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Open AccessArticle

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

MSD International, 8 Biomedical Grove, #04-01/05 Neuros Building, Singapore 138665, Singapore
p53Lab, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore 138648, Singapore
Institute of Chemical and Engineering Sciences (ICES), Agency for Science, Technology and Research (A*STAR), 8 Biomedical Grove, #07, Neuros Building, Singapore 138665, Singapore
Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore
Merck & Co., Inc., West Point, PA 19486, USA
Merck & Co., Inc., Kenilworth, NJ 07033, USA
Merck & Co., Inc., Boston, MA 02115, USA
Authors to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Molecules 2019, 24(12), 2292;
Received: 22 May 2019 / Revised: 14 June 2019 / Accepted: 16 June 2019 / Published: 20 June 2019
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., Phe3, Trp7, and Cba10) 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 Phe3 and Cba10 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., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-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., R84 to Aib4 and S511 to Aib11) 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–NH2 exhibited high-affinity target binding (Mdm2 Kd = 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-Glu5, d-Gln8, and d-Leu9) 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 that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed. View Full-Text
Keywords: peptide permeability; stapled peptide; macrocyclic peptide; D-amino acid; helix-breaker peptide permeability; stapled peptide; macrocyclic peptide; D-amino acid; helix-breaker
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Partridge, A.W.; Kaan, H.Y.K.; Juang, Y.-C.; Sadruddin, A.; Lim, S.; Brown, C.J.; Ng, S.; Thean, D.; Ferrer, F.; Johannes, C.; Yuen, T.Y.; Kannan, S.; Aronica, P.; Tan, Y.S.; Pradhan, M.R.; Verma, C.S.; Hochman, J.; Chen, S.; Wan, H.; Ha, S.; Sherborne, B.; Lane, D.P.; Sawyer, T.K. Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties. Molecules 2019, 24, 2292.

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