Folding Then Binding vs Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase

Abstract

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.

Cyclic peptides can engage a single binding pocket through highly divergent modes

“…Indeed, a subset of the peptides also demonstrates a high degree of preorganization in the unbound state. This preorganization is likely a strong contributor to the high affinities that are routinely observed in this and other naïve RaPID screens (26).…”

Enhancing glycan stability via site-selective fluorination: modulating substrate orientation by molecular design

“…To explore this notion, a computational docking study was performed using the HYDE binding assessment calculation in LeadIT/SeeSAR 21 to predict the bound conformations of these fluorinated probes in human α-amylases and α-glucosidases. Based on the sequence alignment and structural superimposition of salivary -amylase (PDB ID: 1SMD) 22 and pancreatic -amylase (PDB ID: 5U3A), 23 it is clear that both structures display a high degree of sequence and structural similarity, especially at the active site. As 5U3A (pancreatic -amylase) has the highest resolution (0.95 Å), it was used for these docking studies.…”

Structural diversity inde novocyclic peptide ligands from genetically encoded library technologies

“…Since more of the mRNA CPs have secondary structures (82% vs 37.5%) and a higher ‘compactness’ score (1.20 vs 0.94), more involved calculations/simulations are likely required to determine representative properties from the chemical structures alone. While CPs can form secondary structures upon binding, folding in solution prior to target binding may occur, [ 42 ] in particular for CP libraries composed of longer peptides given the greater propensity for adopting secondary structure. Determining the structure of these CPs in solution would aid in this analysis.…”

“…The L Y initiated library showed little conservation amongst the enriched sequences, [ 41 ] but identified piHA‐L5(d10Y) (Ac‐YGHSHIRFGYSYHVSYCG‐NH 2 ), a distinct sequence from piHA‐Dm, [ 42 ] with K i = 14 nM for HPA (note that position 10 was L DOPA, abbreviated to ‘d’ in the original manuscript. The d10Y mutant was only 5‐fold less potent and was used in the majority of subsequent experiments).…”