Discovery of indolylacryloyl-derived oxacins as novel potential broad-spectrum antibacterial candidates.

The emergence of serious bacterial resistance towards clinical oxacins poses a considerable threat to global public health, necessitating the development of novel structural antibacterial agents. Seven types of novel indolylacryloyl-derived oxacins (IDOs) were designed and synthesized for the first time from commercial 3,4-difluoroaniline via an eight-step procedure. The synthesized compounds were characterized by modern spectroscopic techniques. All target molecules were evaluated for antimicrobial activities. Most of the prepared IDOs showed a broad antibacterial spectrum and strong activities against the tested strains, especially ethoxycarbonyl IDO 10d (0.25-0.5 µg/mL) and hydroxyethyl IDO 10e (0.25-1 µg/mL) exhibited much superior antibacterial efficacies to reference drug norfloxacin. These highly active IDOs also displayed low hemolysis, cytotoxicity and resistance, as well as rapid bactericidal capacity. Further investigations indicated that ethoxycarbonyl IDO 10d and hydroxyethyl IDO 10e could effectively reduce the exopolysaccharide content and eradicate the formed biofilm, which might delay the development of drug resistance. Preliminary exploration of the antibacterial mechanism revealed that active IDOs could not only destroy membrane integrity, resulting in changes in membrane permeability, but also promote the accumulation of reactive oxygen species, leading to the production of malondialdehyde and decreased bacterial metabolism. Moreover, they exhibited the capability to bind with DNA and DNA gyrase, forming supramolecular complexes through various noncovalent interactions, thereby inhibiting DNA replication and causing bacterial death. All the above results suggested that the newly developed indolylacryloyl-derived oxacins should hold great promise as potential multitargeting broad-spectrum antibacterial candidates to overcome drug resistance.

Generating Concentration Gradients across Membranes for Molecular Dynamics Simulations of Periodic Systems.

The plasma membrane forms the boundary between a living entity and its environment and acts as a barrier to permeation and flow of substances. Several computational means of calculating permeability have been implemented for molecular dynamics (MD) simulations-based approaches. Except for double bilayer systems, most permeability studies have been performed under equilibrium conditions, in large part due to the challenges associated with creating concentration gradients in simulations utilizing periodic boundary conditions. To enhance the scientific understanding of permeation and complement the existing computational means of characterizing membrane permeability, we developed a non-equilibrium method that enables the generation and maintenance of steady-state gradients in MD simulations. We utilize PBCs advantageously by imposing a directional bias to the motion of permeants so that their crossing of the boundary replenishes the gradient, like a previous study on ions. Under these conditions, a net flow of permeants across membranes may be observed to determine bulk permeability by a direct application of J=PΔc. In the present study, we explore the results of its application to an exemplary O2 and POPC bilayer system, demonstrating accurate and precise permeability measurements. In addition, we illustrate the impact of permeant concentration and the choice of thermostat on the permeability. Moreover, we demonstrate that energetics of permeation can be closely examined by the dissipation of the gradient across the membrane to gain nuanced insights into the thermodynamics of permeability.

Ultrastructural examination of cryodamage in Paracentrotus lividus eggs during cryopreservation.

This study examinates the challenges of cryopreserving sea urchin (Paracentrotus lividus) eggs, a task hindered by factors like low membrane permeability and high sensitivity to cryoprotective agents (CPAs). While successful cryopreservation has been achieved for some marine invertebrates, eggs remain problematic due to their unique characteristics. The study explores the impact of various CPAs and cryopreservation techniques on sea urchin eggs, employing scanning and transmission electron microscopy to analyze cellular damage. The findings reveal that exposure to low CPA concentrations (0.5 M) did not induce significant damage to eggs. However, high concentrations (3 M) proved highly detrimental. Every cryopreservation approach investigated in this study resulted in irreversible damage to the sea urchin eggs, rendering them nonviable for future use. The research sheds light on the importance of understanding the structural alterations induced by CPAs and cryopreservation methods. This knowledge is essential for refining cryopreservation methods, potentially paving the way for successful preservation of these challenging cells.

Gaussian accelerated molecular dynamics simulations facilitate prediction of the permeability of cyclic peptides.

