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
]. 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.
). 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.
). 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.
). 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.
). 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.
Table 1. N-methylpyrazole-based compounds tested for inhibition of nitric oxide production in RAW264.7 cells (RAW/DAN) and A172 cells (A172/DAN) and iNOS protein expression in compound-treated RAW264.7 cells (RAW/Cytoblot). Data are representative IC50 values (more ...)
Interestingly, a counter screen for biochemical enzyme inhibition revealed that compound 1 failed to reduce iNOS activity, indicating that the compound might not directly inhibit the enzyme (data not shown). A tertiary 384-well microplate-based in-cell western blot assay (cytoblot) in RAW264.7 cells utilizing an iNOS-specific antibody determined that compound 1 inhibited LPS/IFN-γ-induced iNOS protein expression with an IC50 of 1.5 μM, a value comparable to that calculated from the NO detection assay (Supplemental Fig. ). To confirm the cytoblot findings, we performed immunoblot analysis and demonstrated that 10 μM compound 1 blocked iNOS expression in RAW264.7 cells. This effect was not seen when cells were treated with vehicle or compound 2, but was seen when global protein translation was blocked in cells using cycloheximide (Fig. , top panel). In contrast to the effects of compound 1 on endogenous iNOS, heterologous expression of recombinant iNOS in HEK293 cells was unperturbed by either compound 1 or compound 2, whereas expression was reduced in cells treated with cycloheximide (Fig. , bottom panel). These data indicate that compound 1 acts upstream in the iNOS pathway and reduces NO production in cells by reducing iNOS protein levels.
Next, we tested the effects of compound on cellular iNOS mRNA transcript levels. A172 cells and RAW264.7 cells were stimulated with proinflammatory factors and treated with compounds at a final concentration of 10 μM for 6 hours before isolating the RNA and performing real-time quantitative PCR to determine whether iNOS mRNA levels were affected. As seen in Fig. (), a statistically significant reduction in iNOS transcripts was measured following treatment with compound 1 relative to either compound 2 or vehicle (DMSO). The reduction of iNOS mRNA caused by compound 1 relative to compound 2 ranged from approximately 4.7-fold in RAW264.7 macrophages (p = 0.01) to 13.7-fold in A172 cells (p = 0.045). As seen for iNOS protein, compound 2 had no statistical effect on iNOS mRNA levels relative to vehicle (Fig. ). These results indicate that compound 1 reduces NO production in cytokine-stimulated cells, at least in part, by reducing cellular iNOS mRNA levels.
To identify the mechanism whereby compound 1 inhibited iNOS expression and to identify the biological pathway(s) impacted by compound 1, we then profiled global gene expression in A172 cells simultaneously treated with cytokine-stimulation media and compound. A significant difference was defined as ≥ 2-fold change (up or down) in mRNA transcript levels with a p value ≤0.05. In cells stimulated with cytokines for 30 minutes, no significant differences in gene expression were observed between cells treated with compound 1 or compound 2 at 10 μM. However, as compared to the 30 minute time-point, after 6 hours of incubation with proinflammatory cytokines and vehicle, approximately 3.8% of genes profiled exhibited altered expression, consistent with the well documented activation of inflammatory signaling pathways [11
]. Strikingly, at 6 hours there was virtually no difference in the gene expression profile (< 0.1 % of transcripts) of cells treated with compound 2 and cytokine as compared to cytokine treatment alone. In contrast, cytokine stimulated cells treated with compound 1 displayed a significantly different gene expression profile at the 6 hour time-point. A subset of genes with altered expression in compound 1 treated cells were common to those induced by cytokine stimulation. As anticipated from previously described qPCR results, iNOS
mRNA levels, which were induced by cytokine stimulation, were reduced 4.7-fold by coincubation with compound 1 (but unaffected by compound 2 treatment). Notably, in addition to iNOS
, the transcripts of several other inflammatory mediators normally upregulated by cytokines, (TNFα, IL1β, MCP-2
) were downregulated by compound 1 relative to compound 2 or vehicle (GEO submission GSE7806
). Gene expression was not universally reduced however, for example, transcripts of IL15
(2.4-fold), chemokine orphan receptor 1 (CMKOR1,
11.2-fold) and ATP-binding cassette protein 1 (ABCA1,
3.5-fold) were increased by compound 1 treatment (GEO submission GSE7806
Interestingly, in addition to altering inflammatory gene expression, compound 1 also affected the mRNA levels of multiple genes involved in the UPR. The transcription factors XBP-1
(3.3-fold), and CHOP
(22-fold) were significantly induced in compound 1-treated cells. Molecular chaperones ERdj4
(24-fold) and BiP
(2.7-fold) and ER to Golgi transporters SEC23B (2-fold) and SEC31L1 (4-fold) were also significantly elevated by compound 1. Further, Herpud1/ Herp, a membrane-associated protein which functions in ER associated degradation was upregulated 9.7-fold (Fig.
