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

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 focus...

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Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632053/
https://www.ncbi.nlm.nih.gov/pubmed/31226791
http://dx.doi.org/10.3390/molecules24122292
Descripción
Sumario: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(3), Trp(7), and Cba(10)) 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(3) and Cba(10) 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(7) to d-Trp(7)) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp(7)-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., R8(4) to Aib(4) and S5(11) to Aib(11)) 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(2) exhibited high-affinity target binding (Mdm2 K(d) = 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(5), d-Gln(8), and d-Leu(9)) 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-Trp(7)-modification, a highly potent ATSP-7041 analog (Mdm2 K(d) = 30 nM; cellular EC(50) = 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.