Cell penetration of oxadiazole-containing macrocycles

Abstract

Passive membrane permeability is an important property in drug discovery and biological probe design. To elucidate the cell-penetrating ability of oxadiazole-containing (Odz) peptides, we employed the Chloroalkane Penetration Assay. The present study demonstrates that Odz cyclic peptides can be highly cell-penetrant depending on the position of specific side chains and the chloroalkane tag. Solution NMR shows that Odz cyclic peptides adopt a β-turn conformation. However, despite observing high cell penetration, we observed low passive permeability in experiments with artificial membranes. These findings highlight the complexity of controlling cell penetration for conformationally sensitive macrocycles and suggest that Odz cyclic peptides may provide a framework for designing cell-penetrant cyclic peptides.

Fig. 1. (a) Cyclosporine A and sanguinamide A. Various features enable these naturally occurring cyclic peptides to have high bioavailability. (b) Features of the oxadiazole-grafted (Odz) macrocycle may promote cell penetration. (c) Schematic showing PAMPA setup. A solution of peptide in donor well is separated from the acceptor well by an artificial membrane. After incubation, the degree to which a peptide can permeate through the artificial membrane and accumulate in the acceptor well is measured using LC-MS. (d) Schematic showing setup of the chloroalkane penetration assay (CAPA). Cells expressing HaloTag enzyme are pulsed with chloroalkane-tagged peptide (ct-peptide). Then, cells are chased with chloroalkane-tagged dye (ct-dye) which reacts with any unreacted HaloTag present after the pulse step. After washing, the cells are analysed using flow cytometry to measure the extent of labelling by the ct-dye, which is inversely proportional to the amount of ct-peptide that penetrated to the cytosol during the pulse step.

Fig. 2. (a) Previous scope of the Odz cyclic peptides. (b) Scope of the chloroalkane-tagged Odz macrocycles. ct represents the chloroalkane carboxylic acid which underwent amide coupling with the lysine side chain. Lys(ct) refers to the lysine side chain that has chloroalkane-tag coupled to it.

Fig. 3. CAPA results comparing the Odz cyclic peptide (1), its homodetic counterpart (8), and control small molecule ct-W. CP₅₀ value shown is after 4 hours of incubation; data for 30 min incubation are shown in Supporting Information. The structures highlight the key differences between cyclic peptides 1 and 8 and show the extra amide bond in the homodetic cyclic peptide. Depiction of compound 8 does not represent its solution conformation. Error bars show standard error of the mean from three independent trials. CP₅₀ values and standard errors of the mean are derived from three independent curve fits from three independent trials.

Fig. 4. (a) CAPA results comparing the Odz macrocycle 1 and compounds which change the location of the chloroalkane tag (compounds 2, 3, and 5). CP₅₀ value shown is after 4 hours of incubation; data for 30 min incubation are shown in Supporting Information. (b) CAPA results comparing the Odz cyclic peptide 1 and analogs with one or two Asp residues (6, 7). CP₅₀ value shown is after 4 hours of incubation. (c) Lysate stability results showing no degradation for compounds 1–7 in a HeLa cell lysate after 4 hours of incubation. Error bars show standard error of the mean from three independent trials. CP₅₀ values and standard errors of the mean are derived from three independent curve fits from three independent trials.

Fig. 5. Variable-temperature NMR (VT NMR) of Odz macrocycles. (a) Structure of compound 1a is shown with specific backbone amide-NH that had low temperature coefficients (T_coeff) shown in red. Based on their T_coeff, the backbone amide NH groups of Phe and Gly are likely to be involved in intramolecular hydrogen bonding. (b) NMR spectra of compound 1a in DMSO-d₆ showing the change in chemical shift as the sample is heated form 25 °C to 45 °C. (c) T_coeff values of compounds 1a to 7a and compound A. Backbone amides with T_coeff above −4 ppb K⁻¹ were interpreted as participating in intramolecular hydrogen bonding, and backbone amides with T_coeff below −4 ppb K⁻¹ were interpreted as being relatively solvent-exposed.

Fig. 6. Solution structures of compound 1a to 7a in DMSO-d₆. Side chains are omitted for clarity.