Despite their widespread use as therapeutics, clinical development of small molecule drugs remains challenging. Among the many parameters that undergo optimization during the drug development process, increasing passive cell permeability (i.e., log(P)) can have some of the largest impact on potency. Cyclic peptides (CPs) have emerged as a viable alternative to small molecules, as they retain many of the advantages of small molecules (oral availability, target specificity) while being highly effective at traversing the plasma membrane. However, the relationship between the dominant conformations that typify CPs in an aqueous versus a membrane environment and cell permeability remain poorly characterized. In this study, we have used Gaussian accelerated molecular dynamics (GaMD) simulations to characterize the effect of solvent on the free energy landscape of lariat peptides, a subset of CPs that have recently shown potential for drug development (Kelly et al., JACS 2021). Differences in the free energy of lariat peptides as a function of solvent can be used to predict permeability of these molecules, and our results show that permeability is most greatly influenced by N-methylation and exposure to solvent. Our approach lays the groundwork for using GaMD as a way to virtually screen large libraries of CPs and drive forward development of CP-based therapeutics.

Study on the effect of different high-voltage electric field polarization process parameters on the vitality of dried chili pepper seeds.

To study the effect of different high-voltage electric field polarisation treatment process parameters on the viability of seeds of dried chili peppers. In this study, a high-voltage electrostatic polarisation treatment system was constructed to carry out experiments on the effects of different high-voltage electric field polarisation treatment process parameters on the viability of dried chili seeds. Conduct one-way tests to determine the preferred polarisation method and the preferred interval for output voltage and polarisation time. Two-factor, five-level central combination test with output voltage and polarization time as test factors and seed conductivity as a response indicator. Determining the better combination of parameters for output voltage and polarization time; Conducting seed germination trials to validate the effectiveness of the polarisation process. The results of the one-way test showed that: Negative-voltage polarisation was more effective than positive-voltage polarisation and alternating positive-negative-voltage polarisation in promoting seed vigor, with a better output voltage in the range of 10-14 kV, and a better polarisation time in the range of 20-40 s; The results of orthogonal tests showed that: Under the condition of negative voltage polarisation treatment, the output voltage of 12.08 kV and polarisation time of 30.32 s was the better parameter combination, at which the seed conductivity was minimum 159.87 uS/(cm g). Analyzing the function of cell membrane selective semi-permeability by seed conductivity change and revealing the mechanism of seed viability enhancement by high voltage electric field polarisation treatment; In the seed germination test, compared with the control group, seed germination potential increased by 9.09%, germination rate increased by 20.45%, germination index increased by 3.49, and vigor index increased by 41.66 under high-voltage electrostatic polarisation treatment, and all vigor indexes were significantly improved. The results of this study can provide a basis for the selection of processes and parameters for subsequent high-voltage electric field polarisation treatment of crop seeds.

Application of cuminaldehyde and ciprofloxacin for the effective control of biofilm assembly of Pseudomonas aeruginosa: A combinatorial study.

Pseudomonas aeruginosa is widely associated with biofilm-mediated antibiotic resistant chronic and acute infections which constitute a persistent healthcare challenges. Addressing this threat requires exploration of novel therapeutic strategies involving the combination of natural compounds and conventional antibiotics. Hence, our study has focused on two compounds; cuminaldehyde and ciprofloxacin, which were strategically combined to target the biofilm challenge of P. aeruginosa. The minimum inhibitory concentration (MIC) of cuminaldehyde and ciprofloxacin was found to be 400 µg/mL and 0.4 µg/mL, respectively. Moreover, the fractional inhibitory concentration index (FICI = 0.62) indicated an additive interaction prevailed between cuminaldehyde and ciprofloxacin. Subsequently, sub-MIC doses of cuminaldehyde (25 µg/mL) and ciprofloxacin (0.05 µg/mL) were selected for an array of antibiofilm assays which confirmed their biofilm inhibitory potential without exhibiting any antimicrobial activity. Furthermore, selected doses of the mentioned compounds could manage biofilm on catheter surface by inhibiting and disintegrating existing biofilm. Additionally, the test combination of the mentioned compounds reduced virulence factors secretion, accumulated reactive oxygen species and increased cell-membrane permeability. Thus, the combination of cuminaldehyde and ciprofloxacin demonstrates potential in combating biofilm-associated Pseudomonal threats.

Multiparametric Analysis of Apoptosis by Flow Cytometry.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process. This approach to analyzing multiple apoptotic characteristics simultaneously yields far more information than single-parameter assays. While more informative than single-parameter assays, these multicolor methods can still be analyzed on relatively simple flow cytometers, making them widely accessible.

Klebicin E, a pore-forming bacteriocin of Klebsiella pneumoniae, exploits the porin OmpC and the Ton system for translocation.

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.

What influences the activity of Degrader-Antibody conjugates (DACs).

