Passive Membrane Permeability Research Articles

Determining small-molecule permeation through lipid membranes.

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( https://doi.org/10.5281/zenodo.6511116 ). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2-3 d.

Conformational Effects on the Passive Membrane Permeability of Synthetic Macrocycles.

Macrocyclic compounds (MCs) can have complex conformational properties that affect pharmacologically important behaviors such as membrane permeability. We measured the passive permeability of 3600 diverse nonpeptidic MCs and used machine learning to analyze the results. Incorporating selected properties based on the three-dimensional (3D) conformation gave models that predicted permeability with Q2 = 0.81. A biased spatial distribution of polar versus nonpolar regions was particularly important for good permeability, consistent with a mechanism in which the initial insertion of nonpolar portions of a MC helps facilitate the subsequent membrane entry of more polar parts. We also examined effects on permeability of 800 substructural elements by comparing matched molecular pairs. Some substitutions were invariably beneficial or invariably deleterious to permeability, while the influence of others was highly contextual. Overall, the work provides insights into how the permeability of MCs is influenced by their 3D conformational properties and suggests design hypotheses for achieving macrocycles with high membrane permeability.

Property-Driven Development of Passively Permeable Macrocyclic Scaffolds Using Heterocycles.

Passive membrane permeability is a fundamental challenge in the development of bioactive macrocycles. To achieve this objective, chemists have resorted to various strategies, the most common of which is deployment of N-methylated amino acids and/or D-amino acids. Here we investigate the effect of heterocyclic grafts on the passive membrane permeability of macrocycles and report the structural consequences of iterative amino acid replacement by azole rings. Through stepwise substitution of amino acid residues for heterocycles, we show that lipophilicity and PAMPA permeability of a macrocycle can be vastly improved. Overall, changes in permeability do not scale linearly as more heterocycles are incorporated, underscoring the subtleties of conformation-property relationships in this class of molecule. NMR analysis and molecular dynamics simulations provide insights into the structural consequences of the added heterocycles and these frameworks can now be applied as macrocyclic scaffolds for drug discovery.

Property‐Driven Development of Passively Permeable Macrocyclic Scaffolds Using Heterocycles**

AbstractPassive membrane permeability is a fundamental challenge in the development of bioactive macrocycles. To achieve this objective, chemists have resorted to various strategies, the most common of which is deployment of N‐methylated amino acids and/or D‐amino acids. Here we investigate the effect of heterocyclic grafts on the passive membrane permeability of macrocycles and report the structural consequences of iterative amino acid replacement by azole rings. Through stepwise substitution of amino acid residues for heterocycles, we show that lipophilicity and PAMPA permeability of a macrocycle can be vastly improved. Overall, changes in permeability do not scale linearly as more heterocycles are incorporated, underscoring the subtleties of conformation‐property relationships in this class of molecule. NMR analysis and molecular dynamics simulations provide insights into the structural consequences of the added heterocycles and these frameworks can now be applied as macrocyclic scaffolds for drug discovery.

Piperine Derivatives Enhance Fusion and Axonal Transport of Mitochondria by Activating Mitofusins

Piperine (1-piperoylpiperidine) is the major pungent component of black pepper (Piper nigrum) and exhibits a spectrum of pharmacological activities. The molecular bases for many of piperine’s biological effects are incompletely defined. We noted that the chemical structure of piperine generally conforms to a pharmacophore model for small bioactive molecules that activate mitofusin (MFN)-mediated mitochondrial fusion. Piperine, but not its isomer chavicine, stimulated mitochondrial fusion in MFN-deficient cells with EC50 of ~8 nM. We synthesized piperine analogs having structural features predicted to optimize mitofusin activation and defined structure-activity relationships (SAR) in live-cell mitochondrial elongation assays. When optimal spacing was maintained between amide and aromatic groups the derivatives were potent mitofusin activators. Compared to the prototype phenylhexanamide mitofusin activator, 2, novel molecules containing the piperidine structure of piperine exhibited markedly enhanced passive membrane permeability with no loss of fusogenic potency. Lead compounds 5 and 8 enhanced mitochondrial motility in cultured murine Charcot-Marie-Tooth disease type 2A (CMT2A) neurons, but only 8 improved mitochondrial transport in sciatic nerve axons of CMT2A mice. Piperine analogs represent a new chemical class of mitofusin activators with potential pharmaceutical advantages.

