Chemical inhibitors of NF-κB have been widely sought for potential use as therapeutics for autoimmunity, inflammation, and cancer 13
. However, the most pharmaceutically tractable of the NF-κB-activating targets, the IKKs, represent a shared component of nearly all known NF-κB activation pathways and thus lack selectivity. In this regard, NF-κB activity is required for innate immunity and host-defense against microorganisms and various viral and bacterial pathogens. In addition to impaired host defense, broad-spectrum suppression of NF-κB pathways may reduce basal NF-κB activity and interfere with the function of NF-κB as a survival factor, leading to potentially toxic side effects. For example, IKK-β knockout mice die at mid-gestation from uncontrolled liver apoptosis 27
. In addition to potentially providing for novel therapeutic agents, development of pathway-selective inhibitors could lead to highly useful research tool compounds for interogating which pathways are important for specific cellular responses.
Using a chemical biology strategy, we devised chemical library screens for inhibitors that selectively inhibit the NF-κB activation pathway induced by PKCs. This pathway is uniquely involved in acquired immunity (rather than innate immunity), and has been linked to numerous autoimmune diseases and some types of lymphomas and lymphocytic leukemia 28
. NF-κB is also induced via PKC by many growth factor receptors. In this reagard, PKC hyperactivity has been associated with some solid tumors 29
, and thus the pathway interrogated here may also be relevant to a variety of malignancies. The NF-κB activation pathway linked to PKCs is known to involve proteins unique to this pathway among the nine known NF-κB activation pathways – namely, CARMA (Bimp)-family proteins, Bcl-10, and MALT (reviewed in 12
). Upon phosphorylation of CARMA1 by PKC in the context of antigen receptor signaling, these proteins form a complex, which recruits TRAF6, an E3 ligase that binds Ubc13, resulting in lysine 63-linked poly-ubiquitination of IKKγ/NEMO, resulting in IKK activation 30
. Caspase-8 is also recruited, resulting in proteolytic processing of c-FLIP, an event required for antigen receptor-induced activation of NF-κB 11
. The components of this complex required for IKK activation may not be completely known and an active complex has not been reconstituted in vitro using purified components, thus making biochemical screens difficult. For this reason, a cell-based strategy for chemical library screening was the only practical option.
Using HEK293 cells containing an NF-κB-driven reporter gene stimulated by PMA/Ionomycin, followed by an orthogonal screen in which we measured levels of the protein product of an endogenous NF-κB target gene (e.g. IL-8) secreted by these same cells, we screened 114,889 compounds, finding only one that had the desired properties, namely CID-2858522. This substituted 2-aminobenzimidazole compound potently inhibits NF-κB reporter gene activity and IL-8 production induced by PKC activators in HEK293 cells, with IC50 < 0.1 uM, while failing to inhibit NF-κB reporter gene activation by agonists of the other NF-κB activation pathways (). CID-2858522 also suppressed anti-IgM-stimulated proliferation of murine B-lymphocytes, as expected for an antagonist of the NF-κB activation pathway activated by B-cell antigen receptors. Because CID-2858522 inhibits NF-κB activation induced by phorbol esters and antigen receptors, it cannot be argued that the compound somehow interferes with uptake of PMA or other PKC-activating phorbol esters. Also, CID-2858522 did not inhibit PKC-mediated phosphorylation of various endogenous substrates in intact cells, arguing against a direct or indirect inhibitory effect on PKCs.
The observation that CID-2858522 only partially suppressed CD3/CD28- or PMA/Ionomysin-induced production of IL-2 by Jurkat T cells is consistent with the fact that NF-κB is only one of several transcriptional regulators of the IL-2 gene, which also include NF-κB, NFAT, and AP-1 20
. We documented that CID 2858522 inhibited NF-κB while failing to suppress AP-1 or NFAT reporter gene activity induced by PKC. Furthermore, given that a variety of NF-κB-activating cytokines were elaborated upon stimulation of cultured lymphocytes with antibodies cross-linking CD3 (TCR) or surface IgM (BCR), it is perhaps not surprising that CID-2858522 only partially suppressed anti-IgM-induced proliferation of primary B-cells and had little effect on anti-CD3/CD28-induced T-cell proliferation. In contrast, an IKK inhibitor essentially completely suppressed lymphocyte proliferation at concentrations of ~ 5 μM, consistent with its ability to neutralize nearly all known NF-κB activation pathways. CID-2858522 also inhibited anti-IgM-induced expression of the endogenous NF-κB target gene, TRAF1, in CLL B-cells. In this regard, the TRAF1 gene promoter contains four NF-κB target sites and a TATA-box, but essentially no other recognizable transcriptional elements 22
, thus making it a good surrogate marker of NF-κB activity in primary cells.
Although the mechanisms involved in antigen receptor-mediated NF-κB activation (upstream of PKC activation) in T cells and B cells are distinct, the downstream events following PKC activation share great similarity. Knockout mice models showed that CARMA1, Bcl-10 and MALT1 are required for antigen receptor-induced NF-κB activation and proliferation of both T cells and B cells 16, 31
. However, CARMA1 mutant mice exhibited normal T but impaired B cell development 32
and MALT1 deficiency has only mild effects on B cell activation MALT1 33
, indicating that the signal transduction apparati by which antigen receptors stimulate NF-κB downstream of PKC activation in T cells versus B cells are not identical. In this regard, it is also possible that antigen receptors and other upstream activators of PKCs induce NF-κB activation by more than one pathway, with CID-2858522 inhibiting only one of them. In this regard, it will be interesting to explore whether various lymphocyte subsets differ in their reliance on the NF-κB-activation pathways targeted by CID-2858522.
The mechanism by which CID-2858522 suppresses PKC-induced NF-κB activity remains to be determined. We mapped at least one site of action of this compound downstream of PKCs and upstream of IKK-β. PKCs induce phosphorylation of CARMA1, an event that was not inhibited by CID-2858522. This compound also neither inhibited PMA-induced recruitment of Bcl-10, MALT, TRAF6, Caspase-8, or IKKγ to CARMA1/CARMA3, nor did it inhibit caspase-8 or FLIP proteolytic processing. The active compound however selectively inhibited IKK-β phosphorylation induced by PkC activators but not TNFα, suggest CID-2858522 acts upstream of IKK-β. However, we cannot exclude the possibility that CID-2858522 was more than one site of action within the PKC-driven pathway for NF-κB activation, including acting at steps downstream of IKKβ.
The CARMA family proteins includes 3 members in mammals, which each contain a N-terminal CARD domain followed by a coiled-coil domain, a PDZ domain, a SH3 domain, and a C-terminal guanylate kinase-like (GUK) domain 34-36
. Predominantly expressed in spleen, thymus, and peripheral blood leukocytes (PBL), CARMA1 has been definitively implicated in antigen receptor signaling. In contrast, CARMA3 is expressed in broad range of tissues but not in spleen, thymus, or PBL 37
and CARMA2 is expressed only in placenta. Suppression of selected members of the CARMA family could provide another plausible explanation for partial inhibition by CID-2858522 of events such as IL-2 production by CD3/CD28- or PMA/Ionomycin-stimulated Jurkat cells.
In summary, using a chemical biology approach, we have identified the first selective chemical inhibitor of the PKC-initiated NF-κB activation pathway. This compound and its active analogs provide novel research tools for elucidating the role of this NF-κB pathway in cellular responses, and may pave the way for future therapeutic applications of specific inhibitors of selective pathways involved in pathogenic activation of NF-κB.