Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Abstract

Aim: Ca 2+ -release-activated Ca 2+ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors. Methods: HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca 2+ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP. Results: A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca 2+ influx with IC 50 of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca 2+ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca 2+ influx inhibition. The N,N′-disubstituted or N′-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity. Conclusion: 1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca 2+ influx inhibition, immune inhibition and low cytotoxicity.

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca 2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

“…The urea analogue 12 was the most potent of a number of analogues reported 35 as specific inhibitors of Orai1, but was not active in our assay. Finally, the extensively-studied biphenyl analogue Synta66 (13) [36][37][38] has been demonstrated to be a potent (IC 50 26 nM by patch-clamp assay) and selective Orai1 inhibitor.…”

“…The percentage inhibition of the maximum peak height for each concentration of each compound normalized to its corresponding DMSO control was calculated and plotted separately for peak 1 (a measure of the release of endoplasmic reticulum calcium store) by addition of CPA to assess potential non-specific effects on Ca 2+ homeostasis and peak 2 (a measure of store-operated Ca 2+ influx). 35,54 Where the % inhibition of peak 2 (SOCE) did not exceed 10% at 100 µM in initial assessments, these compounds were defined as not active.…”

Store-Operated Crac Channel Inhibitors: Opportunities and Challenges

“…Furthermore, this compound could specifically reduce ORAI1/STIM1-mediated Ca 2+ entry, while exhibited no inhibitory effect on other ORAI channels. These results indicated that [128].…”

“…A series of 1-phenyl-3-(1-phenylethyl)urea derivatives has been recently identified as CRAC channel inhibitors. As the lead compound, compound 22 could inhibit Ca 2+ influx with IC 50 of 3.25 ± 0.17 μM in HEK293 cells stably co-expressing ORAI1 and STIM1 [128]. The Ca 2+ influx assay and electrophysiological experiments showed that compound 22 could partially inhibit Ca 2+ entry in constitutively opened CRAC channels which were formed by ORAI1-SS (monomer ORAI1 covalently linked with two S336-485 domains) and completely inhibit the Ca 2+ entry and the current mediated by the opened STIM1free V102A channel (a mutant of ORAI1), which is a constitutively opened CRAC channel, even in the absence of STIM1.…”

“…A total of 40 derivatives have been synthesized, and their primary structure-activity relationships (SARs) study showed that the alkyl substituent on the α-position of the N-phenylethyl group is vital for their inhibition on Ca 2+ influx [128]. Notably, among these derivatives, compound 23 exhibited low cytotoxicity and relatively improved inhibition of IL-2 production in the Jurkat cell line [128].…”