Identification of Orai1 Channel Inhibitors by Using Minimal Functional Domains to Screen Small Molecule Microarrays

Abstract

Store-operated calcium (SOC) channels are vital for activation of the immune cells, and mutations in the channel result in severe combined immunodeficiency in human patients. In lymphocytes, SOC entry is mediated by the Orai1 channel, which is activated by direct binding of STIM1. Here we describe an alternative approach for identifying inhibitors of SOC entry using minimal functional domains of STIM1 and Orai1 to screen a small-molecule microarray. This screen identified AnCoA4, which inhibits SOC entry at submicromolar concentrations and blocks T cell activation in vitro and in vivo. Biophysical studies revealed that AnCoA4 binds to the C terminus of Orai1, directly inhibiting calcium influx through the channel and also reducing binding of STIM1. AnCoA4, unlike other reported SOC inhibitors, is a molecule with a known binding site and mechanism of action. These studies also provide proof of principle for an approach to ion channel drug discovery.

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

“…This process leaves a "STIM1-free" but partially active Orai1 P245L that contributes to the measured current and therefore to an apparent reduction of the slow inactivation process. Two other mutants, G98S and L138F, which have also been associated with tubular aggregate myopathy and congenital miosis syndromes [65], share the defect that we identified in P245L of constitutive channel opening [43,53,65]. Although the constitutive conductance of Orai1 P245L may be less pronounced in heterozygous patients than in heterologous overexpression experiments due to formation of mutant and WT heteromultimers, based on results presented here and on previous findings, we propose that STIM1-independent constitutive channel activation of Orai1 channels is likely the common factor that underlies the disease state.…”

“…But how is CAD-CBD interaction transmitted to the channel gate? Our finding that mutations that are expected to disrupt the P245 proline bend of TM4 open the channel suggests that the rearrangement of TM1 that has been proposed to underlie gating [31,52,53] may be triggered by a conformational change in TM4. We do not know whether mutations to P245 induce a conformational change in Orai1 that is similar to that induced by STIM1.…”

“…However, the finding that STIM1 binding to P245L Orai1 maximally activates the channel indicates that the mechanism that couples STIM1 binding with pore opening is preserved. Strikingly, the P245 bend of TM4 is situated in the same membrane plane as residues in each of the other helices (Supplementary information, Figure S9) that have been implicated in gating: residue G98 in TM1, which is thought to pivot to open the pore [53], residue H134 in TM2 whose mutation constitutively open the channel independently of STIM1 [54] and residue G183 in TM3, whose mutation prevents STIM1-dependent channel activation [55], but allows for channel opening by 2-APB, indicating a defect in STIM1 coupling but not in gating (Supplementary information, Figure S9). …”

Localized Store-Operated Calcium Influx Represses CD95-Dependent Apoptotic Effects of Rituximab in Non-Hodgkin B Lymphomas

“…To date, intense drug discovery efforts have focused on developing small-molecule CRAC-channel antagonists (40)(41)(42) or specific Orai1 Abs (43) to treat autoimmune diseases and chronic inflammation. We propose using these compounds in association with RTX or, more generally, with drugs inducing a CD95-dependent apoptotic pathway, to constitute a new therapeutic opportunity for patients.…”

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…For experiments with CaM and rSK2, a previously described HEK293 cell line with an inducible mCherry-STIM1-T2A-myc-Orai1 was used (Sadaghiani et al, 2014).…”