Enhancing the Therapeutic Potential of an Anti-Leukemic Peptide.

Despite ongoing success in the treatment of childhood acute lymphoblastic leukemia (ALL), patients harboring translocations involving the genetic locus 11q23 continue to have a poor prognosis. The majority of 11q23 translocations result in the formation of a functional fusion protein consisting of the N-terminus of MLL fused to the C-terminal portion of one of more than 30 fusion partners. This translocation results in a functional chimeric protein critical for leukemogenesis. Recently, our lab has identified an interaction between two common MLL fusion partners, AF4 and AF9. Through a series of mapping experiments we identified a small region of human AF4 to be sufficient for its interaction with AF9. Based on these studies, a synthetic peptide (PFWT) that mimics the AF9 binding site on AF4 was developed. Treatment of leukemia cells that express the MLL-AF4 protein with PFWT results in apoptosis with no observable affects on CD34+ hematopoietic stem cells, suggesting the AF9-AF4 interaction is a promising chemotherapeutic target. To improve upon the therapeutic potential of PFWT, we developed a high-throughput enzyme-linked colorimetric assay to identify peptidomimetics that block the AF9-AF4 interaction. A combinatorial peptide library was synthesized in which each position of the 10-mer sequence was substituted with either the α- or corresponding β-amino acid. β-Amino acids which are similar to α-amino acids but contain an additional carbon in their backbone were chosen because of their resistance to proteases (2¹⁰= 1024 possible unique sequences). To date, 30 of the peptides screened compare favorably with PFWT for disrupting the AF4-AF9 interaction. Sequencing of the peptides by MS/MS revealed substitutions at the N- and C-terminal ends are well tolerated. In addition, peptides can incorporate as many as three β-amino acids and still retain biological activity. These data are important for establishing a sequence with improved pharmacokinetic properties as compared to PFWT and serve as the first step in our design towards an optimal peptide sequence for drug development. They also validate the utility of a high-throughput assay system for drug screening. Future studies for the identified peptides include determining their biological half-lives, AF9 binding affinities, and ability to induce apoptosis in leukemia cells.