Grid inhomogeneous solvation theory for cross-solvation in rigid solvents.

Grid Inhomogeneous Solvation Theory (GIST) has proven useful to calculate localized thermodynamic properties of water around a solute. Numerous studies have leveraged this information to enhance structure-based binding predictions. We have recently extended GIST toward chloroform as a solvent to allow the prediction of passive membrane permeability. Here, we further generalize the GIST algorithm toward all solvents that can be modeled as rigid molecules. This restriction is inherent to the method and is already present in the inhomogeneous solvation theory. Here, we show that our approach can be applied to various solvent molecules by comparing the results of GIST simulations with thermodynamic integration (TI) calculations and experimental results. Additionally, we analyze and compare a matrix consisting of 100 entries of ten different solvent molecules solvated within each other. We find that the GIST results are highly correlated with TI calculations as well as experiments. For some solvents, we find Pearson correlations of up to 0.99 to the true entropy, while others are affected by the first-order approximation more strongly. The enthalpy-entropy splitting provided by GIST allows us to extend a recently published approach, which estimates higher order entropies by a linear scaling of the first-order entropy, to solvents other than water. Furthermore, we investigate the convergence of GIST in different solvents. We conclude that our extension to GIST reliably calculates localized thermodynamic properties for different solvents and thereby significantly extends the applicability of this widely used method.

Impact of Cyclization and Methylation on Peptide Penetration through Droplet Interface Bilayers.

Cell-penetrating peptides enter cells via diverse mechanisms, such as endocytosis, active transport, or direct translocation. For the design of orally delivered cell-penetrating peptides, it is crucial to know the contribution of these different mechanisms. In particular, the ability of a peptide to translocate through a lipid bilayer remains a key parameter for the delivery of cargos. However, existing approaches used to assess translocation often provide discrepant results probably because they have different sensitivities to the distinct translocation mechanisms. Here, we focus on the passive permeation of a range of hydrophobic cyclic peptides inspired by somatostatin, a somatotropin release-inhibiting factor. Using droplet interface bilayers (DIB), we assess the passive membrane permeability of these peptides and study the impact of the peptide cyclization and backbone methylation on translocation rates. Cyclization systematically improved the permeability of the tested peptides while methylation did not. By studying the interaction of the peptides with the DIB interfaces, we found membrane insertion and peptide intrinsic diffusion to be two independent factors of permeability. Compared to the industrial gold standard Caco-2 and parallel artificial membrane permeability assay (PAMPA) models, DIBs provide intermediate membrane permeability values, closer to Caco-2. Even for conditions where Caco-2 and PAMPA are discrepant, the DIB approach also gives results closer to Caco-2. Thereupon, DIBs represent a robust alternative to the PAMPA approach for predicting the permeability of peptides, even if the latter present extremely small structural differences.

Mechanistic Insights into Passive Membrane Permeability of Drug-like Molecules from a Weighted Ensemble of Trajectories.

Passive permeability of a drug-like molecule is a critical property assayed early in a drug discovery campaign that informs a medicinal chemist how well a compound can traverse biological membranes, such as gastrointestinal epithelial or restrictive organ barriers, so it can perform a specific therapeutic function. However, the challenge that remains is the development of a method, experimental or computational, which can both determine the permeation rate and provide mechanistic insights into the transport process to help with the rational design of any given molecule. Typically, one of the following three methods are used to measure the membrane permeability: (1) experimental permeation assays acting on either artificial or natural membranes; (2) quantitative structure–permeability relationship models that rely on experimental values of permeability or related pharmacokinetic properties of a range of molecules to infer those for new molecules; and (3) estimation of permeability from the Smoluchowski equation, where free energy and diffusion profiles along the membrane normal are taken as input from large-scale molecular dynamics simulations. While all these methods provide estimates of permeation coefficients, they provide very little information for guiding rational drug design. In this study, we employ a highly parallelizable weighted ensemble (WE) path sampling strategy, empowered by cloud computing techniques, to generate unbiased permeation pathways and permeability coefficients for a set of drug-like molecules across a neat 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine membrane bilayer. Our WE method predicts permeability coefficients that compare well to experimental values from an MDCK-LE cell line and PAMPA assays for a set of drug-like amines of varying size, shape, and flexibility. Our method also yields a series of continuous permeation pathways weighted and ranked by their associated probabilities. Taken together, the ensemble of reactive permeation pathways, along with the estimate of the permeability coefficient, provides a clearer picture of the microscopic underpinnings of small-molecule membrane permeation.

