The protective role of type I IFNs in viral infections has been well established. More recently, type I IFNs have been clearly indicated in the pathogenesis of lupus and other autoimmune diseases (7
). One key goal is to identify a small molecule inhibitor that blocks type I IFN-mediated biological activity effectively, and that is responsible for the pathogenesis of autoimmune disease without compromising IFN-dependent anti-viral activity. We describe here an integrated chemical genomics-based drug discovery approach and its application in the screening for compounds that reverse a gene signature associated with the activation of the IFN-α pathway. Using these chemical probes, we identified the signaling nodes or the cross-talk pathways that can modulate the IFN-α responses. Compounds targeting HDAC, JAK/ STAT, and NF-κb pathways inhibited IFN-α responses. However only compounds targeting JAK/STAT and NF-κb inhibited IFN-α without markedly compromising anti-viral responses. Potentially, compounds targeting these pathways could be useful therapeutically for patients with SLE and other autoimmune conditions with INF-α involvement.
The major signaling pathway activated by type I IFNs involves sequential phosphorylation of the tyrosine residues of the JAK and STAT proteins, however, more and more evidence demonstrates that JAK-STAT signaling alone is not sufficient to explain all the biological effects of type I IFNs. The PI3k and p38 kinase pathways have emerged as critical additional components of IFN-induced signal transduction (6
). There also is emerging evidence that modulation of the function of a distinct STAT protein might account for a specific response. For example, a recent report showed that IFN-α and IFN-β-mediated activation of STAT 4 is required for IFN-γ production during viral infection (30
). However, STAT1 negatively regulates IFN-α dependent induction of IFN-γ (31
). It is not surprising that JAK inhibitors were identified in our assay and demonstrate strong inhibitory potency toward the IFN gene signature. However, despite the complexity of the IFN system, we are able to identify a concentration that effectively inhibits IFN-α related biological activity that contributes to the pathogenesis of the disease and yet retains IFN-α –dependent anti-viral activity. These data suggest that developing drugs that target JAK/ STAT signaling is an attractive direction for the treatment of autoimmune disease.
Besides STAT proteins, type I IFNs also activate other transcription factors. Among them, NF-κb is the most important transcription factor activated by IFNs. The key regulator of NF-κb is the signalsome, which comprises the scaffold protein NEMO and the two kinase inhibitors of NF-κb, Iκb kinase (IKK1) and IKK2. IKK2 is particularly important because it phosphorylates the NF-κb inhibitor Iκb, which is subsequently ubiq-uitinated by the SCFβtrcp
ligase system, leading to the degradation of the kinase and activation of p50-p65 dimer (32
). In addition to this major pathway for the p50–p65 activation, there is an alternative NF-κb pathway, again involving the IKKs, but leading to the activation of two other NF-κb proteins, p100 and RELB (32
). NF-κb positively and negatively regulates IFN-induced gene expression as well as antiviral activity. A recent report showed that NF-κb positively induced antiviral activity toward VSV (33
), whereas another report suggests that NF-κb suppressed both antiviral and immunomodulatory actions of IFN against the influenza virus (34
). The data presented here indicate that IKK2 inhibitors exhibit only small effects on IFN-dependent anti-HSV-1 activity, which is consistent with a previous observation that efficient replication of HSV-1 involves activation of the NF-κb pathway (35
). Interestingly, the IKK2 inhibitor that was identified in our assay has been shown to block inflammation in human airway smooth muscle and in a rat model of asthma (36
). Taken together, inhibitors of the NF-κb signaling pathway may present attractive approaches for the treatment of autoimmune disease.
The requirement for HDAC as positive regulators of IFN-α – and cytokine-induced gene expression has been well established. The deacetylase protein HDAC1 can interact with both the STAT1 and STAT 2 subunits of ISGF3. Whilst the inhibition of deacetylase activity has no effect on IFN-α signaling that leads to STAT phosphorylation, nuclear translocation, the assembly of the ISGF3 or the ISGF3 DNA binding, inhibition of HDAC does target downstream events required for IFN-stimulated gene expression (37
). All these data support a model that deacetylase enzyme may serve as a transcriptional coactivator for ISGF3. In addition, the IFN anti-viral response also requires HDAC activity. The anti-viral response against HCV, EMCV, and VSV were impaired in the presence of HDAC inhibitors (38
). In fact, treatment with HDAC inhibitors increased the viral cytopathic activity, most likely through inhibition of autocrine IFNs. Consistent with previous findings, the HDAC inhibitor Apicilin 1a also was identified in our primary screen and showed strong inhibition of the IFN-α gene signature. However, in our in vitro
HSV-1 assay, it also blocked IFN-α dependent anti-viral activity significantly. These results not only validate our screening approach, but also highlight the importance of HDAC pathway on viral replication. Among the ISG blocked by HDAC inhibitors will be genes crucial for antiviral response. Because the Apicilin 1a significantly impaired innate anti-viral immunity, this HDAC inhibitor is not considered suitable for therapy.
The approach that we developed here () can be adapted readily to screen a large library of small molecular compounds that modulate other cytokine signaling pathways. The unique gene signature sets of multiple cytokine pathways and the ones of the chemical probes identified in this study can be used to provide the necessary landmarks for screening, characterization, and optimization of the resulted active compounds generated in the gene expression profiling process. The use of molecular profiling throughout the drug discovery and development process is likely to increase dramatically over the next few years. This will be based on the clear advantages of multivariant biomarker approaches including the ability to provide a broad view of the biological state of a cell or tissue, the increased predictive power of monitoring multiple parameters simultaneously, and the power of correlating specific molecular phenotypes to clinical, histopathological, or disease model endpoints. It is clear that the increased use of molecular profiling will continue to make an important contribution to drug discovery and development efforts worldwide and, hopefully, will lead to lower failure rates, faster progression through the development process, and increasingly precise tests to match the right medicine with the right patient.
In summary, this is the first time that a large collection of well-annotated small molecule inhibitors targeting multiple in-tracellular signaling pathways has been evaluated using a combined chemical genomic approach. Our data suggest that targeting NF-κb and JAK/STAT signaling pathways may provide potential therapeutic benefit to type I interferon-related diseases such as the SLE, Sjö-gren’s syndrome among others. In this regard, our finding provides an important proof of principle that demonstrates that small molecule inhibitors that target these two signaling pathways represent potential drug candidates for IFN-α –associated autoimmune diseases.