De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized. In this work, we present cocrystallization, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar K i to the active site of human pancreatic α-amylase. We show that a short stably folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilization of the bound conformation is provided through interactions with the target protein after binding. These three structures also reveal a surprising functional convergence to present a motif of a single arginine sandwiched between two aromatic residues in the interactions of the peptide with the key catalytic residues of the enzyme, despite little to no other structural homology. Our results suggest that intramolecular hydrophobic interactions are important for priming binding of small macrocyclic peptides to their target and that high rigidity is not necessary for high affinity.

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in-depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α-amylase (HPA). Notably, incorporation of l-DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (K =480 pm) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural-product-derived motif, already specific for the catalytic side-chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.

The 57‐mer full‐length GPR15L(25‐81) peptide has been identified as the principal endogenous agonist of the G protein‐coupled receptor GPR15. Its main activity resides in the C‐terminal 11‐mer GPR15L(71‐81), which has full efficacy but ~40‐fold lower potency than the full‐length peptide. Here, we systematically investigated the structure–activity relationship of GPR15L(71‐81) by truncations/extensions, alanine‐scanning, and N‐ and C‐terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time‐resolved Förster resonance energy transfer‐based Gi cAMP functional assay. We show that the C‐terminal α carboxyl group and the residues Leu78, Pro75, Val74, and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71‐81), C‐terminally amidated GPR15L(71‐81), and GPR15L(25‐81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer‐based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.

No abstract

The front cover picture shows a lariat noncanonical peptide scaffold presenting one half of a natural‐product‐derived warhead to human pancreatic α‐amylase. The folding of this peptide into an unusually stable 310 helix in solution is driven by a templating effect from the macrocyclic portion on the linear tail. This allows the peptide to place an l‐DOPA side chain in such a way that it forms a strong chelate interaction with one of the enzyme's catalytic carboxylate groups. The resulting nonapeptide inhibitor has remarkable potency, with Ki=480 pm. As the placement of catalytic carboxylates is conserved across all retaining glycosidases, if both halves of this warhead could be incorporated into such a peptide scaffold in the correct folded orientation, then the resulting inhibitor should be active across this class of enzymes. Its specificity could be tuned by varying the peptide side chains. More information can be found in the full paper by S. A. K. Jongkees et al. on page 2333 in Issue 23, 2017 (DOI: 10.1002/cbic.201700457).