Modification of Peptides and Phage-displayed peptides as a tool in cancer therapy
S. Kalhor-Monfared, M.-R. Jafari, J. T. Patterson, P. Kitov, J. J. Dwyer, J. M. Nuss and R. Derda*
Antibody-based therapy has shown tremendous promise in hard-to-treat cancers because antibodies have predictable pharmacokinetics and toxicity, while genetic selection techniques can be readily used to tune the specificity of such drug candidates. Still, significant variability in bio-distribution limits the efficacy of such therapeutics. Our program addresses this challenge by developing genetically-encoded libraries of peptide-derived ligands that combine the advantages of small-molecule drugs and antibody therapeutics. Their major advantage is improved bio-distribution due to small size. Similarly to antibodies, these molecules can be optimized via in vitro selection. Our technology is based on phage-displayed peptide libraries that are modified through multi-step, site-specific organic reactions and convert biosynthesized peptide libraries into libraries with cyclic topology and exceptional diversity (up to 10^9). One of the embodiments of such reactions described in this presentation is macrocyclization of peptides and phage-displayed peptides using small fluorinated compounds (perfluoroarenes). Macrocylization can improve the conformational stability and serum stability of peptides and potentially equip it with properties, such as cell-permeability that would not be accessible to unmodified peptides.
Macrocylization of linear peptides occurs through a selective aromatic nucleophilic substitution (SNAr) reaction on Cys residue of unprotected peptides with a general structure of Xn-Cys-Xm-Cys-Xl, where X is any amino acid and m=1-15. To make this reaction biocompatible and not toxic to bacteriophage, we optimized these reactions to occur rapidly in buffered solvent (pH=8.5) containing only small amounts of organic co-solvents (acetonitrile or DMF). Measurement of the reaction rates of fAr derivatives by 19F NMR with a model thiol donor (β-mercaptoethanol) in aqueous buffers identified decafluoro-diphenylsulfone (DFS) as the most reactive SNAr electrophile. This bivalent electrophile forms macrocycles when reacted with peptides containing two Cys residues. We also tested six peptide hormones—oxytocin, urotensin II, salmon calcitonin, melanin-concentrating hormone, somatostatin-14, and atrial natriuretic factor (1-28), as well as peptides displayed on M13 phage which formed successfully cyclic peptides with DFS. Rates up to 180 M-1s-1 make this reaction one of the fastest Cys-modifications to date.
Shiva Kalhor-Monfared, Mohammad-Reza Jafari, James T. Patterson, Pavel I Kitov, John J. Dwyer, John J. Nuss and Ratmir Derda “Rapid Biocompatible Macrocyclization of Peptides with Decafluorosulfone” Chem. Sci., 2016, in press, DOI: 10.1039/C5SC03856A