Cyclic peptides and macrocycles in general have recently attracted attention as alternative chemotypes to interfere with key therapeutic targets, such as class B G-protein-coupled receptors or mode of actions like protein–protein interactions [Citation1]. A crucial challenge is thereby the design of cyclic peptides with the ability to passively cross cell membranes to modulate intracellular targets and/or oral bioavailability. Although cyclization and selective methylation of backbone amides tends to improve the passive membrane permeability of cyclic peptides (in addition to a better metabolic stability) [Citation2], examples with sufficiently high permeability and aqueous solubility for oral administration are unfortunately rare. The most prominent example (although with low solubility) is cyclosporine A (CsA) [Citation3], where its ability to adopt conformations with different polar surfaces depending on the environment is thought to be essential for its unusually high permeability given its size [Citation4–6]. In order to design cell-permeable cyclic peptides, a detailed understanding of their conformational behavior in polar and apolar environments, the underlying structural determinants and their connection to permeation is necessary.
Permeation mechanism may depend on molecule size
Physics-based methods typically employ the solubility-diffusion theory [Citation7] to estimate the passive membrane permeability of compounds, modeling explicitly the membrane crossing with molecular dynamics (MD) simulations [Citation8]. The permeation process is described by three steps: partitioning of the permeant from the aqueous phase to the membrane, diffusion of the permeant through the membrane and partitioning of the permeant from the membrane to the aqueous phase. Each of these stages can be the rate-limiting step. For flexible molecules, additional steps emerge by the necessity that a ‘permeable’ conformation (and neutral protonation state) is adopted. When treating the ‘barrier region’ (such as the membrane interior) as homogeneous, the diffusion is assumed to depend linearly on the size or volume, respectively, of the permeant and is estimated using the Stokes–Einstein relation [Citation9]. However, even for very small molecules such as water or methanol this may not be an appropriate assumption [Citation10]. Indeed, molecules of different size and complexity may permeate by different mechanisms and thus, have different rate-limiting steps. For very small molecules, the motion of the lipid tails creates temporary cavities within the membrane interior, leading to fractional diffusion of the permeant [Citation10]. In addition, the dipole moment is preferentially aligned with the dipole of the lipid head groups, which requires the molecule to turn within the membrane, that is, to ‘flip-flop’ across the interior [Citation11]. This alignment of the dipole moment was also found to occur for larger drug-like molecules [Citation12]. The combination of solubility-diffusion theory with a kinetic model for permeation based on MD simulations of spontaneous partitioning improved the agreement between calculated and experimental permeability coefficients substantially for six known drug molecules, identifying the ‘flip-flopping’ across the membrane interior as the rate-limiting step for drug-like compounds [Citation12].
Interestingly, if the same linear relationship of diffusion with size is assumed for molecules in the range of 0–1200 Da, two distinct relationships between diffusion and volume of the molecules can be observed (see Figure 5 in [Citation13]). This hints at a different diffusion behavior within the membrane for large molecules beyond Lipinski's ‘rules of five’ [Citation14]. The size of compounds like CsA implies that a single molecule spans a large fraction of the membrane width, and the membrane integrity is likely disturbed upon integration. For comparison, the thickness of a DPPC membrane in the liquid crystalline phase is around 3.6 nm, while the length of the backbone of CsA in the single-molecule crystal conformation [Citation15] (termed the ‘closed’ conformation) is around 1.5 nm (1.9 nm including side chains). It is therefore unlikely that CsA ‘flip-flops’ across the membrane, but rather ‘slides’ from one side to the other. Thus, the adoption of a permeable conformation and partitioning to the membrane (potentially occurring concertedly) may become rate-limiting for this class of compounds.
Permeability cliffs
As mentioned above, partitioning to the membrane requires for large molecules like CsA that a conformation can be adopted, which is also populated in the membrane interior. In this closed conformation, the polar groups are shielded from the apolar environment, typically through intramolecular hydrogen bonds. As such a chameleonic behavior is not straightforward to design, many of the known ‘beyond rules of five’ compounds with decent to good passive permeability are natural products [Citation16]. Due to the limited number of those known, the types of molecules in studies that search for simple relationships between structural features and permeability differ substantially from each other [Citation17,Citation18]. This may simplify the detection of correlations. More challenging and potentially more informative are therefore ‘permeability cliffs’, that is, structurally very similar molecules with large differences in permeability. Such cases [Citation19,Citation20] show that the ability to adopt a closed conformation in an apolar environment is a necessary but not sufficient condition for permeability. For example, side chains can heavily influence the population of the closed conformation in water, which was found to correlate with permeability for a series of closely related peptides [Citation20].
As the adoption of a closed conformation in a polar environment is important, rigidification of the backbone scaffold can be beneficial for permeability [Citation20]. However, the backbone should also not be too rigid as this may negatively affect solubility and prevent interactions with the intracellular target. In addition, NMR studies of CsA showed that the peptide is not completely rigid in chloroform [Citation6], which is in line with MD simulations [Citation5]. Some conformational flexibility might therefore be important to reduce the entropic cost of insertion into the membrane.
