Thomas
Stadelmann
a,
Govindan
Subramanian
b,
Sanjay
Menon
b,
Chad E.
Townsend
c,
R. Scott
Lokey
c,
Marc-Olivier
Ebert
*a and
Sereina
Riniker
*a
aDepartment of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1-5, 8093 Zurich, Switzerland. E-mail: marc-olivier.ebert@org.chem.ethz.ch; sriniker@ethz.ch
bVeterinary Medicine Research & Development, Zoetis, 333 Portage Street, Bldg. 300, Kalamazoo, Michigan 49007, USA
cDepartment of Chemistry and Biochemistry, University of California, Santa Cruz, California 93064, USA
First published on 12th August 2020
Cyclic octadepsipeptides such as PF1022A and its synthetic derivative emodepside exhibit anthelmintic activity with the latter sold as a commercial drug treatment against gastrointestinal nematodes for animal health use. The structure–permeability relationship of these cyclic depsipeptides that could ultimately provide insights into the compound bioavailability 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. Hence, any understanding gained on these depsipeptides would serve as a prototype for future design strategies. In previous nuclear magnetic resonance (NMR) studies, two macrocyclic core conformers of emodepside were detected, one with all backbone amides in trans-configuration (hereon referred as the symmetric conformer) and the other with one amide in cis-configuration (hereon referred as the asymmetric conformer). In addition, these depsipeptides were also reported to be ionophores with a preference of potassium over sodium. In this study, we relate the conformational behavior of PF1022A, emodepside, and closely related analogs with their ionophoric characteristic probed using NMR and molecular dynamics (MD) simulations and finally evaluated their passive membrane permeability using PAMPA. We find that the equilibrium between the two core conformers shifts more towards the symmetric conformer upon addition of monovalent cations with selectivity for potassium over sodium. Both the NMR experiments and the theoretical Markov state models based on extensive MD simulations indicate a more rigid backbone for the asymmetric conformation, whereas the symmetric conformation shows greater flexibility. The experimental results further advocate for the symmetric conformation binding the cation. The PAMPA results suggest that the investigated depsipeptides are retained in the membrane, which may be advantageous for the likely target, a membrane-bound potassium channel.
The cyclic octadepsipeptide PF1022A (1) (Scheme 1) is a natural product, consisting of two repetitions of D-lactic acid, N-methyl L-leucine, D-phenyllactic acid and N-methyl L-leucine and has therefore a C2 symmetry axis. PF1022A demonstrates pharmacological activity against nematodes.9 Its synthetic derivative, emodepside (2) (Scheme 1), containing additional morpholine rings at the para position of the phenyllactic acid aromatic rings, exhibits increased anthelmintic activity10 and is a commercial drug effective against a number of gastrointestinal nematodes in cats. PF1022A and emodepside belong to a subfamily of cyclic depsideptides that have all the backbone amides methylated and possess only apolar side chains. This means that no hydrogen bond donors are present and thus, the formation of intramolecular hydrogen bonds is not possible. Yet, the ability to adopt a conformation, which maximizes the number of intramolecular hydrogen bonds is thought to be essential for passive membrane permeation of cyclic peptides.11–17 Nevertheless, some members of this subfamily of cyclic depsipeptides were found to be permeable or can be easily incorporated into a lipid membrane.18 Thus, to exploit their potential as therapeutics, it is important to establish a better understanding of the relationship between structure (conformational behavior) and permeability.
