Targeting a DNA Binding Motif of the EVI1 Protein by a Pyrrole–Imidazole Polyamide

• Published in: Biochemistry (Easton) 2011-12, Vol.50 (48), p.10431-10441
• Main Authors: Zhang, Yi, Sicot, Géraldine, Cui, Xiaohui, Vogel, Marion, Wuertzer, Charles A, Lezon-Geyda, Kimberly, Wheeler, John, Harki, Daniel A, Muzikar, Katy A, Stolper, Daniel A, Dervan, Peter B, Perkins, Archibald S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Line, Transformed; Cell Line, Tumor; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; Drug Delivery Systems - methods; Growth Inhibitors - chemistry; Growth Inhibitors - metabolism; Growth Inhibitors - pharmacology; Humans; Imidazoles - chemistry; Imidazoles - metabolism; Imidazoles - pharmacology; MDS1 and EVI1 Complex Locus Protein; Molecular Sequence Data; Mutagenesis, Site-Directed; Myeloid Cells - drug effects; Myeloid Cells - metabolism; Myeloid Cells - pathology; Nylons - chemistry; Nylons - metabolism; Nylons - pharmacology; Protein Binding - genetics; Proto-Oncogenes - genetics; Pyrroles - chemistry; Pyrroles - metabolism; Pyrroles - pharmacology; Rats; Retroviridae - genetics; Transcription Factors - antagonists & inhibitors; Transcription Factors - chemistry; Transcription Factors - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi200962u

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole–imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure–function studies on both EVI1 and the t(3;21)­(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1–7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole–imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.