Cyclisation strategies for stabilising peptides with irregular conformations

Abstract

Cyclisation is a common synthetic strategy for enhancing the therapeutic potential of peptide-based molecules. While there are extensive studies on peptide cyclisation for reinforcing regular secondary structures such as α-helices and β-sheets, there are remarkably few reports of cyclising peptides which adopt irregular conformations in their bioactive target-bound state. In this review, we highlight examples where cyclisation techniques have been successful in stabilising irregular conformations, then discuss how the design of cyclic constraints for irregularly structured peptides can be informed by existing β-strand stabilisation approaches, new computational design techniques, and structural principles extracted from cyclic peptide library screening hits. Through this analysis, we demonstrate how existing peptide cyclisation techniques can be adapted to address the synthetic design challenge of stabilising irregularly structured binding motifs.

Fig. 1. Selected examples of common peptide cyclisation chemistries used for conformational stabilisation. Comprehensive lists of examples can be found in reviews that focus on stabilisation of common secondary structures.

Fig. 2. Examples of rational stabilisation of irregular peptide conformation. A) The impact of hydrocarbon-stapling on the conformation of the ESp peptide from virulence factor exoenzyme S (ExoS), which has a combination of helical and irregular structure. Depending on linker length, cyclisation of the helical region can either preserve (β_ss12, PDB 4N84) or distort (β_RS8, PDB 4N7Y) the native conformation. B) The impact of linker length on the binding of stapled peptides targeting TNKS2. Differences in the electron density maps (2F_obs − F_calc, shown as yellow mesh) of peptides cp4n4m5 and cp4n2m3 (PDB 5BXU/5BXO), especially near the staple at the region indicated by the red arrows, suggests differences in flexibility. C) Structure of Pep2A with a urea-bridged bis-triazole linker (red) that constrains the extended conformation of an intrinsically disordered region of the hepatocyte nuclear factor 1β transcription factor (HNF1β). D) Comparison of binding poses for linear peptide H3_1-21K4M (white) and lactam cyclised peptide, macrocycle 31 (orange). Macrocycle 31 has an opposite backbone direction to the linear peptide and is partially protruding out from the binding pocket. The N- and C-terminus are labelled in grey and orange for H3_1-21K4M and macrocycle 31 respectively (PDB 2V1D/6S35), with the lactam staple in magenta.

Fig. 3. Selected examples of macrocyclic tethers for constraining β-strand conformations. A) HIV protease inhibitors based on a known hexapeptide inhibitor, replacing a central Phe with Tyr to enable cyclisation to the C-terminus. B) Calpain protease inhibitors cyclised by histidine alkylation and click.

Fig. 4. Amide backbone peptidomimetic isosteres based on heterocycles. A) Three different monocyclic surrogates for amides that reinforce β-strand conformations. B) GSK3β inhibitor incorporating a bicyclic surrogate for a Thr-Thr dipeptide motif.

Fig. 5. Peptides arising from computational design using MD simulations (A – Voelz and co-workers, B – Crowe Jr and coworkers) and docking software (C – Grossmann and co-workers).

Fig. 6. Bound structures and corresponding 2D representations of cyclic peptides selected through mRNA display. A) Structures of 3.1C bound to BRD3-BD1 (PDB 6U4A) and 3.2C bound to BRD3-BD2 (PDB 6ULP) derived through RaPID screens. The key intramolecular hydrogen bonds are shown on the 2D representations in red. B) Structures of KD2 bound to K-Ras (PDB 6WGN) and piHA-Dm bound to α-amylase (PDB 5KEZ) derived through RaPID screens mediated by a water molecule (shown as a red sphere). The intermolecular hydrogen bonds to water are show in red, while for clarity, the intramolecular hydrogen bonds are not drawn in this instance.

Fig. 7. Bound structures and 2D representations of cyclic peptides selected through phage display. A) Structures of urokinase-type plasminogen activator (uPA) inhibitors derived from phage display libraries (PDB 2NWN/3QN7/4GLY/6A8G/6A8N, left) and a 2D structure of one example UK18 that was cyclised with a 1,3,5-trisubstituted aromatic linker (right). All the structures feature irregular loops spread across the protein surface, anchored by an arginine residue in the substrate pocket (highlighted in red on the 2D structure). Figure adapted from McAllister et al. B) Structures of peptide M21 stabilised by a mesitylene core bound to proinflammatory cytokine tumour necrosis factor-alpha (TNFα) dimer (PDB 4TWT). The α-helical loop interacts with one monomer while the short irregular loop with two proline residues interacts with the other monomer.

Tianyi Gao, Quynh Ngoc Vu, Tiancheng Huang, Haritha Krishna Sudhakar and Reginald Young (pictured from left to right)

Yaw Sing Tan

Yu Heng Lau