Some of the known members of the subfamily of fully backbone N-methylated cyclic depsipeptides with varying core ring sizes are shown in Scheme 1. The smallest members consist only of one N-methylated amino acid and one hydroxy acid (n = 1). For example, 3,6-di-(propan-2-yl)-4-methyl-morpholine-2,5-dione (3) is a natural product and was identified as a potential precursor of the cyclic hexadepsipeptide enniatin B (5).19,20 It showed moderate antioxidant and antimicrobial activity.21,22 Structurally, both the amide and the ester bond in the six-membered ring are in cis-configuration.20 The next larger members consist of two repetitions of an amino acid and a hydroxy acid (n = 2). In NMR studies of cyclo-(N-methyl L-leucine hydroxyisovaleric acid)2 (4) in chloroform, a C2-symmetric conformation was observed.23,24 In contrast to enniatin B (5) (n = 3), it showed no activity against mycobacteria.24 Enniatin B consists of three N-methyl L-valine and three D-hydroxyisovaleric acids and adopts, based on NMR studies, a C3-symmetric conformation with all amides in trans-configuration in chloroform.25 It is a well-known antibacterial, anthelmintic, antifungal, herbicidal and insecticidal compound.26 Due to the lipophilic nature of 5, it can be easily incorporated into lipid bilayers of cell membranes. Enniatin B was found to be ionophoric, i.e. it can carry mono and divalent cations through membranes with a selectivity for K+ over Na+.25,26 Further, it can form stable complexes with cations in solution. A 1:
1 as well as a 2
:
1 sandwich (peptide
:
cation ratio) complex were observed.27 A 3
:
2 complex was proposed as well but with lower stability than the 1
:
1 and the 2
:
1 complexes.27 Enniatin B showed decent permeability (log
Pe = −4.73) in a passive artificial membrane permeability assay (PAMPA)28 and a permeability of 6.1 × 10−4 cm s−1 in a Caco-2 permeability assay.18,29 Beauvericin (6) belongs, like 5, to the enniatin family. It consists of three alternating N-methyl L-phenylalanine and D-hydroxyisovaleric acid residues and was observed in NMR experiments to adopt a C3-symmetric conformation with all amides in trans-configuration in chloroform.1,306 shows cytotoxic, apoptotic, anticancer, anti-inflammatory, antimicrobial, insecticidal and nematocidal activities and is able to transport cations, particularly Ca2+ through lipid bilayers.31 The passive membrane permeability of 6 was determined to be 5.8 × 10−4 cm s−1 in a Caco-2 permeability assay,29 which is similar to the permeability of 5. Verticilide (7) is a cyclic octadepsipeptide (n = 4) such as PF1022A (1) and emodepside (2), and consists of four repetitions of N-methyl L-alanine and four repetitions of D-2-hydroxyheptanoic acid.327 was found to be a ryanodine-binding inhibitor and appears in NMR experiments in chloroform as two – not further studied – conformations in a ratio of 3
:
4.32 Simplification of the NMR spectra of 7 was observed after the addition of a 100-fold excess of KSCN and only one conformer was detected.32
The investigated compounds are only poorly soluble in water. Therefore, methanol and chloroform were chosen as simple mimics for a polar environment and the cell membrane, respectively. In both solvents, the NMR spectra of PF1022A (1) revealed two main conformations that interconvert slowly on the NMR time-scale (Scheme 2). The conformer ratio of 2 has been reported to be 4:
1 in methanol and 3
:
1 in chloroform in previous studies.9,33 The major conformation is characterized by a single cis-amide bond between the D-lactic acid and the N-methyl-L-leucine residue and is thus named asymmetric, whereas all amide bonds are trans in the minor conformation, thus named symmetric.9,33 The crystal structure of 1 apparently shows the asymmetric conformation, however, the data was not deposited with the CCDC (CCDC code MORJEI).34 The crystal structure of 2, on the other hand, is the symmetric conformation (CCDC code DOMZOW).35
Different side-chain and backbone modifications of 1 have been investigated in the literature.33,36–38 An interesting compound with regard to its conformational behavior is the bis-aza analog of PF1022A (8) (Scheme 3), where the asymmetric conformation is stabilized with a 100:
7 conformer ratio in chloroform.36 The conformation solved in the crystal structure is also asymmetric (CCDC code QOXDOW).36 The biological activity of 8 was found to be weaker by a factor of 5–10 compared to 1.36 For another modification with a turn-inducing element consisting of two prolines (D-Pro-L-Pro) (9) (Scheme 3), it was reported that the symmetric conformation is stabilized such that only this conformer is present in solution.37 Furthermore, the biological activity of 9 was found to be higher by a factor of 2 compared to 1.37 These observations led to the hypothesis that the propensity for the symmetric conformation is crucial for anthelmintic activity. However, for a third modification of 1, in which the four peptide bonds were replaced by thiopeptide bonds (10) (Scheme 3), the activity was also increased 2.5 times compared to 1.38 In this case, the increased activity was attributed to a more rigid asymmetric conformation due to the N-methyl-thioamides, which stabilize the cis-amide bond between D-thiolactic acid and N-methyl-L-leucine.33,38 Based on the published data, no clear correlation between activity and conformational preference for the symmetric or asymmetric structure can be found, especially if it is considered that an increase or decrease of activity by a factor of 2 is mostly within the accuracy of experiment. Additionally, no experimental membrane permeability data for 1, 2 and 8–10 is reported in the literature.
