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. 2021 Nov 23;12(12):1942–1947. doi: 10.1021/acsmedchemlett.1c00508

Ring Size as an Independent Variable in Cyclooligomeric Depsipeptide Antiarrhythmic Activity

Abigail N Smith †, Daniel J Blackwell ‡, Bjorn C Knollmann ‡, Jeffrey N Johnston †,*
PMCID: PMC8667307  PMID: 34917258

Abstract

graphic file with name ml1c00508_0006.jpg

Hit-to-lead studies employ a variety of strategies to optimize binding to a target of interest. When a structure for the target is available, hypothesis-driven structure–activity relationships (SAR) are a powerful strategy for refining the pharmacophore to achieve robust binding and selectivity characteristics necessary to identify a lead compound. Recrafting the three-dimensional space occupied by a small molecule, optimization of hydrogen bond contacts, and enhancing local attractive interactions are traditional approaches in medicinal chemistry. Ring size, however, is rarely able to be leveraged as an independent variable because most hits lack the symmetry required for such a study. Our discovery that the cyclic oligomeric depsipeptide ent-verticilide inhibits mammalian cardiac ryanodine receptor calcium release channels with submicromolar potency provided an opportunity to explore ring size as a variable, independent of other structural or functional group changes. We report here that ring size can be a critical independent variable, suggesting that modest conformational changes alone can dramatically affect potency.

Keywords: Cyclic depsipeptide, Macrocycle, Mirror-image natural product, Bro5, Arrhythmia

Macrocyclic peptide drug candidates have become increasingly popular in the last few decades, largely stemming from their ability to interact with “undruggable” targets.1 Several macrocyclic, peptidic compounds2 have already proven to be successful drugs, such as the well-known antibiotic vancomycin, and the immunosuppressant cyclosporine. In this larger size regime (>500 MW), a majority of current macrocyclic drug candidates are natural products or close variants of natural products.3 Relative to small molecules, there is less flexibility to guide the design of these noncanonical drug candidates to optimize both binding and bioavailability. Studies that further the understanding of macrocyclic peptides and their optimization against biological targets are therefore of great value. Despite these challenges, chemists have made strides to develop non-natural macrocyclic drug candidates, utilizing chemical modifications inspired by nature such as amide N-permethylation,4 replacement of amides with esters,5 thioesters/thioamides,6 or ketones,7 incorporation of d-amino acids and nonproteinogenic amino acids,8 side chain modification, and the use of peptide stapling techniques.9,10 Additionally, macrocyclization is increasingly applied to small molecule drug development,2,11 where single-carbon homologue SAR can be applied to optimize pharmacophore presentation (Figure 1).12 However, natural products still continue to dominate the examples of macrocycles that are cell permeable, water-soluble, and even orally bioavailable. Notable contributions to the development of “drug-like” non-natural macrocycles and metrics to quantify these factors are beginning to emerge, highlighting the importance of these compounds in drug discovery.13,14 Peptide and depsipeptide macrocycles, particularly, offer an expansive landscape of ring sizes and the impact of ring size on permeability and solubility is developing slowly.

Figure 1.

Figure 1

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Compare/contrast of ring-size modifications for SAR in medicinal chemistry (conventional approach) to an analogous study using oligomeric ring sizes (this work).

Considering cyclic depsipeptide natural products, nature appears to leverage ring size to fine-tune biological activity. Several prominent examples include valinomycin and montanastatin,15 bassianolide and enniatin C,16 beauvericin and bassiatin,16 and the antimycins.17 Although natural products are ripe with examples of diverse depsipeptides varying in ring size, few studies provide insight into the effect of a systematic variation of ring size and correlation of biological activity at a discrete target. Even then, ring size is usually only explored as a parameter when the ring analogue is also a natural product. We speculated that the ability to vary only ring size while leaving functionality and other features unchanged might be realized with cyclic oligomeric depsipeptides (CODs).

