Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains

Abstract

Rational design of compounds to mimic the functional domains of BCL-2 family proteins requires chemical reproduction of the biologic complexity afforded by the relatively large and folded surfaces of BCL-2 homology (BH) domain peptide alpha-helices. Because the intermolecular handshakes of BCL-2 proteins are so critical to controlling cellular fate, we undertook the development of a toolbox of peptidic ligands that harness the natural potency and specificity of BH alpha-helices to interrogate and potentially medicate the deregulated apoptotic pathways of human disease. To overcome the classic deficiencies of peptide reagents, including loss of bioactive structure in solution, rapid proteolytic degradation in vivo, and cellular impermeability, we developed a new class of compounds based on hydrocarbon stapling of BH3 death domain peptides. Here we describe the chemical synthesis of Stabilized Alpha-Helices of BCL-2 domains or SAHBs, and the analytical methods used to characterize their secondary structure, proteolytic stability, and cellular penetrance.

Figure 22.1

Approaches to covalent α-helical stabilization have included the use of (A) lactam bridges, (B) disulfide bridges, (C) ruthenium-catalyzed ring closing metathesis (RCM) of O-allyl serine residues, and (D) RCM of α,α-disubstituted non-natural amino acids bearing alkyl tethers.

Figure 22.2

Synthetic scheme for generating the chiral α,α-disubstituted non-natural amino acids used to staple bioactive peptides.

Figure 22.3

Design considerations for installing hydrocarbon staples derive from structural data (e.g., BAK BH3/BCL-X_L ΔC; PDB 1BXL [Sattler et al., 1997]) that highlight key interacting surfaces to be avoided. Several (i, i + 4) residues indicated as red balls on the noninteracting surface of the BAK BH3 helix are amenable to replacement with the S5 non-natural amino acid.

Figure 22.4

Synthetic scheme for the generation of SAHBs by Fmoc-based solid-phase peptide synthesis and ruthenium-catalyzed olefin metathesis.

Figure 22.5

Circular dichroism spectra demonstrate the increased α-helicity of SAHBs compared to the corresponding unmodified BH3 peptides (Walensky et al., 2006).

Figure 22.6

SAHBs exhibit marked protease resistance compared with unmodified BH3 peptides as assessed by (A) in vitro trypsin degradation assay, (B) ex vivo, and (C) in vivo serum stability assays (Walensky et al., 2004).

Figure 22.7

Cellular uptake of FITC-BID SAHB and a point mutant derivative, but not the unmodified FITC-BID BH3 peptide, is readily demonstrated by (A) FACS analysis and (B) confocal microscopy of FITC-peptide treated cells in culture. The cellular fluorescence of FITC-SAHB treated cells, as reflected by a shift of the FACS profile to the right compared to FITC-BID BH3–treated cells, corresponds to the intracellular cytosolic localization of FITC-BID SAHB observed by confocal microscopy. (Walensky et al., 2004)