The mechanism of action of 1, 2 and related compounds is not yet fully understood. Initially, their anthelmintic activity was attributed to the binding of a presynaptic latrophilin receptor.10 More recently, binding to the calcium-activated potassium channel SLO-1 was proposed to be involved in the activity of 2, possibly in combination with the latrophilin receptor.39–42 No crystal structure of 1 or 2 bound to one of these proteins is available. PF1022A and derivatives were reported to be ionophores with selectivity for K+ over Na+,43 similar to enniatin B. However, the ion carrier property across lipid bilayers does not appear to be related to the anthelmintic activity, because the enantiomer of PF1022A (i.e. all D- and L-residues switched) exhibited the same ionophoric ability but no anthelmintic activity.43
In this study, the interplay between the macrocyclic core conformational behavior of PF1022A, emodepside and related compounds with their ionophoric nature and their passive membrane permeability was investigated to enhance our understanding for the rational design of such cyclic octadepsipeptides with improved profiles. For this, we characterized the conformational behavior of 1, 2 and 8 and the effect of monovalent cations on the conformational ensembles using solution NMR measurements and extensive MD simulations in chloroform and methanol. With this data, we want to explore how the cyclic depsipeptides interact with cations and determine a plausible coordination mode. The complexation with a cation could be an effective mechanism to bury the polar groups and thus, may be a crucial step for the incorporation of the depsipeptides into the membrane. The passive membrane permeability is assessed with PAMPA with and without the addition of potassium.
Compound | Conformer ratio in CD3OH (asymmetric![]() ![]() |
Conformer ratio in CDCl3 (asymmetric![]() ![]() |
---|---|---|
a The variability is likely due to residual cation content originating from synthesis, workup and purification that differs from batch to batch. | ||
PF1022A (1) | 5![]() ![]() ![]() ![]() ![]() ![]() |
3![]() ![]() ![]() ![]() |
Emodepside (2) | 7![]() ![]() |
7![]() ![]() |
Bis-aza analog (8) | 12![]() ![]() |
10![]() ![]() ![]() ![]() |
For all the investigated peptides, exchange peaks (EXSY peaks) could be detected in ROESY spectra recorded in chloroform-d. In CD3OH, EXSY peaks could only be detected for 8 but low intensity and limited resolution did not allow further analysis. Besides the expected EXSY cross-peaks between the major asymmetric and the minor symmetric conformer for 1 and 2, additional EXSY peaks are present, which indicate that more than the two known conformations are populated in solution. At least two additional low-intensity conformers could be identified (see ESI†). Using the volumes of the EXSY cross-peaks it is possible to calculate the site-to-site exchange rates k1 and k2 between the magnetic sites in the interconverting conformers (Fig. 1). The additional two low-intensity conformers were neglected in the calculation of the exchange rates since their intensity was close to the noise level and their corresponding diagonal peaks were partially buried under other, more intense signals. The calculated site-to-site rates are summarized in Table 2.