Because of their oligomeric nature, CODs provide an effective platform to probe ring-size modified pharmacophores wherein size is manipulated while maintaining the functionality inherent to the biologically active molecule. This is an attribute unique to only oligomeric molecules and something rarely studied in natural product drug design (Figure 1). Examples of the synthesis of non-natural analogues of cyclic depsipeptides include decabassianolide (analogue of bassianolide),18 a 12-mer and 24-mer of beauvericin,19 a tetradepsipeptide and cyclohexadepsipeptide analogue of valinomycin,20 and a 12-mer of bassianolide.19 More generally, there has been noteworthy work with the synthesis of ring-size analogues in the field of oligolides21,22 as well as cyclic peptides.23−25 Yet, still absent from most studies is a large range (>3-mer) of ring sizes, other structural functionality left unchanged, or the evaluation of these compounds against a distinct biological target.

We recently reported26 a study of the fungal COD natural product (−)-nat-verticilide and its enantiomer, (+)-ent-verticilide (9, Chart 1), to determine their activity against mammalian cardiac ryanodine receptor (RyR2), inspired by the natural product’s activity with insect ryanodine receptor.27,28 Although the natural product had no effect, the non-natural enantiomer significantly reduced RyR2-mediated spontaneous calcium release.29 This discovery positioned us to advance a structure–activity study to better understand ring size and its effect on biological activity in the absence of any functional group changes. This is particularly significant in cardiovascular disease, where macrocyclic compounds are rare in therapeutic development.30 In this work, we report the synthesis and activity of ring-size congeners based on the hit compound, ent-verticilide.

Chart 1. ent-Verticilide and Its Ring-size Analogues.

Chart 1

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Results and Discussion

Synthesis of Ring-Size Analogues

We prepared five non-natural ring-size congeners of ent-verticilide (24-membered COD), including 6-, 12-, 18-, 30-, and 36-membered CODs (Chart 1). ent-Verticilide and the 18-membered COD were prepared using methodology previously reported.31,32 The 6-, 12-, 30-, and 36-membered macrocycles were synthesized through a complementary route developed for this purpose (Scheme 1). The synthesis of the depsipeptide unit begins with an enantioselective Henry reaction using hexanal (1) and nitromethane, followed by MOM protection of the subsequent alcohol. Nitroalkane 2 was then subjected to Mioskowski–Nef conditions33 to afford the α-hydroxy acid, which was protected as benzyl ester 3. This hydroxy heptanoic ester was then deprotected and coupled with N-methyl-d-alanine to arrive at the key didepsipeptide unit (5).34 Selective deprotections, followed by condensative couplings, provided monomers of varying lengths, which were globally deprotected and cyclized to furnish the CODs of increasing ring size (Scheme 1).

Scheme 1. Synthesis of the Didepsipeptide Monomer Using an Enantioselective Henry Reaction.

Scheme 1

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Activity of Ring-size Analogues

The activity of the ring size analogues was examined in cardiomyocytes isolated from a CASQ2 gene knockout mouse, a validated model of severe catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans that exhibits pathologically increased RyR2 activity. Ventricular cardiomyocytes were isolated, permeabilized with saponin, and incubated with vehicle (DMSO) or 25 μM of selected compound. RyR2 activity was measured in the form of calcium sparks, which are elementary Ca2+ release events generated by spontaneous openings of intracellular RyR2 Ca2+ release channels. Importantly, saponin selectively permeabilizes the sarcolemmal membrane, leaving the SR membrane intact and ensuring equivalent access of the compounds to the SR membrane where RyR2 resides.

