Genetically encoded discovery of perfluoroaryl macrocycles that bind to albumin and exhibit extended circulation in vivo

Abstract

Peptide-based therapeutics have gained attention as promising therapeutic modalities, however, their prevalent drawback is poor circulation half-life in vivo. In this paper, we report the selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry, with decafluoro-diphenylsulfone (DFS). Testing of the binding of the selected peptides to albumin identified SICRFFC as the lead sequence. We replaced DFS with isosteric pentafluorophenyl sulfide (PFS) and the PFS-SICRFFCGG exhibited KD = 4–6 µM towards human serum albumin. When injected in mice, the concentration of the PFS-SICRFFCGG in plasma was indistinguishable from the reference peptide, SA-21. More importantly, a conjugate of PFS-SICRFFCGG and peptide apelin-17 analogue (N3-PEG6-NMe17A2) showed retention in circulation similar to SA-21; in contrast, apelin-17 analogue was cleared from the circulation after 2 min. The PFS-SICRFFC is the smallest known peptide macrocycle with a significant affinity for human albumin and substantial in vivo circulation half-life. It is a productive starting point for future development of compact macrocycles with extended half-life in vivo.

Phage-encoded bismuth bicycles enable instant access to targeted bioactive peptides

“…A few enriched sequences without this motif are known to us from other screenings as suspected non-target binders. An analysis of the most frequently occurring dipeptide motifs 37 further confirmed the pronounced appearance of HP and PQ/PM within the top 25 hit sequences (Fig. 2b–e ).…”

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

“…159 A promising advantage of developing peptide ligands using these perfluoro compounds is their ability to be directly detected for binding and in vivo analysis through 19 F NMR. 160 Another similar strategy that takes advantage of S N Ar reactivity involves using 2,4-difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB, Table 3) to cyclize phage libraries with two cysteines. 161 Although it is slower than the reaction with DFS, the kinetics are sufficient for effective phage labeling with a second order rate constant of 9.2 M −1 s −1 .…”

“…Both linkers have proven useful in the phage-assisted identification of cyclic peptides, with the resulting peptides (Table S1, Peptides 94 and 95) binding with micromolar affinity to human serum albumin and Bclxl. 160,161 In addition to simply creating macrocyclic peptides on phage, cyclization of peptides with organic linkers also allows for further functionalization of peptide libraries. By incorporation of additional moieties into the linkers, additional properties can be given to the peptide libraries that facilitate selections for certain proteins.…”

“…The first of these applications took advantage of a decafluoro-diphenylsulfone (DFS, Table ) to react with cysteines at extremely quick kinetics with rates upward of 180 M –1 s –1 . A promising advantage of developing peptide ligands using these perfluoro compounds is their ability to be directly detected for binding and in vivo analysis through 19 F NMR . Another similar strategy that takes advantage of S N Ar reactivity involves using 2,4-difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB, Table ) to cyclize phage libraries with two cysteines .…”

“…Although it is slower than the reaction with DFS, the kinetics are sufficient for effective phage labeling with a second order rate constant of 9.2 M –1 s –1 . Both linkers have proven useful in the phage-assisted identification of cyclic peptides, with the resulting peptides (Table S1, Peptides 94 and 95) binding with micromolar affinity to human serum albumin and Bcl-xl. , …”