Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

Abstract

Cyclic peptide display screening techniques can identify drug leads and biological probes with exceptional affinity and specificity. To date, however, the structural and functional diversity encoded in such peptide libraries remains unexplored. We have used the Random nonstandard Peptide Integrated Discovery (RaPID) system to develop cyclic peptide inhibitors of several acetyllysine-binding bromodomains from the Bromodomain and Extra-Terminal domain (BET) family of epigenetic regulators. These peptides have very high affinities for their targets and exhibit extraordinary selectivity (up to 10 6 -fold), making them the highest-affinity and most specific BET-binding molecules discovered to date. Crystal structures of 13 distinct peptide-bromodomain complexes, which all target the acetyllysine-binding pocket, reveal remarkable diversity in both peptide structure and binding mode, and include both α-helical and β-sheet type structures. The peptides can exhibit a high degree of structural preorganization and bivalent binding of two BDs by one peptide was common, flagging the potential for a new direction in inhibitor design that could bring stronger discrimination between BET-family paralogues. Our data demonstrate for the first time the enormous potential held in these libraries to provide a wide array of modes against a single target, maximizing the opportunity to attain high potency and specificity ligands to a wide variety of proteins.

De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex

“…It is intriguing that all the Vps29-binding peptides have been selected for the presence of this Pro-Leu dipeptide and that this peptide has also evolved to mediate binding of endogenous ligands of the retromer complex such as TBC1D5, VARP, and RidL. The de novo macrocyclic peptide screening has therefore inadvertently identified an evolutionarily conserved binding mechanism, and previous screens of Bromodomain and Extra-Terminal (BET) domain–binding peptides also uncovered sequence preferences that partly mimicked known endogenous ligands ( 73 ). Given that Vps29 is also a component of the retriever complex, a Retromer-related assembly containing homologous subunits Vps35L and Vps26C ( 74 , 75 ), these cyclic peptide inhibitors may also provide tools for studies of retriever.…”

Identification of photocrosslinking peptide ligands by mRNA display

“…Pleasingly, after completing a mock selection round, whilst 3.9% of the input DNA was recovered following standard affinity panning, almost no input DNA (0.0069%) was recovered following washes with 5 M guanidine (Supplementary Figure S4). This confirmed that the guanidine washes were sufficient to dissociate a tight binding peptide (K D = 0.49 nM) from its target protein 27 , most likely due to target denaturation.…”

“…To test the feasibility of our proposed strategy, we first confirmed that treating biotinylated proteinbound beads with guanidine chloride (up to 8 M) did not disrupt the interaction between biotin and streptavidin (Supplementary Figure S3). Subsequently, we used a model mRNA template encoding a previously identified non-covalent peptide ligand (3.1B) of the first bromodomain of BRD3, BRD3-BD1, 27 to confirm the effectiveness of our denaturing washes in removing non-covalent ligands.…”

“…We aimed to identify photocrosslinking peptide ligands for the second bromodomain (BD2) of BRD3 from the bromodomain and extraterminal domain (BET) family of transcriptional coregulators, [28][29][30] against which we have recently identified a series of potent non-covalent cyclic peptides. 27 We constructed an mRNA displayed peptide library (>10 12 members) containing six to twelve random amino acids, flanked by an initiator codon and a C-terminal cysteine prior to a GSGSGSlinker. Peptides were initiated with N-chloroacetyl-D-tyrosine (by flexizyme-mediated reprogramming of the start codon), to produce macrocyclic peptides through reaction with the cysteine.…”

De novomacrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

“…It is intriguing that all of the Vps29-binding peptides have been selected for the presence of this Pro-Leu dipeptide, and that this peptide has also evolved to mediate binding of endogenous ligands of the Retromer complex such as TBC1D5, VARP and the bacterial effector RidL. The de novo macrocyclic peptide screening has therefore inadvertently identified an evolutionarily conserved binding mechanism, and interestingly previous screens of BET domain-binding peptides also uncovered sequence preferences that partly mimicked known endogenous ligands 111 . Given that Vps29 is also a component of the Retriever complex, a Retromer-related assembly containing homologous subunits Vps35L/C16orf62 and Vps26C/DSCR3 112, 113 , these cyclic peptide inhibitors may provide valuable tools for studies of Retriever.…”