Optimization of Cyclic Peptide Property Using Chromatographic Capacity Factor on Permeability of Passive Cell Membrane and Human Induced Pluripotent Stem Cell-Derived Intestinal Membrane

Cyclic peptides have attracted increasing attention as a privileged class of molecules addressing undruggable targets. Cell permeability of cyclic peptides has remained a challenging issue owing to their molecular properties. Various efficiency metrics have emerged to assess this issue. Among them, the lipophilic permeability efficiency (LPE) metric is the difference between an experimental 1,9-decadiene-water partition coefficient at pH 7.4 (log Ddec/w) and calculated octanol/water partition coefficients (ALogP). This metric provides insight into how structural changes affect permeability. Here, we demonstrate the chromatographic capacity factor (log k’) of cyclic peptides using reversed-phase liquid chromatography as an alternative to log Ddec/w, which enables efficient and reliable experimental lipophilicity for the adoption of LPE in early drug discovery. The log k’ indicates the passive membrane permeability of cyclic peptides and can be used to optimize passive membrane permeability in combination with other parameters. In addition, intestinal membrane permeability of cyclic peptides on human induced pluripotent stem cell-derived intestinal epithelial cells was achieved with log k’ and high passive membrane permeability, although cyclic peptides are P-glycoprotein substrates. These approaches could facilitate optimization of properties of cyclic peptides for oral administration and contribute to the successful discovery and development of cyclic peptides.

Simulation Models for Prediction of Bioavailability of Medicinal Drugs-the Interface Between Experiment and Computation.

The oral drug bioavailability (BA) problems have remained inevitable over the years, impairing drug efficacy and indirectly leading to eventual human morbidity and mortality. However, some conventional lab-based methods improve drug absorption leading to enhanced BA, and the recent experimental techniques are up-and-coming. Nevertheless, some have inherent drawbacks in improving the efficacy of poorly insoluble and low impermeable drugs. Drug BA and strategies to overcome these challenges were briefly highlighted. This review has significantly unravelled the different computational models for studying and predicting drug bioavailability. Several computational approaches provide mechanistic insights into the oral drug delivery system simulation of descriptors like solubility, permeability, transport protein-ligand interactions, and molecular structures. The in silico techniques have long been known still are just being applied to unravel drug bioavailability issues. Many publications have reported novel applications of the computational models towards achieving improved drug BA, including predicting gastrointestinal tract (GIT) drug absorption properties and passive intestinal membrane permeability, thus maximizing time and resources. Also, the classical molecular simulation models for free solvation energies of soluble-related processes such as solubilization, dissolutions, supersaturation, and precipitation have been used in virtual screening studies. A few of the tools are GastroPlusTM that supports biowaiver for drugs, mainly BCS class III and predicts drug compounds' absorption and pharmacokinetic process; SimCyp® simulator for mechanistic modelling and simulation of drug formulation processes; pharmacodynamics analysis on non-linear mixed-effects modelling; and mathematical models, predicting absorption potential/maximum absorption dose. This review provides in silico-experiment annexation in the drug bioavailability enhancement, possible insights that lead to critical opinion on the applications and reliability of the various in silico models as a growing tool for drug development and discovery, thus accelerating drug development processes.

A New Amino Acid for Improving Permeability and Solubility in Macrocyclic Peptides through Side Chain-to-Backbone Hydrogen Bonding.