![]() | ||
Fig. 1 Schematic drawing of the magnetization transfer pathways used for the analysis of the EXSY data for 1, 2 (left) and 8 (right). In the symmetric conformation of 1 and 2, one of the two chemically equivalent amide bonds can flip into a cis-configuration to reach the asymmetric conformation (amide bond between Lac15 and Mle26, see Scheme 1). In this process, magnetization is transferred via two different site-to-site pathways (A ↔ B and A ↔ C with kAB = kAC = k1 and kBA = kCA = k2), each leading to a separate set of EXSY cross-peaks. During a transition from the symmetric to the asymmetric conformation, each nucleus in a symmetric pair undergoes either pathway equally likely. In the reverse process from the asymmetric to the symmetric conformation a nucleus at site B will always follow A ↔ B whereas a nucleus at site C will always follow A ↔ C. As a consequence, the site-to-site exchange rates k1 and k2 for 1 and 2 differ from the mechanistic exchange rates: k′1 = 2 × k1 and k′2 = k2 where K = k′1/k′2. In 8, the C2 symmetry is broken by the two additional nitrogen atoms in the backbone and only a single magnetization transfer pathway has to be considered. Therefore for 8k1 = k'1 and k2 = k'2. |
Compound | k 1 [s−1] | k 2 [s−1] | k ex [s−1] |
---|---|---|---|
PF1022A (1) | 0.16 | 0.09 | 0.25 |
Emodepside (2) | 0.12 | 0.06 | 0.18 |
Bis-aza analog (8) | 0.17 | 0.02 | 0.19 |
The site-to-site exchange rates of 1, 2 and 8 are comparable and are about twice as high compared to the exchange rate reported for cyclosporine A (kex ≈ 0.1 s−1).44 This is plausible as the smaller ring size of the cyclic octadepsipeptides (24-membered ring) compared to cyclosporine A (33-membered ring) increases the ring strain. Since these results are based on a single mixing time, no direct error estimate can be given. From the comparison of the cross-peak intensities on both sides of the diagonal, errors about 20% can be assumed.
In 1H and 13C NMR spectra of the investigated cyclic octadepsipeptides, the signals for the symmetric conformer were generally found to be broader. To quantify this additional exchange broadening, presumably originating from processes on the millisecond to microsecond range, 13C T2 relaxation time measurements of 1 in CDCl3 were performed (Fig. 2). It is clearly visible that the symmetric conformer has shorter T2 relaxation times for the backbone carbons compared to the asymmetric conformer. This indicates greater backbone flexibility on the μs to ms timescale for the symmetric conformer. To the best of our knowledge, this is the first time that such behavior was observed for a cyclic depsipeptide.
Markov state models (MSMs)47–50 are a powerful tool to analyze the conformational dynamics in MD simulations. Here, we generated core-set Markov models of PF1022A (1) in chloroform using common nearest neighbor (CNN) based clustering49,51–53 and the PyEMMA package.54 This procedure has been used successfully with other cyclic peptides.12 The MSMs were constructed separately for the asymmetric and the symmetric conformations (but with the same TICA space55). For the asymmetric subset, only two unconnected conformational states could be identified, whereby one arose from a single simulation and was considered as noise. Therefore, the backbone with the asymmetric configuration appears to be relatively rigid. In contrast, the backbone with the symmetric configuration shows substantially more flexibility, and seven conformational states could be observed (Fig. 3). This is in line with the NMR experiments, where shorter T2 relaxation times were observed for the symmetric conformer, indicating higher flexibility on the μs–ms time scale.
The conformational states 3 and 5, as well as 6 and 7, are in principle the same, rotated by 180° due to the C2 symmetry of the symmetric conformation. This allows for an easy check of convergence. It can be seen in Fig. 3 (and Table S8 in the ESI†) that the model is not yet fully converged. Note that the starting structure of the simulation corresponds to state 7. Conversion from state 7 to state 6 is essentially a complete reorientation of the entire backbone. Thus, very long simulations (>10 μs) would be needed to obtain the same population for state 6. Nevertheless, the results also indicate that the conformational space for the symmetric conformation is already sampled quite extensively.