The targeted protein degradation (TPD) technology employing proteolysis-targeting chimeras (PROTACs) has been widely applied in drug chemistry and chemical biology for the treatment of cancer and other diseases. PROTACs have demonstrated significant advantages in targeting undruggable targets and overcoming drug resistance. However, despite the efficient degradation of targeted proteins achieved by PROTACs, they still face challenges related to selectivity between normal and cancer cells, as well as issues with poor membrane permeability due to their substantial molecular weight. Additionally, the noteworthy toxicity resulting from off-target effects also needs to be addressed. To solve these issues, Degrader-Antibody Conjugates (DACs) have been developed, leveraging the targeting and internalization capabilities of antibodies. In this review, we elucidates the characteristics and distinctions between DACs, and traditional Antibody-drug conjugates (ADCs). Meanwhile, we emphasizes the significance of DACs in facilitating the delivery of PROTACs and delves into the impact of various components on DAC activity. These components include antibody targets, drug-antibody ratio (DAR), linker types, PROTACs targets, PROTACs connections, and E3 ligase ligands. The review also explores the suitability of different targets (antibody targets or PROTACs targets) for DACs, providing insights to guide the design of PROTACs better suited for antibody conjugation.

Macrocycles and macrocyclization in anticancer drug discovery: Important pieces of the puzzle.

Increasing disease-related proteins have been identified as novel therapeutic targets. Macrocycles are emerging as potential solutions, bridging the gap between conventional small molecules and biomacromolecules in drug discovery. Inspired by successful macrocyclic drugs of natural origins, macrocycles are attracting more attention for enhanced binding affinity and target selectivity. Due to the conformation constraint and structure preorganization, macrocycles can reach bioactive conformations more easily than parent acyclic compounds. Also, rational macrocyclization combined with sequent structural modification will help improve oral bioavailability and combat drug resistance. This review introduces various strategies to enhance membrane permeability in macrocyclization and subsequent modification, such as N-methylation, intramolecular hydrogen bonding modulation, isomerization, and reversible bicyclization. Several case studies highlight macrocyclic inhibitors targeting kinases, HDAC, and protein-protein interactions. Finally, some macrocyclic agents targeting tumor microenvironments are illustrated.

The antimicrobial property of JY-1, a complex mixture of Traditional Chinese Medicine, is linked to it abilities to suppress biofilm formation and disrupt membrane permeability.

The substantial increase of infections, caused by novel, sudden, and drug-resistant pathogens, poses a significant threat to human health. While numerous studies have demonstrated the antibacterial and antiviral effects of Traditional Chinese Medicine, the potential of a complex mixture of traditional Chinese Medicine with a broad-spectrum antimicrobial property remains underexplored. This study aimed to develop a complex mixture of Traditional Chinese Medicine (TCM), JY-1, and investigate its antimicrobial properties, along with its potential mechanism of action against pathogenic microorganisms. Antimicrobial activity was assessed using a zone of inhibition assay and the drop plate method. Hyphal induction of Candida albicans was conducted using RPMI1640 medium containing 10% FBS, followed by microscopic visualization. Quantitative real-time PCR (RT-qPCR) was employed to quantify the transcript levels of hyphal-specific genes such as HWP1 and ALS3. The impact of JY-1 on biofilm formation was evaluated using both the XTT reduction assay and scanning electron microscopy (SEM). Furthermore, the cell membrane integrity was assessed by protein and nucleic acid leakage assays. Our results clearly showed that JY-1 significantly inhibits the vegetative growth of Candida spp. and Cryptococcus spp. In addition, this complex mixture is effectively against a wide range of pathogenic bacteria, including Staphylococcus aureus, Vancomycin-resistant enterococci, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae. More interestingly, JY-1 plays a direct anti-viral role against the mammalian viral pathogen vesicular stomatitis virus (VSV). Further mechanistic studies indicate that JY-1 acts to reduce the expression of hyphal specific genes HWP1 and ALS3, resulting in the suppression of the hyphal formation of C. albicans. The antimicrobial property of JY-1 could be attributed to its ability to reduce biofilm formation and disrupt the cell membrane permeability, a process resulting in microbial cell death and the release of cellular contents. Taken together, our work identified a potent broad-spectrum antimicrobial agent, a complex mixture of TCM which might be developed as a potential antimicrobial drug.

Insights on Structure-Passive Permeability Relationship in Pyrrole and Furan-Containing Macrocycles.