The compounds were first screened by incubating cardiomyocytes for 10 min at 25 μM concentration and compared to vehicle (DMSO) conditions. The 18- and 24-membered rings significantly inhibited RyR2-mediated Ca2+ spark frequency, however, the 6-, 12-, 30-, and 36-membered rings appeared to have no effect (Figure 2A). To ensure there was no delayed binding effect with the inactive rings, longer incubation times for two of the macrocycles were investigated. Extending the incubation time to 60 min did not enable any measurable inhibition of spark frequency in either the 12- or 30-membered rings (Figure 2B), suggesting that ring size is a necessary component for activity. Incubation with the 12- or 18-membered linear precursors also demonstrated no inhibition of spark activity, despite clear inhibition with the 18-membered ring, supporting the importance of a cyclic structure for activity (Figure 2C). To compare potency, concentration–response curves were generated for the 18- and 24-membered rings. Both compounds inhibited RyR2 with similar potency (Figure 2D,E). The natural (d,l) versions of the 6-,18-, 24-, 30-, and 36-macrocycles were also prepared, but none of these were active in the assay.

Figure 2.

Figure 2

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Biological screen for cardiac ryanodine receptor (RyR2) activity. Note that “ent-24” = 9, and “ent-18” refers to structure 8 in Chart 1, which is also the enantiomer of a natural product. (A) Calcium spark frequency in permeabilized murine cardiomyocytes was recorded as an index of RyR2 activity. Compounds were screened at 25 uM concentration after 10 min incubation. * = p < 0.001 by one-way ANOVA with Tukey’s posthoc test. (B) Incubation time was extended to 60 min for the 12- and 30-membered rings. (C) The seco-acid precursors to 12- and 18-membered rings were tested at 25 μM. (D) Concentration response curve for the 18-membered ring and (E) 24-membered ring. Cells were incubated for 30 min.

Structure–Activity Relationship

Of the five ring-size variants prepared, the 6-, 12-, 30-, and 36-membered ring did not alter calcium spark frequency (Figure 3). However, the 18-membered variant (8) was a potent inhibitor. It is important to note that because the plasma membrane of the cells is permeabilized, cell permeability is removed as a factor contributing to loss of activity. However, several hypotheses for the activity results are proposed by considering the impact of ring size on the conformation and degree of expected rigidity for this ent-verticilide series. The presentation of the methyl and pentyl side chains was expected to deviate significantly in the 6-membered ring variant (6), but as ring size increases, the conformational mobility is predicted to increase. This is expected to reach a point of limited returns, however, if the COD flexibility begins to work against the tightest binding conformation. This could be the case with the 30- and 36-membered macrocycles. The 6- and 12-membered rings, on the other hand, mirror the difficulties that small molecules display in binding to large, complex targets. The lack of activity from these two ring sizes suggests that ent-verticilide could be interacting with RyR2 over a large surface area, whereas the smallest two ring sizes cannot achieve a similar interaction.

Figure 3.

Figure 3

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Activity summary of ring-size analogues.

We also considered lipophilicity as a second factor. Larger molecular weight compounds can be more lipophilic than their small molecule counterparts. This increase in lipophilicity usually results in a corresponding decrease in aqueous solubility. However, a “chameleon-like” behavior has been attributed to macrocyclic compounds, describing their ability to adopt conformations that bury hydrophilic or hydrophobic functionality based on their environment.35 This attribute of molecules well outside of the traditional realm of drug-likeness endows them with both the lipophilicity needed to cross cell membranes as well as aqueous solubility. These two properties are crucial for activity with intracellular targets along with bioavailability. In this ring-size series, both the AlogP36 values and the topical polar surface areas (TPSA)37,38 were calculated to quantify the relative lipophilicity for each compound. These calculated values follow the expected trend: as the ring size increases, lipophilicity increases (Table 1). In these initial studies, however, a corresponding decrease in solubility was not observed. Each ring size was soluble in the aqueous buffer used in the calcium sparks assays at 25 μM, and no complications with precipitation were observed.39 This was further verified by kinetic solubility studies (see Supporting Information).40 Additionally, ent-verticilide maintains activity in vivo,26 indicating some level of aqueous solubility. This increase in lipophilicity could, however, still be a factor contributing to the loss of activity. If the target binding surface includes numerous hydrogen bonding sites, the pharmacophore could have surpassed a nonpolar threshold in the larger macrocycles. Furthermore, increases in lipophilicity could reduce the target specificity, resulting in a loss of activity through off-target binding.41 The smaller ring sizes (6- and 12-membered rings) may suffer from increased rigidity, an effect noted in studies of a decapeptide analogue of cyclosporine.42