![]() | ||
Fig. 4 Titration of 5 mM PF1022A (1) with a KSCN solution in CD3OH: Hα region of 1H NMR spectra. Chemical shift changes were observed for the symmetric conformation, best seen for the signal of the Hα proton in residue Phl37 (blue labels). In addition, a change in the ratio between the symmetric and asymmetric conformation is observed. Also the asymmetric conformation shows small changes in chemical shift at high salt concentrations. The titration plot for emodepside (2) can be found in the ESI.† |
Salt | PF1022A (1) | Emodepside (2) | Bis-aza analog (8) |
---|---|---|---|
KSCN | 7![]() ![]() ![]() ![]() |
7![]() ![]() ![]() ![]() |
12![]() ![]() ![]() ![]() |
NaSCN | 5![]() ![]() ![]() ![]() |
7![]() ![]() ![]() ![]() |
— |
NH4SCN | 7![]() ![]() ![]() ![]() |
— | — |
CsSCN | 7![]() ![]() ![]() ![]() |
— | — |
The changes in asymmetric:
symmetric ratio upon addition of monovalent salt are comparable between 1 and 2 for KSCN and NaSCN. Consequently, the affinities of the two peptides for the cations are expected to be very similar. Therefore, for subsequent titrations only PF1022A (1) was used. In contrast, the bis-aza analog (8), which predominantly adopts the asymmetric conformer, required a much higher salt concentration to observe a shift in the conformer ratio (Fig. 5).
The titration data of PF1022A (1) with KSCN and CsSCN (as well as 2 with KSCN) can only be explained by a model containing at least two different ion-bound symmetric species, which are in fast exchange with the unbound symmetric conformation. In the case of a simple mixture of the free depsipeptide and only a 1:
1 complex, the observed chemical shift is expected to change from the value of the free conformer towards that of the ion-bound conformer. However, we do not observe this asymptotic behavior. Instead, first the chemical shift drops with increasing salt concentration, then reaches a minimum and increases again at high concentrations. This indicates that at least a third symmetric species, which interacts with the ion, is populated. We propose a mixture of a 2
:
1 (peptide
:
cation ratio) and a 1
:
1 complex in solution, as was reported for enniatin B (5) and beauvericin (6).25,27 Such a mixture was already postulated for PF1022A (1) but not supported by any experimental data.56 Normally, fitting of the equilibrium constants K1 and K2 is straightforward using the measured change in chemical shift in dependence of the salt concentration.57 However, this system is more complicated due to the pre-equilibrium between the free asymmetric and symmetric conformers, and possibly additional species such as a 2
:
1 complex with one symmetric and one asymmetric conformer, or an asymmetric ion-bound conformer. We fitted our data with a model containing the free peptide in its symmetric conformation, the symmetric 1
:
1 complex, and the symmetric 2
:
1 complex. Instead of explicitly considering the pre-equilibrium, we have used the total concentration of all symmetric species obtained from integration of the 1H spectra. We interpret the results only qualitatively since similar fits may be achieved with different fitting parameters. Fig. 6 clearly shows that the change in the asymmetric
:
symmetric ratio can be used to qualitatively measure the cation affinity of the symmetric conformer. The order of affinities with Cs+ > K+ > Na+ > NH4+ is in agreement with those reported in literature,43 where alkali metals from Li+ to Cs+ were tested. If the change in chemical shift is plotted as a function of the salt concentration while keeping the peptide concentration constant, it can be observed that the change in chemical shift at high salt concentration is ordered by cation size. One could therefore speculate that the backbone of the depsipeptide has to adapt more extensively to accommodate smaller ions. This, in turn, leads to larger chemical shift changes in these complexes.
![]() | ||
Fig. 6 Titration of 5 mM PF1022A (1) (top) and 5 mM emodepside (2) (bottom) with different monovalent cations (CsSCN in grey, KSCN in blue, NaSCN in orange and NH4SCN in red) in CD3OH while the total volume was kept constant. The titration with CsSCN was only done up to 125 mM due to solubility issues. (Left): Change of the concentration of the symmetric conformation upon the addition of the corresponding salt. The data points were fitted with a damped logistic growth function (for details see ESI†). (Right): Change of the chemical shift of the Phl37/Phm37 Hα proton as a function of the salt concentration (for details of the fit, see ESI†). The plots were generated with R.58 |
A consistent pattern is visible when comparing the plots on the left side and on the right side of Fig. 6. A higher salt concentration is needed to achieve a 1:
1 ratio between the asymmetric and the total symmetric species than for a 50% change in chemical shift. The apparent lag increases with decreasing ion affinity. One can show that this behavior can already be reproduced by two coupled equilibria (ion independent conformational change and formation of the 1
:
1 complex). Its observation alone does not imply any cooperative phenomena or the presence of higher order complexes. Without further knowledge about the relative stabilities of the 2
:
1 and 1
:
1 complexes for each metal, a more detailed analysis is not possible at this stage.
