NFκB signaling and MRTF/SRF signaling are not often related to each other. However, one protein, pirin, has been identified in both regulatory pathways with the potential to modulate gene transcription. Pirin is a non-heme iron-binding protein with a variety of proposed functions. Our lab discovered pirin as the main target of a series of small molecule compounds, CCG-1423 and related compounds. These compounds were originally discovered in a screen to find novel inhibitors for the MRTF/SRF signaling pathway, but pirin was identified as the main target. Evidence of pirin involvement in MRTF/SRF signaling is limited. However, there has been research linking pirin to NFκB signaling as a transcriptional co-activator. Specifically, it was found that Fe(III)-pirin binds to the p65 subunit of NFκB. In this dissertation, in Chapter 2, I first focused on confirming that pirin interacts with and modulates p65 signaling through binding to the p65/DNA complex in cells. However, after extensive investigation of pirin’s potential interaction with p65, I was not able to detect complex formation. Furthermore, knockdown of pirin did not alter p65 response gene expression. Further supporting the lack of binding to the p65/DNA complex, pirin immunofluorescence in NIH 3T3 fibroblasts showed primarily cytoplasmic localization and colocalization with the endoplasmic reticulum. Another proposed function of pirin is as a qeurcetinase. This research confirms pirin’s quercetinase activity and shows inhibition of enzymatic activity through CCG compounds. In Chapter 3, I focus on the development of a direct MRTF-A/SRF inhibitor peptide. The CCG compounds bind to pirin and do not show engagement with proteins in the MRTF/SRF signaling pathway. However, a 21-amino acid peptide from the basic region 1 of MRTF-A is sufficient for disrupting MRTF-A binding to SRF in vitro. Here, I show that we can use this peptide as a starting point to develop a direct inhibitor binding to SRF. By using Amplified Luminescence Proximity Homogenous Assay (ALPHA) I was able to detect that a single amino acid change of Tyr to Phe can decrease peptide IC50 almost 3-fold. Additionally, I found the shortest peptide sequence without loss in inhibitory capacity to be a 14-mer peptide. Unfortunately, in a cellular reporter assay, peptides did not show inhibition of MRTF/SRF signaling. Therefore, further improvement of peptides will be necessary to develop them as functional MRTF/SRF inhibitors in cells.
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