Project Summary/Abstract
The long-term goals of this proposal are to explore the protein dimerization interface as an area for therapeutic
intervention. Protein dimerization/oligomerization is a recurring theme in biology representing the mechanism
by which hundreds of proteins regulate key cellular processes such as enzymatic activity and signal transduction.
This non-covalent protein homo- or heterodimerization is mediated by hydrophobicity and both shape and charge
complementarity. Once thought to be undruggable, dimer interfaces are emerging as an area for powerful
therapeutic intervention for inflammatory diseases, pain, genetic diseases, cancer, and other diseases. The goal
of this proposal is to develop a clinically relevant small protein dimerization inhibitor. As a model, we will use
our dimerization coiled-coil (cc) inhibitor of Bcr-Abl. Bcr-Abl is an example of a protein that must dimerize to
enable its oncogenic activity. Bcr-Abl, results from an abnormal chromosomal translocation, manifests as a
constitutively active tyrosine kinase and causes of 95% of chronic myeloid leukemias (CML). We build on our
novel, computationally designed Bcr coiled-coil mutant (ccmut) that selectively dimerizes with Bcr-Abl and inhibits
its activity. When virally delivered as a gene, ccmut is effective against wild-type and mutant forms of Bcr-Abl.
Our ccmut specifically favors heterodimerization with Bcr-Abl to disrupt Bcr-Abl dimerization, a necessary step for
oncogenesis, and thus represents a novel therapeutic strategy. We have also fused ccmut to a non-toxic cell-
penetrating peptide with known leukemia cell specificity and showed that it disrupts Bcr-Abl dimerization and
enters and kills leukemia cells. For this proposal, we will explore peptide stapling technologies (to increase
proteolytic stability) and native chemical ligation to synthesize 2 shorter stapled peptides into a longer therapeutic
protein domain. We will first computationally model possible staple locations that maintain target affinity. These
stapled versions (CPP-St-ccmut) are predicted to enter cells, resist serum proteolysis, bind to Bcr-Abl and inhibit
its activity. We will then test the activity of our constructs with and without TKI ponatinib to test “multidomain
targeting” of Bcr-Abl in CML cell lines, CML patient samples, and a CML animal model. Aims are as follows: Aim
1: Computationally design and synthesize with solid state peptide synthesis and native chemical ligation, a
leukemia-specific, stapled cc inhibitor (CPP-St-ccmut) against Bc-Abl. Aim 2: Determine cell internalization,
binding, and apoptotic ability of CPP-St-ccmut candidates in leukemic cell lines including those with clinically
relevant mutations that are resistant to TKIs, and cells derived from patient samples, alone and in combination
with ponatinib. Aim 3: Demonstrate efficacy of CPP-St-ccmut with and without ponatinib in a simple, pre-clinical
mouse model of CML (syngeneic mouse model using intravenously injected BaF/3 cells expressing drug-
resistant Bcr-Abl variants, including compound mutants). Our goal is to develop a stapled protein domain
targeting the protein-protein dimerization interface Bcr-Abl kinase.