It is known that valinomycin, a cyclic dodecadepsipeptide, as well as some crown ethers can bind cations even in an apolar solvent.59–61 This ability is an indirect evidence that the ion-bound complex may exist inside the membrane interior, i.e. that ion transport across a membrane is possible. To assess if the cyclic octadepsipeptides are also able to bind cations in an apolar solvent, KSCN was added to a solution of 1 in chloroform and sonicated for several hours. In subsequent NMR measurements, only the symmetric conformation could be detected in solution (Fig. 7), which indicates ion binding.
The same effect was achieved by mixing a solution of emodepside (2) in chloroform with a saturated aqueous KSCN solution and letting the solution stand until phase separation had occurred (Fig. 8). These results demonstrate that PF1022A and emodepside can carry cations from a polar phase into an apolar environment.
![]() | ||
Fig. 9 (Top): Snapshot of the 1![]() ![]() ![]() ![]() |
The ion-bound conformation in the MD simulations is, however, dependent on the system setup. In simulations with two molecules of PF1022A (1) in chloroform (1 μs) in presence of a single potassium ion, both a 1:
1 and a 2
:
1 complex (Fig. 9) could be observed over the course of the simulation, whereby the 1
:
1 complex did not adopt a cavitand-like structure.
To verify the cavitand-like structure of the 1:
1 complex experimentally, we first aimed to crystalize PF1022A (1) in the presence of KSCN. Crystallization attempts with equimolar salt and peptide concentration led to separate crystals of KSCN and 1, in which 1 is crystallized in the asymmetric conformation with one co-crystallized methanol molecule (Fig. 10). The structure agrees well with the asymmetric crystal structure of the bis-aza analog (8) (CCDC code QOXDOW), justifying the use of the latter as starting structure in the MD simulations of 1. By increasing the KSCN concentration to a 10-fold excess in methanol, an ion-bound complex of 1 could be crystallized. The crystal structure revealed a 2
:
3 complex (peptide
:
cation), with co-crystallized methanol and one water molecule (Fig. 11). The ion-bound peptide crystallized in the symmetric conformation as observed in the NMR experiments and the MD simulations. This complex is likely not the major structure present in solution. In an MD simulation, the 2
:
3 complex showed very low stability.
![]() | ||
Fig. 10 Crystal structure of PF1022A (1) (CCDC number: 2004078†) crystallized in the asymmetric conformation. Carbon atoms are colored in grey, nitrogen atoms in light blue and oxygen in red. The ellipsoids represent 50% of probability level and hydrogen atoms are shown with a radius of 0.3 Å. One methanol molecule is co-crystalized and disordered. The figure was created with Mercury.63 |
![]() | ||
Fig. 11 (Left): Crystal structure of a 2![]() ![]() |
Since the crystallization experiments were not able to confirm the cavitand-like structure, we next turned to NMR to answer this question. The most straightforward evidence would be a through-space correlation between the two aromatic rings, which should be very close in the cavitand-like structure. However, this correlation is not experimentally accessible in these cyclic depsipeptides due to the C2 symmetry of the symmetric conformer. One possible solution for this issue is to break the C2 symmetry by introducing a substitution in the aromatic ring of one of the two phenyllactic acids. The PF1022A analog 11 contains an iodine substituent in para-position at one of the aromatic rings (Fig. 12), and exhibits the same conformational behavior and ionophoric properties as 1 (experimental results summarized in the ESI†). With 11, it should be possible to observe ROESY correlations between the two aromatic rings, if the cavitand-like structure is present in solution. However, such correlations were not observed (Fig. 12). Therefore, the cavitand-like structure is likely an artifact of the setup in the MD simulation with a single peptide and potassium ion. This is further supported by the observation that no cavitand-like structure was adopted in the MD simulations with two peptides and a potassium ion (see discussion above).
