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Inhibition of inducible nitric oxide synthase expression by a novel small molecule activator of the unfolded protein response.

Symons KT, Massari ME, Dozier SJ, Nguyen PM, Jenkins D, Herbert M, Gahman TC, Noble SA, Rozenkrants N, Zhang Y, Rao TS, Shiau AK, Hassig CA - Curr Chem Genomics (2008)

Bottom Line: Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR.Further, erstressin activates the transcription of multiple genes involved in the UPR.These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

No MeSH data available.


Compound 1, a synthetic 3-trifluoromethyl-N-methylpyrazole, MW=349.76, inhibits cellular iNOS activity. Compound 2, 3-methyl-N-methylpyrazole, MW=295.79, a structural analog of Compound 1, is inactive in cellular iNOS inhibition assays.
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Figure 1: Compound 1, a synthetic 3-trifluoromethyl-N-methylpyrazole, MW=349.76, inhibits cellular iNOS activity. Compound 2, 3-methyl-N-methylpyrazole, MW=295.79, a structural analog of Compound 1, is inactive in cellular iNOS inhibition assays.

Mentions: To identify small molecules that reduce the production of iNOS-derived NO, we developed a homogeneous forward chemical genetic screen in the murine macrophage cell line RAW 264.7. Stimulation of RAW 264.7 cells with bacterial lipopolysaccharide (LPS) and IFN-γ results in the increased expression of a host of inflammatory genes including iNOS [10]. The effect of compounds on iNOS activity was quantified by indirectly measuring the production of NO from cells. This approach enables not only the detection of compounds that inhibit iNOS directly, but also compounds that act upstream in the iNOS-NO axis. Using this assay, we screened a 650,000 compound library using a fully automated ultra high-throughput robotic system. Compounds were tested in single point at 10 μM and hits were confirmed in 7-point dose response. A total of 330 compounds (0.05% hit rate) demonstrated significant reduction in NO production (>30% inhibition) without detectable cytotoxicity. One inhibitor identified from this screen, compound 1, shared no structural similarity to previously described iNOS inhibitors (Fig. 1). Several analogs of compound 1 were synthesized and tested in the homogeneous cell-based NO detection and other follow-up assays. These analogs provided a cursory assessment of the structure activity relationship for the series. Replacement of the R1 2-chlorobenzylthio with the 3-methyl derivative (Cmpd 3) was tolerated, though removal of the 2-substituent (Cmpd 4) or addition of a 6-fluoro substituent (Cmpd 5) reduced activity across all assays. Modifications to the R2 oxime through alkylation (Cmpd 6), conversion to a hydrazone (Cmpds 7 and 8) or replacement with a carboxylic acid or amide (Cmpds 9 and 10) greatly reduced or eliminated activity. Two additional structurally related analogs of merit were identified (Cmpds 11 and 12) with R1 sulfones in place of the thio ether and a nitrile in place of the R2 oxime. Compound 12 displayed the most potent in vitro activity profile of the series, and the data indicate that the oxime is not essential for activity of compounds within this chemical series. Strikingly, replacement of the R3 trifluoromethyl with a methyl group (Cmpd 2) resulted in an inactive molecule (Fig. 1 and Table 1). In contrast to compound 1, which inhibited NO production with an IC50 of 2.8 μM, compound 2 was essentially inactive in the NO detection assay (Fig. 2A). Demonstrating that its effects are conserved across species, compound 1 also inhibited NO production in cytokine-stimulated human A172 glioblastoma cells with an IC50 of 4.2 μM. As seen in RAW264.7 cells, compound 2 did not affect NO production in A172 cells (Fig. 2A). The subtle structural difference between compound 1 and compound 2, combined with the significant difference in ability of the two compounds to block cellular iNOS activity, facilitated a pharmacological approach to elucidating the mechanism of action of compound 1.


Inhibition of inducible nitric oxide synthase expression by a novel small molecule activator of the unfolded protein response.

Symons KT, Massari ME, Dozier SJ, Nguyen PM, Jenkins D, Herbert M, Gahman TC, Noble SA, Rozenkrants N, Zhang Y, Rao TS, Shiau AK, Hassig CA - Curr Chem Genomics (2008)

Compound 1, a synthetic 3-trifluoromethyl-N-methylpyrazole, MW=349.76, inhibits cellular iNOS activity. Compound 2, 3-methyl-N-methylpyrazole, MW=295.79, a structural analog of Compound 1, is inactive in cellular iNOS inhibition assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803434&req=5

Figure 1: Compound 1, a synthetic 3-trifluoromethyl-N-methylpyrazole, MW=349.76, inhibits cellular iNOS activity. Compound 2, 3-methyl-N-methylpyrazole, MW=295.79, a structural analog of Compound 1, is inactive in cellular iNOS inhibition assays.
Mentions: To identify small molecules that reduce the production of iNOS-derived NO, we developed a homogeneous forward chemical genetic screen in the murine macrophage cell line RAW 264.7. Stimulation of RAW 264.7 cells with bacterial lipopolysaccharide (LPS) and IFN-γ results in the increased expression of a host of inflammatory genes including iNOS [10]. The effect of compounds on iNOS activity was quantified by indirectly measuring the production of NO from cells. This approach enables not only the detection of compounds that inhibit iNOS directly, but also compounds that act upstream in the iNOS-NO axis. Using this assay, we screened a 650,000 compound library using a fully automated ultra high-throughput robotic system. Compounds were tested in single point at 10 μM and hits were confirmed in 7-point dose response. A total of 330 compounds (0.05% hit rate) demonstrated significant reduction in NO production (>30% inhibition) without detectable cytotoxicity. One inhibitor identified from this screen, compound 1, shared no structural similarity to previously described iNOS inhibitors (Fig. 1). Several analogs of compound 1 were synthesized and tested in the homogeneous cell-based NO detection and other follow-up assays. These analogs provided a cursory assessment of the structure activity relationship for the series. Replacement of the R1 2-chlorobenzylthio with the 3-methyl derivative (Cmpd 3) was tolerated, though removal of the 2-substituent (Cmpd 4) or addition of a 6-fluoro substituent (Cmpd 5) reduced activity across all assays. Modifications to the R2 oxime through alkylation (Cmpd 6), conversion to a hydrazone (Cmpds 7 and 8) or replacement with a carboxylic acid or amide (Cmpds 9 and 10) greatly reduced or eliminated activity. Two additional structurally related analogs of merit were identified (Cmpds 11 and 12) with R1 sulfones in place of the thio ether and a nitrile in place of the R2 oxime. Compound 12 displayed the most potent in vitro activity profile of the series, and the data indicate that the oxime is not essential for activity of compounds within this chemical series. Strikingly, replacement of the R3 trifluoromethyl with a methyl group (Cmpd 2) resulted in an inactive molecule (Fig. 1 and Table 1). In contrast to compound 1, which inhibited NO production with an IC50 of 2.8 μM, compound 2 was essentially inactive in the NO detection assay (Fig. 2A). Demonstrating that its effects are conserved across species, compound 1 also inhibited NO production in cytokine-stimulated human A172 glioblastoma cells with an IC50 of 4.2 μM. As seen in RAW264.7 cells, compound 2 did not affect NO production in A172 cells (Fig. 2A). The subtle structural difference between compound 1 and compound 2, combined with the significant difference in ability of the two compounds to block cellular iNOS activity, facilitated a pharmacological approach to elucidating the mechanism of action of compound 1.

Bottom Line: Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR.Further, erstressin activates the transcription of multiple genes involved in the UPR.These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

No MeSH data available.