Despite the notoriously poor membrane permeability of peptides, many cyclic peptide natural products show high passive membrane permeability and potently inhibit a variety of "undruggable" intracellular targets. A major impediment to the design of cyclic peptides with good permeability is the high desolvation energy associated with the peptide backbone amide NH groups. While several strategies have been proposed to mitigate this deleterious effect, only few studies have used polar side chains to sequester backbone NH groups. We investigated the ability of N,N-pyrrolidinylglutamine (Pye), whose side chain contains a powerful hydrogen-bond-accepting C═O amide group but no hydrogen-bond donors, to sequester exposed backbone NH groups in a series of cyclic hexapeptide diastereomers. Analyses revealed that specific Leu-to-Pye substitutions conferred dramatic improvements in aqueous solubility and permeability in a scaffold- and position-dependent manner. Therefore, this approach offers a complementary tool for improving membrane permeability and solubility in cyclic peptides.

Membrane-like interfaces can facilitate the conformational closure of cyclic peptides for passive membrane permeability

Cyclic peptides have the potential to vastly extend the scope of druggable proteins and to lead to new therapeutics for currently untreatable diseases. However, the design of cyclic peptides with oral bioavailability is challenging and often only achieved via a tedious trial-and-error process. We use molecular dynamics (MD) simulations and Markov state modeling of cyclic peptides to rationalize the relationship between their structure and passive membrane permeability. Previous work has shown that an important determinant for permeability is a peptide's conformational behavior in different environments.

Polar/apolar interfaces modulate the conformational behavior of cyclic peptides with impact on their passive membrane permeability.

Cyclic peptides have the potential to vastly extend the scope of druggable proteins and lead to new therapeutics for currently untreatable diseases. However, cyclic peptides often suffer from poor bioavailability. To uncover design principles for permeable cyclic peptides, a promising strategy is to analyze the conformational dynamics of the peptides using molecular dynamics (MD) and Markov state models (MSMs). Previous MD studies have focused on the conformational dynamics in pure aqueous or apolar environments to rationalize membrane permeability. However, during the key steps of the permeation through the membrane, cyclic peptides are exposed to interfaces between polar and apolar regions. Recent studies revealed that these interfaces constitute the free energy minima of the permeation process. Thus, a deeper understanding of the behavior of cyclic peptides at polar/apolar interfaces is desired. Here, we investigate the conformational and kinetic behavior of cyclic decapeptides at a water/chloroform interface using unbiased MD simulations and MSMs. The distinct environments at the interface alter the conformational equilibrium as well as the interconversion kinetics of cyclic peptide conformations. For peptides with low population of the permeable conformation in aqueous solution, the polar/apolar interface facilitates the interconversion to the closed conformation, which is required for membrane permeation. Comparison to unbiased MD simulations with a POPC bilayer reveals that not only the conformations but also the orientations are relevant in a membrane system. These findings allow us to propose a permeability model that includes both ‘prefolding’ and ‘non-prefolding’ cyclic peptides – an extension that can lead to new design considerations for permeable cyclic peptides.

Cyclosporin Structure and Permeability: From A to Z and Beyond.

Cyclosporins are natural or synthetic undecapeptides with a wide range of actual and potential pharmaceutical applications. Several members of the cyclosporin compound family have remarkably high passive membrane permeabilities that are not well-described by simple structural metrics. Here we review experimental studies of cyclosporin structure and permeability, including cyclosporin-metal complexes. We also discuss models for the conformation-dependent permeability of cyclosporins and similar compounds. Finally, we identify current knowledge gaps in the literature and provide recommendations regarding future avenues of exploration.

Effect of Flexibility, Lipophilicity, and the Location of Polar Residues on the Passive Membrane Permeability of a Series of Cyclic Decapeptides.

Cyclic peptides have received increasing attention over the recent years as potential therapeutics for "undruggable" targets. One major obstacle is, however, their often relatively poor bioavailability. Here, we investigate the structure-permeability relationship of 24 cyclic decapeptides that share the same backbone N-methylation pattern but differ in their side chains. The peptides cover a large range of values for passive membrane permeability as well as lipophilicity and solubility. To rationalize the observed differences in permeability, we extracted for each peptide the population of the membrane-permeable conformation in water from extensive explicit-solvent molecular dynamics simulations and used this as a metric for conformational rigidity or "prefolding." The insights from the simulations together with lipophilicity measurements highlight the intricate interplay between polarity/lipophilicity and flexibility/rigidity and the possible compensating effects on permeability. The findings allow us to better understand the structure-permeability relationship of cyclic peptides and extract general guiding principles.

