Conformational Behavior of Peptides

Structure-Permeability Relationship of Cyclic Peptides

Peptides and peptidomimetics have recently attracted much attention as alternative chemotypes to interfere with key therapeutic targets such as class B G-protein coupled receptors and protein-protein interactions. The preferred route for delivering drugs is oral administration, for which the compound must be able to cross the gut wall by passive diffusion. Therefore, the successful development of peptide-type drugs requires a reliable determination of the membrane permeability of molecules. As experimental assays are time and cost intensive, computational approaches are particularly appealing for this purpose. Summaries and perspectives on future development in this area can be found in Refs. [1, 2, 3].

[1] external pageRiniker, Future Med. Chem. (2019), 11, 637.
[2] external pageLinker et al., CHIMIA (2021), 75, 518.
[3] external pageKamenik et al., ACS Book (2022), Ch. 5, pp. 137-154

Kinetic models based on multi-microsecond molecular dynamics data obtained in polar and apolar environments are employed to rationalize the membrane permeability of cyclic peptides. As a first example the natural product cyclosporine A (CsA) was used [4]. The conformational landscape of cyclic peptides was found to be more complicated than previously assumed, and thus not only thermodynamics but also kinetics are contribute. Due to the increased flexibility of cyclic peptides compared to small organic molecules, different conformations can exhibit largely different polarities. As a result, the populations and interconversion rates between metastable conformational states may become important for permeability. The permeability of cyclosporine E (CsE), a synthetic derivative of CsA, which differs structurally only in one missing backbone N-methylation, is an order of magnitude lower compared to CsA. In line with this, the interconversion time scales in water were found to be an order of magnitude slower [5].

[4] external pageWitek et al., J. Chem. Inf. Model. (2016), 56, 1547.
[5] external pageWitek et al., ChemPhysChem (2017), 18, 3309.

Our approach was further applied to a series of cyclic decapeptides [6]. Six peptides were selected that had all the same backbone methylation patterns and only differed in the two residues at the turns. Nevertheless, they differ in permeability and solubility. While for these compounds the interconversion rates were similar, the simulations showed that the population of the "closed" conformation (i.e. the conformation with four intra-molecular hydrogen bonds between backbone atoms) correlated with the experimentally observed permeability. When extending the study to 24 peptides of the same series [7], we found that not only the composition of the peptides can have a large effect on their properties but also the location of specific residues in the scaffold. Especially for polar residues, subtle structural changes can result in large differences in the conformational behavior and ultimately permeability. Our results showed that flexibility can be compensated by higher lipophilicity (up to a limit), and some degree of polarity can be tolerated when the membrane-permeable conformation is sufficiently populated in water ("pre-folding").

[6] external pageWitek et al., J. Chem. Inf. Model. (2019), 59, 294.
[7] external pageWang et al., J. Med. Chem. (2021), 64, 12761.

Most MD-based work of the conformational ensembles of cyclic peptides focussed on solvent environments. However, during the key steps of the permeation through the membrane, cyclic peptides are exposed to interfaces between polar and apolar regions as well as the sterically crowded envrionment of a lipid bilayer. When including an explicit apolar/polar interface in our approach [8, 9], we found that the interface acts like a "catalyst" for the interconversion between open and closed conformations. The simulations further revealed that not only the conformations but also the orientations are relevant in a membrane. Overall, we identified four steps of the membrane permeation process of cyclic peptides: (1) anchoring with residues in transient gaps between lipid head groups, (2) insertion in the membrane and orienting parallel to the membrane plane, (3) if the peptide enters in an open conformation, interconversion to the closed permeable conformation, and (4) leaflet crossing involving anchoring and rotation.

[8] external pageLinker et al., RSC Adv. (2022), 12, 5782.
[9] external pageLinker et al., J. Med. Chem. (2023), 66, 2773.

Beyond Standard Peptides: Semi-peptidic Macrocycles and Depsipeptides

In depsipeptides, some of the amide groups in the backbone are replaced by ester groups. We studied the cyclic octadepsipeptides such as PF1022A and its synthetic derivative emodepside, which exhibit anthelmintic activity [10]. Emodepside is sold as a commercial drug treatment for animal health use. However, the structure-permeability relationship of these cyclic depsipeptides is not yet well understood. The fully N-methylated amide backbone and apolar sidechain residues do not allow for the formation of intramolecular hydrogen bonds, normally observed in the membrane-permeable conformations of cyclic peptides (see above). In addition, these depsipeptides were also reported to be ionophores with a preference of potassium over sodium. In our study, we related the conformational behaviour of PF1022A, emodepside, and closely related analogs with their ionophoric characteristic probed using NMR and MD simulations and finally evaluated their passive membrane permeability. We found that the equilibrium between the two core conformers shifts upon addition of monovalent cations as only one conformation binds the cation, with selectivity for potassium over sodium. The permeability results suggested that the investigated depsipeptides are actually retained in the membrane, which may be advantageous for the likely target, a membrane-bound potassium channel.

[10] external pageStadelmann et al., Org. Biomol. Chem. (2020), 18, 7110.

Semipeptidic macrocycles typically contain a synthetic linker connecting two ends of a short peptide. Recent work in our group revealed how a change in only a single stereocenter in two semi-peptidic macrocycles leads to a 'permeability cliff' in experimental permeability assays [11]. Such a cliff occurs when two structurally highly similar compounds exhibit strongly different permeability. Using a combination of NMR and MD studies, we could show how the change affects the conformational ensemble of the macrocycles and through this the population of conformations with a maximal number of intramolecular hydro- gen bonds in the apolar (chloroform) environment.

[11] external pageComeau, Ries et al., J. Med. Chem. (2021), 64, 5365.