Table 1. Comparison of CODs by Ring Size, Molecular Weight, Calculated AlogP Values, and Calculated Topical Polar Surface Area (TPSA).

ring size molecular weight (g/mol) calcd AlogPa calcd TPSA (Å2)b
6 (6) 213.27 0.95–1.60 66.76
12 (7) 426.55 3.11–3.97 100.71
18 (8) 669.83 4.59–5.19 151.69
24 (9) 853.11 5.05–5.63 207.06
30 (10) 1066.39 5.41–5.83 248.17
36 (11) 1335.77 5.67–5.98 303.29
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a

AlogP values were calculated using the Virtual Computational Chemistry Laboratory ALOGPS 2.1 program.

b

TPSA values were calculated using QikProp in the Schrödinger software suite.

Remarkably, from this study we discovered another potent ring-size variant, the 18-membered COD. This finding indicates that the full structure of ent-verticilide, a 24-membered cyclic oligomeric depsipeptide, is not critical to its activity. These data support an intriguing hypothesis, where a region, rather than total volume of the molecule, is responsible for its activity. It also indicates that conformation plays an essential role, as the 12-membered ring and the 12-membered linear precursor, exactly “half” variants of ent-verticilide, display no activity.

Conclusion

To the best of our knowledge, this is the first comparative study of a wide series of unnatural ring-size CODs, and the most direct correlation made between ring size as a single variable and its effect on activity at a target known to elicit a pharmacologically relevant effect. Importantly, we disclose an equipotent ring-size congener (18-membered ring) that was uncovered alongside other inactive analogues, most notably the 12- and 30-membered ring-size oligomers. Our finding of another potent COD ring-size congener highlights the value of exploring ring size in SAR studies of oligomeric compounds and complements prior observations of activity among naturally occurring ring-size variants of COD natural products. Studies to further identify biologically active depsipeptide macrocycles, hidden in plain sight so to speak, are ongoing.

Acknowledgments

We are grateful to Prof. Corey Hopkins (Nebraska Medical Center) for assistance with TPSA calculations, as well as discussion and critical feedback. Research reported in this publication was supported by the National Heart, Blood, and Lung Institute of the National Institutes of Health (NIH R01 HL151223 and HL151125 (F31 support for A.N.S.)) and the PhRMA Foundation Postdoctoral Fellowship (D.J.B. support). The Indiana University Mass Spectrometry Facility acknowledges support from the NSF (CHE1726633), and we thank IUMSC for HRMS data acquisition and analysis.

Glossary

Abbreviations

SAR

structure–activity relationship

COD

cyclic oligomeric depsipeptides

RyR

ryanodine receptor

AlogP

partition coefficient which is the ratio of a compound’s concentration in octanol to its concentration in water when the phases are at equilibrium

TPSA

total polar surface area

CASQ2

calsequestrin 2

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00508.

  • Complete experimental details, NMR data (PDF)

Author Present Address

§ Daniel J. Blackwell: Vanderbilt Center for Arrhythmia Research and Therapeutics (VanCART), Vanderbilt University School of Medicine, Medical Research Building IV, Room 1265, 2215B Garland Avenue, Nashville, Tennessee 37232-0575, United States

Author Present Address

∥ Bjorn C. Knollmann: Vanderbilt Center for Arrhythmia Research and Therapeutics (VanCART), Vanderbilt University School of Medicine, Medical Research Building IV, Room 1265, 2215B Garland Avenue, Nashville, Tennessee 37232-0575, United States.

The authors declare no competing financial interest.

This paper was originally published ASAP on November 23, 2021, with an error in Chart 1. The corrected version was reposted on November 30, 2021.

Supplementary Material

ml1c00508_si_001.pdf (5MB, pdf)

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