Macrocycles have recognized therapeutic potential, but their limited cellular permeability can hinder their development as oral drugs. To better understand the structure-permeability relationship of heterocycle-containing, semipeptidic macrocycles, a library was synthesized. These compounds were created by developing two novel reactions described herein: the reduction of activated oximes by LiBH4 and the aqueous reductive mono-N-alkylation of aldehydes using catalytic SmI2 and stoichiometric Zn. The permeability of the macrocycles was evaluated through a parallel artificial membrane permeability assay (PAMPA), and the results indicated that macrocycles with a furan incorporated into the structure have better passive permeability than those with a pyrrole moiety. Compounds bearing a 2,5-disubstituted pyrrole (endo orientation) were shown to be implicated in intramolecular H-bonds, enhancing their permeability. This study highlighted the impact of heterocycles moieties in semipeptides, creating highly permeable macrocycles, thus showing promising avenues for passive diffusion of drugs beyond the rule-of-five chemical space.

Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides.

In this study, we explored the potential of the photoremovable o-nitrobenzyl (oNB) group as a tool to manipulate the membrane permeability and regulate the conformation of linear peptides by means of experimental and computational studies. We found that the introduction of one or more oNB groups markedly increased the permeability and altered the conformation, as compared to the corresponding unmodified peptides. We thoroughly investigated the impact of peptide length, number of oNB group, oNB insertion position, and introduction of N- and C-terminal protecting groups on the passive membrane permeability by means of parallel artificial membrane permeability assay (PAMPA). Photoreaction of peptides containing one or two oNB groups proceeded cleanly in moderate to high yields, releasing the unprotected parent linear peptide. The oNB-modified peptides showed a cis/trans conformational equilibrium, while after photolysis, the unprotected linear peptides showed only the trans-amide conformation. Furthermore, a comprehensive comparison of oNB-modified peptides and N-methylated peptides was conducted, encompassing conformational analysis and physicochemical properties. N-Substituted peptides favored a folded-like structure, which may contribute to the improvement in permeability.

Predicting the intrinsic membrane permeability of Caco-2/MDCK cells by the solubility-diffusion model.

Membrane permeability is one of the main determinants for the absorption, distribution, metabolism and excretion of compounds and is therefore of crucial importance for successful drug development. Experiments with artificial phospholipid membranes have shown that the intrinsic membrane permeability (P0) of compounds is well-predicted by the solubility-diffusion model (SDM). However, using the solubility-diffusion model to predict the P0 of biological Caco-2 and MDCK cell membranes has proven unreliable so far. Recent publications revealed that many published P0 extracted from Caco-2 and MDCK experiments are incorrect. In this work, we therefore used a small self-generated set as well as a large revised set of experimental Caco-2 and MDCK data from literature to compare experimental and predicted P0. The P0 extracted from Caco-2 and MDCK experiments were systematically lower than the P0 predicted by the solubility-diffusion model. However, using the following correlation: log P0,Caco-2/MDCK = 0.84 log P0,SDM - 1.85, P0 of biological Caco-2 and MDCK cell membranes was well-predicted by the solubility-diffusion model.

Phytohemagglutinin from Phaseolus vulgaris enhances the lung cancer cell chemotherapy sensitivity by changing cell membrane permeability.

Chemotherapy is still a prevalent strategy for clinical lung cancer treatment. However, the inevitable emerged drug resistance has become a great hurdle to therapeutic effect. Studies have demonstrated that the primary cause of drug resistance is a decrease in the chemotherapeutic medicine concentration. Several lectins have been confirmed to be effective as chemotherapy adjuvants, enhancing the anti-tumor effects of chemotherapy drugs. Here, we combined phytohemagglutinin (PHA), which has been reported possess anti-tumor effects, with chemotherapy drugs Cisplatin (DDP) and Adriamycin (ADM) on lung cancer cells to detect the sensitivities of PHA as a chemotherapy adjuvant. Our results demonstrated that the PHA significantly enhanced the sensitivity of lung cancer cells to DDP and ADM, and Western blot showed that PHA combined with DDP or ADM enhance cytotoxic effects by inhibiting autophagy and promoting apoptosis. More importantly, we found PHA enhanced the chemotherapeutic drugs cytotoxicity by changing the cell membrane to increase the intracellular chemotherapeutic drugs concentration. Besides, the combination of PHA and ADM increased the ADM concentration in the multidrug-resistant strain A549-R cells and achieved the drug sensitization effect. Our results suggest that PHA combined with chemotherapy can be applied in the treatment of lung cancer cells and lung cancer multidrug-resistant strains, and provide a novel strategy for clinical tumor chemotherapy and a new idea to solve the problem of drug resistance in clinical lung cancer.

Temperature Dependence of Membrane Permeability Parameters for Five Cell Types Using Nonideal Thermodynamic Assumptions to Mathematically Model Cryopreservation Protocols.

