Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo

In this paper, we report selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry. Modification of phage-displayed libraries SXCXnC-phage, n=3-5, where X is any amino acid except for cysteine by decafluoro-diphenylsulfone (DFS), yields genetically-encoded library of octafluoro-diphen-ylsulfone-crosslinked macrocycles (OFS-SXCXnC-phage). Selection from these libraries using albumin as a bait identified a family of significantly enriched perfluoroaryl-macrocycles. Synthesis of perfluoroaryl-macrocycles predicted by phage display and testing their binding properties by 19F NMR and fluorescent polarization identified OFS-macrocycle with SICRFFC sequence as the most potent albumin binder. We observed that OFS-macrocycles slowly react with biological nucleophiles such as glutathione. Replacing decafluoro-diphenylsulfone by nearly isosteric pentafluorophenyl sulfide yielded perfluorophenylsulfide (PFS)-crosslinked macrocycles devoid of undesired reactivity. The augmented lead PFS-macrocycle with SICRFFC sequence exhibited KD = 4-6 M towards human serum albumin and similar affinities towards rat and mouse albumins. When injected in mouse, the PFS-SICRFFCGGG compound was significantly retained in circulation in vivo when compared to control PFS-macrocyclic peptide. The perfluoroaryl-macrocycles with SICRFFC motif are the smallest known peptide macrocycle with significant affinity for human albumin and they are a productive starting point for future development of compact macrocycles with predictable circulation half-life in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504611v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@cd54d2org.highwire.dtl.DTLVardef@7bd9aborg.highwire.dtl.DTLVardef@10f7515org.highwire.dtl.DTLVardef@1cfc49a_HPS_FORMAT_FIGEXP M_FIG C_FIG