Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties

• Published in: Molecules (Basel, Switzerland) Switzerland), 2019-06, Vol.24 (12), p.2292
• Main Authors: Partridge, Anthony W, Kaan, Hung Yi Kristal, Juang, Yu-Chi, Sadruddin, Ahmad, Lim, Shuhui, Brown, Christopher J, Ng, Simon, Thean, Dawn, Ferrer, Fernando, Johannes, Charles, Yuen, Tsz Ying, Kannan, Srinivasaraghavan, Aronica, Pietro, Tan, Yaw Sing, Pradhan, Mohan R, Verma, Chandra S, Hochman, Jerome, Chen, Shiying, Wan, Hui, Ha, Sookhee, Sherborne, Brad, Lane, David P, Sawyer, Tomi K
• Format: Article
• Language: English
• Subjects: Affinity; Affinity chromatography; Amino Acid Sequence - genetics; Amino Acid Substitution - genetics; Amino acids; Amino Acids - chemical synthesis; Amino Acids - chemistry; Cation exchanging; Cell-Penetrating Peptides - chemical synthesis; Cell-Penetrating Peptides - chemistry; Cell-Penetrating Peptides - genetics; Cell-Penetrating Peptides - pharmacology; Cellular structure; Circular Dichroism; D-amino acid; D-Amino acids; Dipeptides - chemistry; Dithiothreitol; E coli; Elution; Glycerol; helix-breaker; Humans; Hydrocarbons; Imidazole; macrocyclic peptide; MDM2 protein; Oligopeptides - chemistry; Peptide Fragments - chemistry; peptide permeability; Peptides; Peptides, Cyclic - pharmacology; Permeability; Permeability - drug effects; Protein Conformation; Protein Structure, Secondary; Proteins; Proto-Oncogene Proteins c-mdm2 - chemistry; Proto-Oncogene Proteins c-mdm2 - genetics; Sodium chloride; stapled peptide
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules24122292

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe , Trp , and Cba ) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically 'break' right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe and Cba resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp to d-Trp ) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp -modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., 8 to Aib and 5 to Aib ) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac-Leu-Thr-Phe-Aib-Glu-Tyr-Trp-Gln-Leu-Cba-Aib-Ser-Ala-Ala-NH exhibited high-affinity target binding (Mdm2 K = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu , d-Gln , and d-Leu ) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed