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Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

ABSTRACT: The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

SUBMITTER: Dixon AS

PROVIDER: S-EPMC3313827 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

Dixon Andrew S AS Miller Geoffrey D GD Bruno Benjamin J BJ Constance Jonathan E JE Woessner David W DW Fidler Trevor P TP Robertson James C JC Cheatham Thomas E TE Lim Carol S CS

Molecular pharmaceutics 20111212 1

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major c ...[more]

PMID: 22136227

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Project description:The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL). As a result of the so-called Philadelphia Chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation, relative to traditional small molecule therapeutics. Here, we iterate a new generation of our inhibitor against Bcr-CC mediated Bcr-Abl assembly that is designed to address these constraints through incorporation of all-hydrocarbon staples spanning i, i + 7 positions in helix α2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to design and characterize single and double staple candidates in silico, evaluating binding energetics and building upon our seminal work modeling single hydrocarbon staples when applied to a truncated Bcr-CC sequence. This strategy enables us to efficiently build, characterize, and screen lead single/double stapled CPP-CCmut3-st candidates for experimental studies and validation in vitro and in vivo. In addition to full-length CPP-CCmut, we model a truncated system characterized by deletion of helix α1 and the flexible-loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems without CPP, with cyclized CPP, and with linear CPP, for a total of six systems which comprise our library. From this library, we present lead stapled peptide candidates to be synthesized and evaluated experimentally as our next-generation inhibitors against Bcr-Abl.

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Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

Publications

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

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