William Darling: The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide

1, 2 They have the potential to occupy conformational space that cannot be covered by traditional small molecule drugs and probes, and thus can address undruggable targets such as protein-protein interactions.3, 4 Cyclic peptides are an important class of bRo5 macrocycles,5, 6 particularly cystine-bridged peptides, which can be designed from naturally occurring bioactive peptide sequences and are readily synthesised. Cyclising bioactive peptides by forming disulfide bonds frequently stabilises the structure, increases potency and selectivity, enhances stability to proteases and can also lead to increased membrane permeability.7, 8 However, such disulfide bridges can be easily reduced or scrambled, which limits the in vivo stability of these peptides.9, 10

To further improve the metabolic stability and retain the potency and selectivity of cystine-bridged peptides, both thioacetal and thioether (lanthionine)-bridged analogues have been explored. A simple protocol for the rebridging of disulfides by the insertion of a methylene group to form a stable thioacetal has recently been reported11 and has been used to prepare biologically active analogues of peptides with increased serum stability.11, 12 The first thioether-bridged analogue of a medicinally relevant peptide was reported in 1997 by Goodman and co-workers, who prepared lanthionine-bridged somatostatin analogues which had greater receptor binding selectivity and a longer half-life in vivo than cystine-bridged peptides.13 

Since then, lanthionine analogues of several other classes of biologically active peptides have been prepared and shown to be highly potent, receptor-selective and resistant to proteolysis.14-20 However, in a few cases, this cyclisation modality resulted in the peptide analogues having reduced or no binding to the target receptor.21, 22 Excitingly, a thioether stabilised angiotensin(1–7) analogue, cAng(1–7) exhibits significantly enhanced vasodilation and plasma stability compared with the naturally occurring linear peptide.23 cAng(1–7) can be delivered orally and via the lung24 and a variant sequence with a D-Lys at the N-terminus, LP2, has completed preclinical and in-human safety studies for treatment of cardiovascular disease25 and colorectal cancer.26 Solid-phase synthesis of lanthionine-bridged peptides using orthogonally protected lanthionine monomers has been established27, 28 with monomers corresponding to all possible lanthionine diastereoisomers being available.27, 29 In a complementary approach, the post-translational modification enzymes have been exploited for the biosynthesis of engineered lanthionine-type therapeutic peptides.30-32 They have also been used to generate phage display libraries19 and lanthipeptide libraries20 in E. coli.

However, what is currently lacking is a molecular level understanding of the effect that these three different bridging modalities - disulfide, methylene thioacetal and thioether - have on the underlying conformational properties of the resulting cyclic peptides, for instance as shown in Figure 1. Replacement of disulfide bonds will cause changes in polarity, hydrophobicity, bond length and bond angles, properties which are typically consistent and well characterised across cystine bridges. These bond length/angles and polarity deviations can cause global conformational changes or peptide-target polarity mismatches which can prove either detrimental, or beneficial, for biological activity and target selectivity.33 Moreover, macrocycles are conformationally flexible. This allows them to adapt to give high-affinity binding to a variety of targets, and can also confer solubility in both aqueous and non-polar environments resulting in enhanced cell permeability.1-6 Understanding the structures of the individual conformers that make up the solution ensemble is vital to understanding these medicinally desirable properties.34-36 Despite this, there have been virtually no studies comparing the effects of different cyclisation modalities on the solution conformation of these peptides. Conformational analyses by NMR of disulfide and lanthionine-bridged sandostatin analogues37 and of disulfide- and lanthionine-bridged peptides binding to death receptor 519 have been reported. In these studies, similar conformations with the backbone atoms overlaying closely were seen for both pairs of peptide analogues, however only a single average conformation was reported for each peptide.

Read full article here