). Changes in the expression of several genes identified in the expression profiling study were confirmed by independent qPCR analysis (Supplemental Figs.
). Similar gene expression signatures have been described for known ER stressors, including tunicamycin and thapsigargin [12
] and these effects were absent in cells treated with compound 2. These and other findings described below suggest that compound 1 may induce the UPR, and as a result, we have named this compound erstressin and its inactive analog compound 2, nostressin.
Fig. (3). Erstressin (Compound 1) induces biochemical markers of ER stress and expression of genes involved in the UPR. Panel A. Erstressin increases mRNA levels of ER stress genes. Heat map of normalized microarray data from A172 cells simultaneously induced with (more ...)
Activation of the UPR gene expression program is initiated by at least three ER transmembrane protein sensors, including ATF-6, IRE-1, and PERK. Under normal conditions, all three proteins are maintained in inactive conformations by Grp78, an HSP70-family chaperone. The binding of accumulated unfolded proteins to Grp78 causes the dissociation of these three complexes and the activation of signal transduction. IRE-1 and PERK are both kinases that self-oligomerize upon release of Grp78. In the case of IRE-1, oligomerization-induced autophosphorylation activates an internal ribonuclease domain that triggers alternative splicing of its mRNA substrate, XBP-1
, encoding a bZIP transcription factor that activates downstream UPR genes. Oligomerized PERK undergoes autophosphorylation and then phosphorylates two substrates, the bZIP transcription factor NRF2 and elongation factor 2α (eIF2α). Cumulatively, these signaling events lead to the UPR gene transcription response [14
To determine if erstressin affects UPR gene expression by activating these pathways, we tested for several early markers of ER stress signaling. For comparison, we also characterized thapsigargin, which induces ER stress by inhibiting the Ca2+
-ATPase resident within the ER, thereby causing protein misfolding indirectly by disrupting the function of Ca2+
-dependent chaperones Grp78, Grp94, and calreticulin [15
]. Immunoblot analysis of protein extracts isolated from RAW264.7 cells treated with 10 μM erstressin for 30 minutes showed elevated eIF2α phosphorylation. This effect by erstressin was less pronounced than that seen in cells treated with 1 μM thapsigargin, but was completely absent in cells treated with 10 μM nostressin (Fig.
). Next, we tested whether the splicing of XBP-1
mRNA by the ER transmembrane ribonuclease IRE-1, was altered by erstressin treatment. PCR amplification of RNA samples isolated from HeLa cells incubated with 20 μM erstressin or 50 nM thapsigargin for 1 hour revealed the presence of alternatively spliced XBP-1
. Once again, 20 μM nostressin did not induce alternative splicing of XBP-1
). These findings suggest that, similar to thapsigargin, erstressin activates multiple UPR signaling pathways within the first 0.5 - 1 hour of compound treatment. The eIF2α phosphorylation was increased by erstressin treatment prior to any detectable changes in transcription (within 30 minutes), suggesting that this effect by erstressin is upstream of changes in UPR gene expression.
To eliminate the possibility that the effects of erstressin on iNOS were simply a consequence of reduced cell viability or general cytotoxicity, we tested the effects of the compound on cytokine induced A172 cells in several assays. Reduced metabolic activity and cell viability as a consequence of the depletion of cellular ATP stores was measured in cells treated with compound. Cellular ATP levels were unaffected at both 6 hours and 18 hours of treatment with either erstressin or nostressin, but substantially reduced in cells treated with the positive control, toxoflavin (Fig. ). In separate experiments, cells were stained with JC-1, a cationic dye which accumulates in the mitochondria under depolarized conditions, and can be used to detect reduced viability and apoptosis in individual cells. As anticipated from the ATP assay, no increase in JC-1-positive cells was observed at 6 hours or 18 hours of treatment with either erstressin or nostressin, even at concentrations of 10 μM or 30 μM (Fig. and data not shown). Similar results were obtained using an independent apoptosis assay (Annexin V) (data not shown). These findings suggest that cytotoxicity is unlikely to account for the observed reduction in iNOS expression and activity caused by erstressin in A172 cells.
To evaluate the potential of erstressin to suppress iNOS expression in vivo
, we determined the effects of erstressin on NO production in a model of inflammatory disease. Administration of LPS to rodents induces a systemic inflammatory response coincident with expression of iNOS [16
]. Consistent with its ability to reduce iNOS activity in cell culture, intraperitoneal administration of erstressin reduced NO metabolites in rat plasma following LPS stimulation by 50% relative to vehicle, p<0.001 (Fig.
). In a separate experiment, the potent iNOS inhibitor, 1400W, was tested as a comparator in the LPS model (Supplemental Fig.
). Although 1400W is a more efficacious compound (76% reduction in NO metabolites; p<0.0001), the results are consistent with an ability of erstressin to downregulate iNOS in vivo
during proinflammatory stimulation, providing for the possibility that a similar mechanism of action to that seen in cell culture experiments might downregulate iNOS in certain inflammatory disease settings.