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

Zhang HZ, Xu XL, Chen HY, Ali S, Wang D, Yu JW, Xu T, Nan FJ - Acta Pharmacol. Sin. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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 IC50 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.

No MeSH data available.


Related in: MedlinePlus

Compound 1 inhibits the CRAC channel by specifically targeting the ORAI1 protein. (A) Ca2+ influx data indicate that Compound 1 decreases the calcium level in the O1S1 opened CRAC channel. (B) Compound 1 partially inhibits the constitutively opened MSS CRAC channel. (C) Compound 1 completely inhibits the constitutively opened V102A CRAC channel. (D) Compound 1 partially inhibits the O1S1 CRAC channel. (E) Compound 1 does not inhibit the O2S1 CRAC channel. CRAC current was recorded, and the data were processed into the leak-subtracted current-voltage relationship at −100 mV. The black, red and blue lines represent the current trace in untreated cells (MSS, n=10; V102A, n=6; O1S1, n=7; O2S1, n=7), the current trace in cells treated with 10 μmol/L of compound 1 (MSS, n=10; V102A, n=6; O1S1, n=9; O2S1, n=7), and the current trace in cells treated with 10 μmol/L of YM58483 (MSS, n=7; O1S1, n=7; O2S1, n=7), respectively.
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fig3: Compound 1 inhibits the CRAC channel by specifically targeting the ORAI1 protein. (A) Ca2+ influx data indicate that Compound 1 decreases the calcium level in the O1S1 opened CRAC channel. (B) Compound 1 partially inhibits the constitutively opened MSS CRAC channel. (C) Compound 1 completely inhibits the constitutively opened V102A CRAC channel. (D) Compound 1 partially inhibits the O1S1 CRAC channel. (E) Compound 1 does not inhibit the O2S1 CRAC channel. CRAC current was recorded, and the data were processed into the leak-subtracted current-voltage relationship at −100 mV. The black, red and blue lines represent the current trace in untreated cells (MSS, n=10; V102A, n=6; O1S1, n=7; O2S1, n=7), the current trace in cells treated with 10 μmol/L of compound 1 (MSS, n=10; V102A, n=6; O1S1, n=9; O2S1, n=7), and the current trace in cells treated with 10 μmol/L of YM58483 (MSS, n=7; O1S1, n=7; O2S1, n=7), respectively.

Mentions: Compound 1 was evaluated to determine the inhibitory mechanism on the CRAC channel (Figure 3). As shown in Figure 3A and 3B, 10 μmol/L of compound 1 effectively decreased the high intracellular calcium level induced by the TG-opened CRAC channels in the ORAI1 and STIM1 stably co-expressed HEK293 cells. To analyze the target protein and inhibitory effect of compound 1, we tested it in constitutively opened CRAC channels that were formed by ORAI1-SS (monomer ORAI1 covalently linked with two S336–485 domains, MSS)25 and the ORAI1 mutant, V102A26. In MSS cells, the Ca2+ influx data showed that 30 μmol/L of compound 1 maximally inhibited 78% of the calcium level mediated by the MSS construct and that the calculated IC50 was approximately 0.2 μmol/L. Patch clamp data confirmed that 10 μmol/L of compound 1 inhibited 54% of the total MSS current. Although the inhibitory effect of compound 1 on the MSS channels was partial, this result indicated that the possible target site of compound 1 was located on ORAI1, the S (336–485) domain, or both, instead of other regions, except 336-485, on STIM1. The ORAI1 mutant, V102A, produces no Ca2+ selective, constitutively opened CRAC channels, even in the absence of STIM126. The inhibitory effect of compound 1 on these STIM1-free V102A mutant channels is shown in Figure 3C and indicates that 10 μmol/L of compound 1 completely, inhibits the calcium level and the current mediated by the opened V102A channel, which further demonstrates that the target protein of compound 1 is ORAI1.


