LMO protein 4 (LMO4) is a transcriptional co-regulatory protein that contributes to development, but also to the progression of breast cancer, although the mechanism or mechanisms involved are only partially known. The generation of inhibitors of LMO4 could form useful research tools for understanding these mechanisms of action and might form the basis of new breast cancer therapies. Previously, the laboratory has worked with β-strand peptide inhibitors of LMO4 based on natural binding partners of LMO4, but those peptides tend to lack specificity for LMO4 over related proteins.
Our hypothesis is that α-helical peptides can be more specific than β-strand peptides. We have designed two methodologies that will be used to identify novel α-helical peptides that bind to LMO4. For an initial screening tool we developed a split EGFP complementation system. EGFP was fragmented and tethered to the N- and C-terminal ends of LMO4 tethered constructs. In the positive control LMO4 interacts with the LIM interaction domain (LID) of its native binding partner LDB1, bringing the two fragments of EGFP together leading to fluorescence. In the negative control LMO4 and the nonsense peptide fail to interact and no fluorescence was detected. For validation we have been developing a yeast two hybrid competition assay in which a series of tethered LMO4-LID constructs were designed to gauge the binding affinity of novel peptides. Although the novel constructs tested thus far were unable to differentiate the binding affinities of previously generated computational LIDs, some constructs may stabilise LMO4 to increase sensitivity in the assay.