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Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

The chameleonic behavior of cyclosporin A (CsA) was investigated through conformational ensembles employing multicanonical molecular dynamics simulations that could sample the cis and trans isomers of N-methylated amino acids; these assessments were conducted in explicit water, dimethyl sulfoxide, acetonitrile, methanol, chloroform, cyclohexane (CHX), and n-hexane (HEX) using AMBER ff03, AMBER10:EHT, AMBER12:EHT, and AMBER14:EHT force fields. The conformational details were discussed employing the free-energy landscapes (FELs) at T = 300 K; it was observed that the experimentally determined structures of CsA were only a part of the conformational space. Comparing the ROESY measurements in CHX-d12 and HEX-d14, the major conformations in those apolar solvents were essentially the same as that in CDCl3 except for the observation of some sidechain rotamers. The effects of the metal ions on the conformations, including the cis/trans isomerization, were also investigated. Based on the analysis of FELs, it was concluded that the AMBER ff03 force field best described the experimentally derived conformations, indicating that CsA intrinsically formed membrane-permeable conformations and that the metal ions might be the key to the cis/trans isomerization of N-methylated amino acids before binding a partner protein.

Constrained, membrane-permeable peptides offer the possibility of engaging challenging intracellular targets. Structure-permeability relationships have been extensively studied in cyclic peptides whose backbones are cyclized from head to tail, like the membrane permeable and orally bioavailable natural product cyclosporine A. In contrast, the physicochemical properties of lariat peptides, which are cyclized from one of the termini onto a side chain, have received little attention. Many lariat peptide natural products exhibit interesting biological activities, and some, such as griselimycin and didemnin B, are membrane permeable and have intracellular targets. To investigate the structure-permeability relationships in the chemical space exemplified by these natural products, we generated a library of scaffolds using stable isotopes to encode stereochemistry and determined the passive membrane permeability of over 1000 novel lariat peptide scaffolds with molecular weights around 1000. Many lariats were surprisingly permeable, comparable to many known orally bioavailable drugs. Passive permeability was strongly dependent on N-methylation, stereochemistry, and ring topology. A variety of structure-permeability trends were observed including a relationship between alternating stereochemistry and high permeability, as well as a set of highly permeable consensus sequences. For the first time, robust structure-permeability relationships are established in synthetic lariat peptides exceeding 1000 compounds.

Recent advances in DNA and mRNA encoding technologies have enabled the discovery of high-affinity macrocyclic peptides and peptide-like ligands against virtually any protein target of interest. Unfortunately, even the most potent biochemical leads from these screening technologies often have weak cellular activity due to poor absorption. Biasing such libraries towards passive cell permeability in the design phase would facilitate development of leads against intracellular targets. We set out to empirically evaluate the intrinsic permeability of thousands of geometrically diverse hexa- and heptapeptide scaffolds by permuting backbone stereochemistry and N-methylation, and by including peptoid and β-amino acid residues at select positions, with the goals of providing a resource for biasing library-based screening efforts toward passive membrane permeability and studying the effects of the backbone elements introduced on a large number of compounds. Libraries were synthesized via standard split-pool solid phase peptide synthesis, and passive permeability was measured in pools of 150 compounds using a highly multiplexed version of the parallel artificial mem-brane permeability assay (PAMPA) under sink conditions. Compounds were identified using CycLS, a high-resolution mass spectrometry-based method that uses stable isotopes to encode stereochemistry and matches MSMS data to virtual fragment libraries based on the expected macrocyclic products. From the compounds that were identified with high confidence, 823 hexameric and 1330 heptameric scaffolds had PAMPA permeability coefficients greater than 1x10-6 cm/s. The prevalence of high permeability compounds in these two libraries suggests that passive permeability is achievable for hexa- and heptapeptides with highly diverse backbone geometries.

Cyclic peptides are attractive molecules as inhibitors with high affinity and selectivity against intracellular protein-protein interactions (PPIs). On the other hand, cyclic peptides generally have low passive cell-membrane permeability, which makes it difficult to discover cyclic peptides that efficiently permeate into cells and inhibit intracellular PPIs. Here, we show that backbone amide-to-ester substitutions are useful for improving membrane permeability of peptides. Permeability in a series of model dipeptides increased upon amide-to-ester substitution. Amide-to-ester substitutions increased permeability in the same manner as amide-to-N-methyl amide substitutions, which are conventionally used for increasing permeability. Furthermore, amide-to-ester substitutions of exposed amides of a cyclic peptide successfully improved permeability. Conformational studies of the cyclic peptides using NMR and molecular mechanics calculations revealed that an amide-to-ester substitution of an exposed amide bond did not affect its low-energy conformation in CDCl3, in contrast with an N-methyl amide substitution. We envision that amide-to-ester substitution will be a potentially useful strategy for rational design of bioactive peptides with high membrane permeability.

As interest in protein-protein interactions and other previously-undruggable targets increases, medicinal chemists are returning to natural products for design inspiration toward molecules that transcend the paradigm of small molecule drugs. These compounds, especially peptides, often have poor ADME properties and thus require a more nuanced understanding of structure-property relationships to achieve desirable oral bioavailability. Although there have been few clinical successes in this chemical space to date, recent work has identified opportunities to introduce favorable physicochemical properties to peptidic macrocycles that maintain activity and oral bioavailability.

Conformational ensembles of eight cyclic hexapeptide diastereomers in explicit cyclohexane, chloroform, and water were analyzed by multicanonical molecular dynamics (McMD) simulations. Free-energy landscapes (FELs) for each compound and solvent were obtained from the molecular shapes and principal component analysis at T = 300 K; detailed analysis of the conformational ensembles and flexibility of the FELs revealed that permeable compounds have different structural profiles even for a single stereoisomeric change. The average solvent-accessible surface area (SASA) in cyclohexane showed excellent correlation with the cell permeability, whereas this correlation was weaker in chloroform. The average SASA in water correlated with the aqueous solubility. The average polar surface area did not correlate with cell permeability in these solvents. A possible strategy for designing permeable cyclic peptides from FELs obtained from McMD simulations is proposed.

Cyclic peptide (CP) natural products provide useful model systems for mapping "beyond-Rule-of-5" (bRo5) space. We identified the phepropeptins as natural product CPs with potential cell permeability. Synthesis of the phepropeptins and epimeric analogues revealed much more rapid cellular permeability for the natural stereochemical pattern. Despite being more cell permeable, the natural compounds exhibited similar aqueous solubility as the corresponding epimers, a phenomenon explained by solvent-dependent conformational flexibility among the natural compounds. When analyzing the polarity of the solution structures we found that neither the number of hydrogen bonds nor the total polar surface area accurately represents the solvation energies of the high and low dielectric conformations. This work adds to a growing number of natural CPs whose solvent-dependent conformational behavior allows for a balance between aqueous solubility and cell permeability, highlighting structural flexibility as an important consideration in the design of molecules in bRo5 chemical space.