De Novo Synthesis and Structural Elucidation of CDR-H3 Loop Mimics.

The binding affinity of antibodies to specific antigens stems from a remarkably broad repertoire of hypervariable loops known as complementarity-determining regions (CDRs). While recognizing the pivotal role of the heavy-chain 3 CDRs (CDR-H3s) in maximizing antibody-antigen affinity and specificity, the key structural determinants responsible for their adaptability to diverse loop sequences, lengths, and noncanonical structures are hitherto unknown. To address this question, we achieved a de novo synthesis of bulged CDR-H3 mimics excised from their full antibody context. CD and NMR data revealed that these stable standalone ß-hairpin scaffolds are well-folded and retain many of the native bulge CDR-H3 features in water. In particular, the tryptophan residue, highly conserved across CDR-H3 sequences, was found to extend the kinked base of these ß-bulges through a combination of stabilizing intramolecular hydrogen bond and CH/π interaction. The structural ensemble consistent with our NMR observations exposed the dynamic nature of residues at the base of the loop, suggesting that ß-bulges act as molecular hinges connecting the rigid stem to the more flexible loops of CDR-H3s. We anticipate that this deeper structural understanding of CDR-H3s will lay the foundation to inform the design of antibody drugs broadly and engineer novel CDR-H3 peptide scaffolds as therapeutics.

Tunable CH/π Interactions within a Tryptophan Zipper Motif to Stabilize the Fold of Long ß-Hairpin Peptides.

The tryptophan zipper (Trpzip) is an iconic folding motif of ß-hairpin peptides capitalizing on two pairs of cross-strand tryptophans, each stabilized by an aromatic-aromatic stacking in an edge-to-face (EtF) geometry. Yet, the origins and the contribution of this EtF packing to the unique Trpzip stability remain poorly understood. To address this question of structure-stability relationship, a library of Trpzip hairpins was developed by incorporating readily accessible nonproteinogenic tryptophans of varying electron densities. We found that each EtF geometry was, in fact, stabilized by an intricate combination of XH/π interactions. By tuning the π-electron density of Trpface rings, CH/π interactions are strengthened to gain additional stability. On the contrary, our DFT calculations support the notion that Trpedge modulations are challenging due to their simultaneous paradoxical engagement as H-bond donors in CH/π and acceptors in NH/π interactions.

Passive Membrane Permeability of Sizable Acyclic ß-Hairpin Peptides.

The recent shift toward increasingly larger drug modalities has created a significant demand for novel classes of compounds with high membrane permeability that can inhibit intracellular protein-protein interactions (PPIs). While major advances have been made in the design of cell-permeable helices, stapled ß-sheets, and cyclic peptides, the development of large acyclic ß-hairpins lags far behind. Therefore, we investigated a series of 26 ß-hairpins (MW > 1.6 kDa) belonging to a chemical space far beyond the Lipinski "rule of five" (fbRo5) and showed that, in addition to their innate plasticity, the lipophilicity of these peptides (log D 7.4 ≈ 0 ± 0.7) can be tuned to drastically improve the balance between aqueous solubility and passive membrane permeability.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into ß-hairpin scaffolds. As a result, a 20-residue long ß-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 µM; Ki of 41 nM).

Folding in Place: Design of ß-Strap Motifs to Stabilize the Folding of Hairpins with Long Loops.

Despite their pivotal role in defining antibody affinity and protein function, ß-hairpins harboring long noncanonical loops remain synthetically challenging because of the large entropic penalty associated with their conformational folding. Little is known about the contribution and impact of stabilizing motifs on the folding of ß-hairpins with loops of variable length and plasticity. Here, we report a design of minimalist ß-straps (strap = strand + cap) that offset the entropic cost of long-loop folding. The judicious positioning of noncovalent interactions (hydrophobic cluster and salt-bridge) within the novel 8-mer ß-strap design RW(V/H)W···WVWE stabilizes hairpins with up to 10-residue loops of varying degrees of plasticity (Tm up to 52 °C; 88 ± 1% folded at 18 °C). This "hyper" thermostable ß-strap outperforms the previous gold-standard technology of ß-strand-ß-cap (16-mer) and provides a foundation for producing new classes of long hairpins as a viable and practical alternative to macrocyclic peptides.

Structure-Property Relationship Study of N-(Hydroxy)Peptides for the Design of Self-Assembled Parallel ß-Sheets.

The design of novel and functional biomimetic foldamers remains a major challenge in creating mimics of native protein structures. Herein, we report the stabilization of a remarkably short ß-sheet by incorporating N-(hydroxy)glycine (Hyg) residues into the backbone of peptides. These peptide-peptoid hybrids form unique parallel ß-sheet structures by self-assembly upon hydrogenation. Our spectroscopic and crystallographic data suggest that the local conformational perturbations induced by N-(hydroxy)amides are outweighed by a network of strong interstrand hydrogen bonds.

Intercepted-Knoevenagel condensation for the synthesis of unsymmetrical fused-tricyclic 4H-pyrans.

4H-Pyrans (4H-Pys) and 1,4-dihydropyridines (1,4-DHPs) are important classes of heterocyclic scaffolds in medicinal chemistry. Herein, an indium(III)-catalyzed one-pot domino reaction for the synthesis of highly functionalized 4H-Pys, and a model of 1,4-DHP is reported. This alternative approach to the challenging Hantzsch 4-component reaction enables the synthesis of fused-tricyclic heterocycles, and the mechanistic studies underline the importance of an intercepted-Knoevenagel adduct to achieve higher chemoselectivity towards these types of unsymmetrical heterocycles.