Passing the Barrier - How Computer Simulations Can Help to Understand and Improve the Passive Membrane Permeability of Cyclic Peptides.

Proteins with large and flat binding sites as well as protein-protein interactions are considered ' undruggable ' with conventional small-molecule drugs. Cyclic peptides have been found to be capable of binding to such targets with high affinity, making this class of compounds an interesting source for possible therapeutics. However, the oftentimes poor passive membrane permeability of cyclic peptides still imposes restrictions on the applicability of cyclic peptide drugs. Here, we describe how computational methods in combination with experimental data can be used to improve our understanding of the structure-permeability relationship. Especially the conformational dynamic and chameleonic nature of cyclic peptides, which we investigate by a combination of MD simulations and kinetic modeling, is important for their ability to permeate passively through the membrane. The insights from such studies may enable the formulation of design principles for the rational design of permeable cyclic peptides.

Recent Experimental Developments in Studying Passive Membrane Transport of Drug Molecules.

The ability to measure the passive membrane permeation of drug-like molecules is of fundamental biological and pharmaceutical importance. Of significance, passive diffusion across the cellular membranes plays an effective role in the delivery of many pharmaceutical agents to intracellular targets. Hence, approaches for quantitative measurement of membrane permeability have been the topics of research for decades, resulting in sophisticated biomimetic systems coupled with advanced techniques. In this review, recent developments in experimental approaches along with theoretical models for quantitative and real-time analysis of membrane transport of drug-like molecules through mimetic and living cell membranes are discussed. The focus is on time-resolved fluorescence-based, surface plasmon resonance, and second-harmonic light scattering approaches. The current understanding of how properties of the membrane and permeant affect the permeation process is discussed.

Online Real-Time Monitoring of Exhaled Breath Particles Reveals Unnoticed Transport of Nonvolatile Drugs from Blood to Breath

We used online secondary electrospray ionization mass spectrometry to measure venlafaxine (VEN), a nonvolatile drug, in the exhaled air of mice intraperitoneally treated with VEN. The breath pharmacokinetic (PK) profile of VEN was recorded, which was in good agreement with that of the blood. Combined with online collection of exhaled breath particles (EBPs), it was shown that VEN existed as part of EBPs rather than gas molecules in the breath. Linear free-energy relationship analysis confirmed that almost completely ionized VEN at physiological conditions unlikely partition from the lung lining fluid (LLF) into breath air. This implies that the occurrence of VEN in exhaled air accompanies the formation of EBPs from the LLF. By comparison with the low breath signals of VEN metabolites, passive membrane permeability and lung/blood partition coefficient are suggested as the main influencing factors for the levels of drugs in the breath. This study advances our knowledge on the mechanism by which nonvolatile drugs are transferred from blood into exhaled breath, providing guidance for breath test-based therapeutic drug monitoring.

Total syntheses and antiproliferative activities of prenostodione and its analogues.

A high-yielding total synthesis of the indole alkaloid prenostodione was completed in 4 steps and 44% overall yield from 1H-indole-3-carboxylic acid. The expedient syntheses of prenostodiones containing distinct substituents at the para position of the phenyl frame underscored the scope of this methodology. The cytotoxic activities of the tert-butyl esters of prenostodione analogues were tested using six tumor cell lines. Preliminary structure-activity studies revealed the importance of the identity of the aromatic substituent at the C-4 position for cytotoxic activity. The IC50 values of these compounds were found to compare satisfactorily with those of the commercially available drugs etoposide and cisplatin. Furthermore, the compounds with, respectively, -OMe (14d) and -NO2 (14f) groups at C-4 were more selective than these control compounds in PC-3, K-562, and MCF-7 cells. Also, computational studies were carried out to determine the ADMET profiles and passive membrane permeabilities of the compounds. The results suggested the promise of 14d and 14f as hit compounds for the development of new anticancer agents.

Geometrically Diverse Lariat Peptide Scaffolds Reveal an Untapped Chemical Space of High Membrane Permeability.

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.