ca 2+ influx pathway. Mediated by Orai1, this Ca 2+ influx has specific and essential roles in biological processes as diverse as lactation to immunity. Although pharmacological inhibitors of this Ca 2+ influx mechanism have helped to define the role of store-operated Ca 2+ entry in many cellular events, the lack of isoform specific modulators and activators of Orai1 has limited our full understanding of these processes.Here we report the identification and synthesis of an Orai1 activity enhancer that concurrently potentiated Orai1 Ca 2+ -dependent inactivation (CDI). This unique enhancer of Orai1 had only a modest effect on Orai3 with weak inhibitory effects at high concentrations in intact MCF-7 breast cancer cells. The Orai1 enhancer heightened vascular smooth muscle cell migration induced by platelet-derived growth factor and the unique store-operated Ca 2+ entry pathway present in skeletal muscle cells. These studies show that IA65 is an exemplar for the translation and development of Orai isoform selective agents. The ability of IA65 to activate CDI demonstrates that agents can be developed that can enhance Orai1-mediated Ca 2+ influx but avoid the cytotoxicity associated with sustained Orai1 activation. IA65 and/or future analogues with similar Orai1-and CDIactivating properties could function to fine-tune physiological processes important in specific disease states, such as cellular migration and immune cell function.

1,2,4-Oxadiazole-Bearing Pyrazoles as Metabolically Stable Modulators of Store-Operated Calcium Entry

“…The identification of a SOCE enhancer among a class of inhibitors has been reported in three previously published classes of modulators, with two of them described by us and represented by pyrtriazoles (AL-2T ( 43 ), NM-3G ( 44 ), Figure 5 ), 35 and biphenyl triazoles (compound 45 , Figure 5 ) 39 and one reported in the literature (IA65 ( 46 ), Figure 5 ). 40 A similar behavior is also shared by 2-APB ( 47 ), a well-known inhibitor of IP3 receptors and TRP channels. The compound is a SOCE modifier in Orai1- and Orai3-expressing cells, acting as SOCE enhancer at low concentrations, while high concentrations induce a transient increase followed by complete inhibition.…”

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells

“…Several chemical inhibitors [ 23 , 24 , 25 , 26 , 27 ] as well as potentiators of SOCE [ 28 , 29 , 30 ] have been reported. However, 2-aminoethyl diphenylborinate (2-APB, Figure 1 ) remains the best characterized compound used to modulate store-operated CRAC channels, despite of its effects on other known targets [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

“…In this study, we used the MDA-MB-231 breast cancer cell line to screen our newly developed 2-APB analogues for their action on SOCE, using a fluorometric imaging plate reader (FLIPR)-based Ca 2+ imaging assay. FLIPR assays with MDA-MB-231 cells have been used in the past for discovery of SOCE modulators [ 26 , 30 , 72 ]. Earlier reports using RNA silencing have shown that SOCE in MDA-MB-231 cells is conducted by STIM1 and Orai1 [ 18 , 26 , 73 ].…”

Store-Operated Calcium Entry as a Therapeutic Target in Acute Pancreatitis: Discovery and Development of Drug-Like SOCE Inhibitors

“…Given that the focus of this study was to identify novel SOCE negative modulators for the treatment of AP, the profile of compound 47 was not investigated further, but its discovery supports the idea that minor structural modifications of SOCE inhibitors can interfere with channel gating and produce activators, as already observed for pyrtriazoles (AL-2T ( 57 ), NM-3G ( 58 ); Figure 5 ) 16 and for another recently described SOCE enhancer (IA65 ( 59 ), Figure 5 ). 22 47 therefore represents an enhancer of SOCE from a third distinct class of modulators and provides grounds to develop models to understand the mechanism by which this occurs.…”