Upon addition of cations, a shift towards the symmetric conformation was observed in the NMR titration experiments, which indicates that only the symmetric conformation can bind tightly to the ions. A preference for cesium over potassium over sodium was found, which is in agreement with previous studies. Furthermore, we could show that these cyclic octadepsipeptides can carry cations into an apolar solvent, like other ionophores. The titration curves indicate a mixture of both 1:
1 and 2
:
1 (2 peptides and 1 cation) complexes. MD simulations suggest the formation of a sandwich complex, like the one observed for enniatin B (5). A cavitand-like structure of the 1
:
1 complex seen in the MD simulations could, however, not be confirmed experimentally using the mono-iodine substituted analog (11). Crystallization of PF1022A (1) with an excess of KSCN in methanol yielded a 2
:
3 complex (2 peptides and 3 potassium ions), where the peptides are in the symmetric conformation, confirming the findings in the NMR experiments and MD simulations.
The fact that the symmetric conformers can bind cations might still be relevant for activity, since the metal bound species may possess a higher propensity for membrane incorporation than the free peptide. This would also be in line with the location of the proposed target, SLO-1, a membrane-bound ion channel. The results of the PAMPA experiments and the ineffective wash-out of PF1022A from CaCo-2 membranes may indeed indicate that the peptides do not permeate but rather incorporate into the membrane. Our extensive NMR and computational characterizations are in this case very important to provide further insight at atomic resolution beyond the scope of PAMPA. In terms of the investigated properties, no significant differences were found between 1 and 2. The ratios between symmetric and asymmetric conformations in solutions as well as their binding affinities towards cations are similar. Thus, the difference in anthelmintic activity between 1 and 2 cannot be directly related to a difference in the conformational behavior or ionophoric property, but likely stems from the effect of the morpholino substitution modulating the potency at the target. The studied bis-aza analog (8), for which the asymmetric conformation is further stabilized, has a significantly lower affinity towards cations, which could be an indication that cation binding may be an important aspect for membrane incorporation, and potentially influence activity. Future studies with cyclic octadepsipeptides that exhibit different cation binding affinities might be able to further elucidate these connections.
The CD3OH signal was suppressed by presaturation or excitation sculpting.6813C-HSQC spectra were recorded with sensitivity enhancement69 and multiplicity editing. TOCSY spectra were recorded with zero quantum filter70 and 80 ms DIPSI271 isotropic mixing except for 1 in chloroform where 80 ms mlev1772 mixing was used. The mixing time for the EASY-ROESY experiments was set to 100 ms if not otherwise stated. For all 2D spectra, the time domain in both dimensions was extended to twice its size by zero filling and apodized with a cos2 or sin function. The baseline of the resulting spectra was corrected with a polynomial of fifth order or using the Whittacker smoother algorithm.73 Processing was done with Bruker TopSpin™ version 4.0 (Bruker Biospin AG) and MestReNova 12.0 (Mestrelab Research). Resonance assignment and integration of ROESY cross-peaks were performed with SPARKY 3.115.7413C T2-relaxation time measurements were done with a series of sensitivity enhanced 13C-CPMG-HSQC spectra75 using a slightly modified version of Bruker standard pulse program hsqct2etf2gpsi with ten different evenly spaced relaxation delays between 15.2 ms and 456 ms. Heating effect compensation was used. Fitting of the exponential decays was done with Prism 8.4 (GraphPad Software).
M = eLtm × M0 | (1) |
![]() | (2) |
![]() | (3) |
L is the difference between the kinetic matrix K containing the site-to-site rate constants and the relaxation matrix R, tm is the mixing time used in the ROESY experiment, and Ri are the auto-relaxation rates of the exchanging sites in the symmetric (A) and asymmetric (B, C) conformations.
As an example, the procedure is described in the following for PF1022A (1).
