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  • The effects of C on the NF


    The effects of C646 on the NF-κB pathway could be explained by the role for p300 that has been described in the regulation of the NF-κB pathway [5]. For instance, acetylations of the p65 NF-κB subunit on lysines 218, 221 and 310 are mediated by the HATs p300 and PCAF [11] and increase transcriptional activity. In contrast, acetylations on lysines 122 and 123 decrease transcriptional activity and are also mediated by p300 [12]. Furthermore, upon DNA binding, the transcriptional activation domain of p65 interacts with the HATs p300 and CBP as co-activators of gene transcription [13]. Thus, p300 plays a crucial role in the regulation of acetylation of specific lysine residues of NF-κB, which then determine NF-κB transcriptional capacity, DNA binding and duration of action. This suggests that inhibition of the HAT p300 using small molecule inhibitors such as C646 may allow for regulation of gene expression via the NF-κB transcription factor [7]. Since the NF-κB pathway is a key factor in inflammatory responses, this calls for investigation of the potential of C646 to suppress these responses. Of particular interest may be applications in inflammatory lung diseases such as Licofelone and chronic obstructive pulmonary disease (COPD), which still pose a major health problem. Interestingly, a role for the NF-κB pathway has been described in these diseases, as reviewed [14]. Increased NF-κB activity has been observed in the bronchial epithelium and peripheral blood mononuclear cells of asthmatic patients [15], [16]. Increased nuclear localization of p65 was observed in sputum macrophages during exacerbations of COPD [17] and also in bronchial biopsies of stable COPD patients [18]. Several lines of research have focused on modulating NF-κB activity as a novel therapeutic strategy for the treatment of asthma and COPD [14]. We hypothesize that pharmacological inhibition of the HAT p300 by small molecule inhibitor C646 will result in inhibition of pro-inflammatory gene expression via inhibition of the NF-κB signaling pathway. For these reasons, we set out to explore the p300 HATi C646 in a model system for inflammatory lung diseases. We observed decreased NF-κB reporter gene activity in RAW-Blue murine macrophages, and decreased pro-inflammatory gene expression in RAW264.7 murine macrophages as well as in murine lung tissue slices. Next to this we set out to quantify changes in lysine acetylation in the cellular substrates of p300. Acetylation of histone H3 and histone H4 was quantified using mass spectrometry in RAW264.7 macrophages. Counterintuitively, C646 increased acetylation on histone H3 residues 18–26, containing H3 K18 and H3 K23. Based on these findings and the structural properties of C646 we investigated its inhibitory potential on recombinant HDACs and found inhibition from 7μM and higher concentrations for type I and II HDACs. Importantly, these findings call for further optimization of the selectivity profile of the p300 HATi C646 and its derivatives.
    Materials and methods
    Discussion The inhibitory effects of C646 on pro-inflammatory gene expression in RAW264.7 macrophages and in murine precision-cut lung tissue slices upon receiving an LPS and IFNγ stimulus are promising for further development of this inhibitor in model systems of inflammatory lung diseases. The results suggest inhibition of NF-κB signaling, in line with the observed decrease in NF-κB promoter activity in a reporter gene assay upon C646 pre-treatment. This would support previous lines of research focusing on reducing NF-κB activity in inflammatory lung diseases [14] and indicate that this is a promising strategy. This is also in line with previous studies indicating inhibition of the NF-κB pathway upon C646 treatment in prostate cancer cells [10], and decreased expression of the NF-κB mediated pro-inflammatory gene COX-2 upon C646 treatment in rats [9].