Abstract
Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.
Highlights
We sought to identify novel peptide sequences that interact at the Bcl-2 homology 3 (BH3) binding groove of Mcl-1
Use of the pan-active anti-apoptotic Bcl-2 inhibitor Sabutoclax as a displacer resulted in a sequence population that was 20% enriched with SB-02, 20% enriched with the non-BH3 peptide sequence SB-03 (NETVNTMLTYYY), and 40% enriched with the non-BH3 peptide sequence SB-04 (NETVELMQAYLH)
Helical wheel plotting of the reverse BH3 (rBH3) peptides, the BH3-like peptide SB-02 and other known BH3 peptides shows that they position a selection of hydrophobic residues opposite of the conserved acidic residue (Fig. 1)
Summary
We sought to identify novel peptide sequences that interact at the BH3 binding groove of Mcl-1. A short peptide derived from the glucokinase rBH3 motif exhibits binding to Mcl-1 comparable to that seen for a 12-residue Noxa A peptide (Table 1) We believe that this sequence represents a unique peptide motif present in native proteins that serves as a new class of Mcl-1-specific binding proteins, much as the BH3-only proteins are able to interact with various anti-apoptotic Bcl-2 family members. This finding will allow for development of improved Mcl-1 specific small molecule and stapled peptidebased therapeutics [16, 17]. Its identification may provide the basis for increased understanding of possible crosstalk taking place between a number of divergent cellular signaling and homeostatic processes and the regulation of apoptosis through Mcl-1
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