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

Zhang HZ, Xu XL, Chen HY, Ali S, Wang D, Yu JW, Xu T, Nan FJ - Acta Pharmacol. Sin. (2015)

Compound 1 inhibits the CRAC channel by specifically targeting the ORAI1 protein. (A) Ca2+ influx data indicate that Compound 1 decreases the calcium level in the O1S1 opened CRAC channel. (B) Compound 1 partially inhibits the constitutively opened MSS CRAC channel. (C) Compound 1 completely inhibits the constitutively opened V102A CRAC channel. (D) Compound 1 partially inhibits the O1S1 CRAC channel. (E) Compound 1 does not inhibit the O2S1 CRAC channel. CRAC current was recorded, and the data were processed into the leak-subtracted current-voltage relationship at −100 mV. The black, red and blue lines represent the current trace in untreated cells (MSS, n=10; V102A, n=6; O1S1, n=7; O2S1, n=7), the current trace in cells treated with 10 μmol/L of compound 1 (MSS, n=10; V102A, n=6; O1S1, n=9; O2S1, n=7), and the current trace in cells treated with 10 μmol/L of YM58483 (MSS, n=7; O1S1, n=7; O2S1, n=7), respectively.
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Related In: Results  -  Collection

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fig3: Compound 1 inhibits the CRAC channel by specifically targeting the ORAI1 protein. (A) Ca2+ influx data indicate that Compound 1 decreases the calcium level in the O1S1 opened CRAC channel. (B) Compound 1 partially inhibits the constitutively opened MSS CRAC channel. (C) Compound 1 completely inhibits the constitutively opened V102A CRAC channel. (D) Compound 1 partially inhibits the O1S1 CRAC channel. (E) Compound 1 does not inhibit the O2S1 CRAC channel. CRAC current was recorded, and the data were processed into the leak-subtracted current-voltage relationship at −100 mV. The black, red and blue lines represent the current trace in untreated cells (MSS, n=10; V102A, n=6; O1S1, n=7; O2S1, n=7), the current trace in cells treated with 10 μmol/L of compound 1 (MSS, n=10; V102A, n=6; O1S1, n=9; O2S1, n=7), and the current trace in cells treated with 10 μmol/L of YM58483 (MSS, n=7; O1S1, n=7; O2S1, n=7), respectively.
Mentions: Compound 1 was evaluated to determine the inhibitory mechanism on the CRAC channel (Figure 3). As shown in Figure 3A and 3B, 10 μmol/L of compound 1 effectively decreased the high intracellular calcium level induced by the TG-opened CRAC channels in the ORAI1 and STIM1 stably co-expressed HEK293 cells. To analyze the target protein and inhibitory effect of compound 1, we tested it in constitutively opened CRAC channels that were formed by ORAI1-SS (monomer ORAI1 covalently linked with two S336–485 domains, MSS)25 and the ORAI1 mutant, V102A26. In MSS cells, the Ca2+ influx data showed that 30 μmol/L of compound 1 maximally inhibited 78% of the calcium level mediated by the MSS construct and that the calculated IC50 was approximately 0.2 μmol/L. Patch clamp data confirmed that 10 μmol/L of compound 1 inhibited 54% of the total MSS current. Although the inhibitory effect of compound 1 on the MSS channels was partial, this result indicated that the possible target site of compound 1 was located on ORAI1, the S (336–485) domain, or both, instead of other regions, except 336-485, on STIM1. The ORAI1 mutant, V102A, produces no Ca2+ selective, constitutively opened CRAC channels, even in the absence of STIM126. The inhibitory effect of compound 1 on these STIM1-free V102A mutant channels is shown in Figure 3C and indicates that 10 μmol/L of compound 1 completely, inhibits the calcium level and the current mediated by the opened V102A channel, which further demonstrates that the target protein of compound 1 is ORAI1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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 IC50 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.

No MeSH data available.


Related in: MedlinePlus