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Abstract
The passive membrane permeation of small-molecule drugs and relatively small hydrophobic peptides is relatively well understood. In contrast, how long polar peptides can directly pass through a membrane has remained a mystery. This process can be achieved with transcellular permeation enhancers, contributing significantly to the oral transcellular absorption of important peptide drugs like semaglutide — the active component in Ozempic, which is used as Rybelsus in a successful oral formulation. Here we now provide, for the first time, a detailed, plausible molecular mechanism of how such a polar peptide can realistically pass through a membrane paired with the permeation enhancer salcaprozate sodium (SNAC). We provide not only simulation results, obtained with scalable continuous constant pH molecular dynamics (CpHMD) simulations, but also experimental evidence (NMR, DOSY, and DLS) to support this unique passive permeation mechanism. Our computational and experimental evidence points toward the formation of permeation-enhancer-filled, fluid membrane defects, in which the polar peptide can be submerged in a process analogous to sinking in quicksand.
Supplementary materials
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Supplementary Information
Description
This file contains Supplementary Methods, Supplementary Figures, Captions of Supplementary Movies, Supplementary Tables, and Supplementary References
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Supplementary Movie 1
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Trajectory of SNAC Exiting a Membrane: The CpHMD trajectory shows a pulling simulation of SNAC through a POPC membrane (32 lipids in each leaflet with 0.15 M NaCl, 310.15 K) at pH 5. SNAC is shown in space-filling mode. Phospholipid head groups in yellow. SNAC is shown in cyan, regardless of its protonation state.
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Supplementary Movie 2
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Unbiased Trajectory of SNAC Aggregation in Water: The 50-ns CpHMD trajectory shows 50 SNAC molecules (cyan) aggregating in water (0.15 M NaCl, 310.15 K) at pH 5. All SNACs are shown in cyan, regardless of their protonation state.
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Supplementary Movie 3
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Unbiased Trajectory of SNAC Aggregation in a Nonpolar Solvent: The 50-ns CpHMD trajectory shows 50 SNAC molecules (cyan) aggregating in dichloromethane. All SNACs are shown in cyan, regardless of their protonation state.
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Supplementary Movie 4
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Unbiased Trajectory of SNAC Aggregation with Semaglutide in a Nonpolar Solvent: The 100-ns CpHMD trajectory shows 50 SNAC molecules (cyan) aggregating with semaglutide (red) in dichloromethane. All SNACs are shown in cyan, regardless of their protonation state.
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Supplementary Movie 5
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Unbiased Trajectory of SNAC Aggregation with Semaglutide in Water: The 100-ns CpHMD trajectory shows 50 SNAC molecules (cyan) aggregating with semaglutide (red) in water (0.15 M NaCl, 310.15 K) at pH 5. All SNACs are shown in cyan, regardless of their protonation state.
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Supplementary Movie 6
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Trajectory of Semaglutide’s Lipid Tail Entering a Membrane: The CpHMD trajectory shows a 100-ns pulling simulation of the terminal carboxylic acid function (highlighted in red) of semaglutide’s lipid tail (S1) into a POP lipid bilayer membrane (32 lipids in each leaflet with 0.15 M NaCl, 310.15 K) at pH 5. See Supplementary Fig. 16 for the structure of S1. Phospholipid head groups are shown in yellow.
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Supplementary Movie 7
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Unbiased Trajectory of Semaglutide and SNAC Interacting with a POPC Membrane: The trajectory shows a 100-ns CpHMD simulation of semaglutide (red, space-filling mode) in the presence of 400 SNACs (cyan, representing the approximate semaglutide:SNAC ratio in a Rybelsus tablet). During the simulation, semaglutide spontaneously lays down on the membrane surface and starts to sink into the membrane, as described in the main text and Fig. 5. All SNACs are shown in cyan, regardless of their protonation state. Phospholipid head groups are shown in yellow (space-filling mode), cholesterol in gray, and phospholipid tails in magenta.
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Supplementary Movie 8
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Unbiased Trajectory of Semaglutide Spontaneously Sinking into a POPC Membrane in the Presence of SNAC: The trajectory shows a 1-μs CpHMD simulation of semaglutide (red, space-filling mode) in the presence of 400 SNAC molecules (shown in cyan). The simulation system was prepared with the semaglutide lipid tail anchored in the membrane as detailed in the main text. During this simulation, semaglutide started to spontaneously sink into the membrane. All SNACs are shown in cyan, regardless of their protonation state. Phospholipid head groups are shown in yellow (space-filling mode), cholesterol in gray, and phospholipid tails in magenta.
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Supplementary Movie 9
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Unbiased Trajectory Showing Membrane Defects Forming and Expanding in the Presence of SNAC: The 1-μs CpHMD trajectory shows SNAC-filled defects forming in the phosphate head group layer (top view, phosphate head groups shown in yellow, SNAC shown in cyan regardless of protonation state, and semaglutide shown in red).
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Supplementary Movie 10
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Unbiased Trajectory Highlighting SNAC/Semaglutide Aggregate Mobility/Fluidity in the Membrane: The 1-μs CpHMD trajectory highlights the movement of select SNAC molecules (shown in space-filling mode in blue, yellow, black, red, and brown), which move dynamically through the membrane and are part of a SNAC/semaglutide membrane aggregate, which forms a membrane defect. Semaglutide is shown in gray, phosphate head groups of the membrane in yellow, and other SNAC molecules in cyan (regardless of their protonation state).
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