Sampling of the closed conformation of large cyclic peptides
Most studies on the permeability of large cyclic peptides have been based on compounds with known closed conformation (from crystal structures and/or NMR solution structures). Computational approaches to predict the permeability of new compounds, whether simple (e.g., minimum 3D PSA [Citation18]) or more sophisticated (such as in [Citation5,Citation19,Citation20]), require the knowledge of the closed conformation. However, identifying such conformations for cyclic peptides ‘beyond hexamers’ using in silico conformer generators becomes very difficult due to the large number of degrees of freedom (although already reduced through cyclization). Knowledge-based conformer generators using systematic search, experience combinatorial explosion in these cases, whereas stochastic approaches become inefficient. Other search strategies, based, for example, on molecular dynamics or Monte Carlo simulations, require a reasonable 3D input structure. As a result, a future challenge for the computational chemistry community is to develop efficient search strategies to identify the closed conformation of new cyclic peptides in a reliable and robust manner.
Conclusion
In order to capitalize on the potential of cyclic peptides and macrocycles in general as therapeutics, design principles and structural descriptors to achieve good passive membrane permeability have to be identified. For this, we need a detailed understanding of the complex conformational behavior of these flexible molecules in different environments as subtle topological differences can cause substantial changes in the conformational ensembles (such as permeability cliffs). First steps have been made into this direction by exhaustive MD simulations combined with kinetic models. Clearly, such computationally intensive approaches cannot be applied in a high-throughput manner, but they may point toward promising directions that can be exploited to obtain simpler descriptors.
Acknowledgments
The author thanks H-J Roth for helpful discussions.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
- Zorzi A Deyle K Heinis C . Cyclic peptide therapeutics: past, present and future . Curr. Opin. Chem. Biol.38 , 24 – 29 ( 2017 ).
- Adessi C Soto C . Converting a peptide into a drug: strategies to improve stability and bioavailability . Curr. Med. Chem.9 , 963 – 978 ( 2002 ).
- Schreiber SL Crabtree GR . The mechanism of action of cyclosporin A and FK506 . Immunol. Today13 , 136 – 142 ( 1992 ).
- Rezai T Bock JE Zhou MV Kalyanaraman C Lokey RS Jacobson MP . Conformational flexibility, internal hydrogen bonding, and passive membrane permeability: successful in silico prediction of the relative permeabilities of cyclic peptides . J. Am. Chem. Soc.128 , 14073 – 14080 ( 2006 ).
- Witek J Keller BG Blatter M Meissner A Wagner T Riniker S . Kinetic models of cyclosporin A in polar and apolar environments reveal multiple congruent conformational states . J. Chem. Inf. Model.56 , 1547 – 1562 ( 2016 ).
- Wang CK Swedberg JE Harvey PJH Kaas Q Craik DJ . Conformational flexibility is a determinant of permeability for cyclosporin . J. Phys. Chem. B122 , 2261 – 2276 ( 2018 ).
- Diamond JM Katz Y . Interpretation of nonelectrolyte partition-coefficients between dimyristoyl lecithin and water . J. Membr. Biol.17 , 121 – 154 ( 1974 ).
- Marrink SJ Berendsen HJC . Simulation of water transport through a lipid membrane . J. Phys. Chem.98 , 4155 – 4168 ( 1994 ).
- Xiang TX Anderson BD . Molecular distributions in interphases: statistical mechanical theory combined with molecular dynamics simulation of a model lipid bilayer . Biophys. J.66 , 561 – 572 ( 1994 ).
- Chipot C Comer J . Subdiffusion in membrane permeation of small molecules . Sci. Rep.6 , 35913 ( 2016 ).
- Comer J Schulten K Chipot C . Diffusive models of membrane permeation with explicit orientational freedom . J. Chem. Theory Comput.10 , 2710 – 2718 ( 2014 ).
- Dickson CJ Hornak V Pearlstein RA Duca JS . Structure–kinetic relationships of passive membrane permeation from multiscale modeling . J. Am. Chem. Soc.139 , 442 – 452 ( 2017 ).
- Pye CR Hewitt WM Schwochert J et al. Nonclassical size dependence of permeation defines bounds for passive adsorption of large drug molecules . J. Med. Chem.60 , 1665 – 1672 ( 2017 ).
- Lipinski CA Lombardo F Dominy BW Feeney PJ . Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings . Adv. Drug Deliv. Rev.46 , 3 – 25 ( 1997 ).
- Loosli HR Kessler H Oschkinat H Weber HP Petcher TJ Widmer A . Peptide conformations. Part 31. The conformation of cyclosporin A in the crystal and in solution . Helv. Chim. Acta68 , 682 – 704 ( 1985 ).
- Dang T Süssmuth RD . Bioactive peptide natural products as lead structures for medicinal use . Acc. Chem. Res.50 , 1566 – 1576 ( 2017 ).
- Alex A Millan DS Perez M Wakenhut F Whitlock GA . Intramolecular hydrogen bonding to improve membrane permeability and absorption in beyond rule of five chemical space . Med. Chem. Commun.2 , 669 – 674 ( 2011 ).
- Sebastiano MR Doak BC Backlund M et al. Impact of dynamically exposed polarity on permeability and solubility of chameleonic drugs beyond the rule of 5 . J. Med. Chem.61 , 4189 – 4202 ( 2018 ).
- Witek J Mühlbauer M Keller BG et al. Interconversion rates between conformational states as rationale for the membrane permeability of cyclosporines . ChemPhysChem18 , 3309 – 3314 ( 2017 ).
- Witek J Wang S Schroeder B et al. Rationalization of the membrane permeability differences in a series of analogue cyclic decapeptides . J. Chem. Inf. Model.59 , 294 – 308 ( 2019 ).