EXSY peak volumes extracted from the ROESY spectrum (shown schematically in Fig. 13) and the magnetic fractions in the 1H NMR spectrum are inserted in matrix A(eqn 4). Site-to-site rates are obtained by taking the logarithm of matrix A and dividing the result by the mixing time (0.1 s) (eqn 5): kAB = kAC = 0.09 s−1 and kBA = kCA = 0.16 s−1 (averaged rates). Calculations were carried out in Mathematica 12.0.78
![]() | (4) |
![]() | (5) |
![]() | ||
Fig. 13 Schematic EXSY spectrum with sites A (symmetric conformation), B and C (asymmetric conformation). A exchanges with B and C but B does not exchange with C. |
System | Starting structure (CCDC code) | Number of simulations | Solvent | Number of solvents | Length of thermalization per step [ps] | Length per MD simulation [μs] |
---|---|---|---|---|---|---|
PF1022A | DOMZOW | 1 | CHCl3 | 329 | 2000* | 10 |
PF1022A | DOMZOW | 10 | CHCl3 | 329 | 2000 | 1 |
PF1022A | DOMZOW | 1 | CH3OH | 637 | 20 | 1 |
PF1022A | QOXDOW | 1 | CHCl3 | 344 | 2000 | 10 |
PF1022A | QOXDOW | 10 | CHCl3 | 344 | 2000 | 1 |
PF1022A + K+ | DOMZOW | 1 | CHCl3 | 328 | 20 | 1 |
PF1022A + K+ | DOMZOW | 1 | CH3OH | 636 | 20 | 10 |
PF1022A + K+ | QOXDOW | 1 | CHCl3 | 343 | 20 | 1 |
2 PF1022A + K+ | DOMZOW | 1 | CHCl3 | 3085 | 2000 | 1 |
2 PF1022A + 3 K+ | 2004087 | 1 | CHCl3 | 389 | 2000 | 1 |
2 PF1022A + 3 K+ | 2004087 | 1 | CH3OH | 765 | 2000 | 1 |
Emodepside | DOMZOW | 1 | CHCl3 | 497 | 2000* | 10 |
Emodepside | DOMZOW | 1 | CH3OH | 989 | 20 | 1 |
Emodepside + K+ | DOMZOW | 1 | CHCl3 | 496 | 20 | 1 |
Emodepside + K+ | DOMZOW | 1 | CH3OH | 988 | 20 | 10 |
Bis-aza analog | DOMZOW | 11 | CHCl3 | 330 | 2000 | 1 |
Bis-aza analog | QOXDOW | 1 | CHCl3 | 350 | 2000* | 10 |
Bis-aza analog | QOXDOW | 10 | CHCl3 | 350 | 2000 | 1 |
Bis-aza analog | QOXDOW | 1 | CH3OH | 688 | 20 | 1 |
Bis-aza analog + K+ | QOXDOW | 1 | CHCl3 | 349 | 20 | 10 |
Bis-aza analog + K+ | QOXDOW | 1 | CH3OH | 688 | 20 | 1 |
PF1022A# | DOMZOW | 1 | CHCl3 | 329 | 2000* | 10 |
PF1022A# | DOMZOW | 10 | CHCl3 | 329 | 2000* | 1 |
PF1022A# | QOXDOW | 1 | CHCl3 | 344 | 2000* | 10 |
PF1022A# | QOXDOW | 10 | CHCl3 | 344 | 2000* | 1 |
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Fig. 14 Chapman–Kolmogorov test for the symmetric conformer of 1 in chloroform with 7 states and a lag time of 10 ns. |
Around 10 mg of 1 was dissolved in methanol together with a tenfold excess of KSCN. The concentrated sample was put in the freezer at −28 °C. After three days transparent crystals were obtained and were given to SMOCC for analysis. The obtained crystal structure was a complex of two peptides with three ions with co-crystalized methanol molecules and one water molecule. Crystal data for C118H198K3N11O36S3 (M = 2560.34 g mol−1): triclinic, space group P1 (no. 1), a = 14.90080(10) Å, b = 15.83070(10) Å, c = 16.84690(10) Å, α = 111.2440(10)°, β = 101.4290(10)°, γ = 100.0950(10)°, V = 3495.22(5) Å3, Z = 1, T = 100.0(1) K, μ(CuKα) = 1.908 mm−1, Dcalc = 1.216 g cm−3, 95494 reflections measured (5.872° ≤ 2Θ ≤ 159.716°), 28
018 unique (Rint = 0.0424, Rsigma = 0.0371) which were used in all calculations. The final R1 was 0.0502 (I > 2σ(I)) and wR2 was 0.1453 (all data).
Footnote |
† Electronic supplementary information (ESI) available. CCDC 2004078 and 2004087. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0ob01447h |
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