Cryopreservation is the process of preserving biological matter at subzero temperatures for long-term storage. During cryopreservation, cells are susceptible to various injuries that can be mitigated by controlling the cooling and warming profiles and cryoprotective agent (CPA) addition and removal procedures. Mathematical modeling of the changing cell volume at different temperatures can greatly reduce the experiments needed to optimize cryopreservation protocols. Such mathematical modeling requires as inputs the cell membrane permeabilities to water and CPA and the osmotically inactive fraction of the cell. Since the intra- and extracellular solutions are generally thermodynamically nonideal, our group has been incorporating the osmotic virial equation to model the solution thermodynamics that underlie the cell volume change equations, adding the second and third osmotic virial coefficients of the grouped intracellular solute to the cell osmotic parameters that must be measured. In our previous work, we reported methods to obtain cell osmotic parameters at room temperature by fitting experimental cell volume kinetic data with equations that incorporated nonideal solution thermodynamics assumptions. Since the relevant cell volume excursions occur at different temperatures, the temperature dependence of the osmotic parameters plays an important role. In this work, we present a new two-part fitting method to obtain five cell-type-specific parameters (water permeability, dimethyl sulfoxide permeability, osmotically inactive fraction, and the second and third osmotic virial coefficients of the intracellular solution) from experimental measurements of equilibrium cell volume and cell volume as a function of time at room temperature and 0 °C for five cell types, namely, human umbilical vein endothelial cells (HUVECs), H9c2 rat myoblasts, porcine corneal endothelial cells (PCECs), the Jurkat T-lymphocyte cell line, and human cerebral microvascular endothelial cells (hCMECs/D3 cell line). The fitting method in this work is based on both equilibrium and kinetic cell volume data, enabling us to solve some technical challenges and expand our previously reported measurement technique to 0 °C. Finally, we use the measured parameters to model the cell volume changes for a HUVEC cryopreservation protocol to demonstrate the impact of the nonideal thermodynamic assumptions on predicting the changing cell volume during freezing and thawing.

Impact of the Unstirred Water Layer on the Permeation of Small-Molecule Drugs.

Over the last two decades, numerous molecular dynamics (MD) simulation-based investigations have attempted to predict the membrane permeability to small-molecule drugs as indicators of their bioavailability, a majority of which utilize the inhomogeneous solubility diffusion (ISD) model. However, MD-based membrane permeability is routinely 3-4 orders of magnitude larger than the values measured with the intestinal perfusion technique. There have been contentious discussions on the sources of the large discrepancies, and the two indisputable, potentially dominant ones are the fixed protonation state of the permeant and the neglect of the unstirred water layer (UWL). Employing six small-molecule drugs of different biopharmaceutical classification system classes, the current MD study relies on the ISD model but introduces the (de)protonation of the permeant by characterizing the permeation free energy of both neutral and charged states. In addition, the role of the UWL as a potential resistance against permeation is explored. The new MD protocol closely mimics the nature of small-molecule permeation and yields estimates that agree well with in vivo intestinal permeability.

Automatic generation of functional peptides with desired bioactivity and membrane permeability using Bayesian optimization.

Peptides are potentially useful modalities of drugs; however, cell membrane permeability is an obstacle in peptide drug discovery. The identification of bioactive peptides for a therapeutic target is also challenging because of the huge amino acid sequence patterns of peptides. In this study, we propose a novel computational method, PEptide generation system using Neural network Trained on Amino acid sequence data and Gaussian process-based optimizatiON (PENTAGON), to automatically generate new peptides with desired bioactivity and cell membrane permeability. In the algorithm, we mapped peptide amino acid sequences onto the latent space constructed using a variational autoencoder and searched for peptides with desired bioactivity and cell membrane permeability using Bayesian optimization. We used our proposed method to generate peptides with cell membrane permeability and bioactivity for each of the nine therapeutic targets, such as the estrogen receptor (ER). Our proposed method outperformed a previously developed peptide generator in terms of similarity to known active peptide sequences and the length of generated peptide sequences.

Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells.

Electric pulses used in electroporation-based treatments have been shown to affect the excitability of muscle and neuronal cells. However, understanding the interplay between electroporation and electrophysiological response of excitable cells is complex, since both ion channel gating and electroporation depend on dynamic changes in the transmembrane voltage (TMV). In this study, a genetically engineered human embryonic kidney cells expressing NaV1.5 and Kir2.1, a minimal complementary channels required for excitability (named S-HEK), was characterized as a simple cell model used for studying the effects of electroporation in excitable cells. S-HEK cells and their non-excitable counterparts (NS-HEK) were exposed to 100 µs pulses of increasing electric field strength. Changes in TMV, plasma membrane permeability, and intracellular Ca2+ were monitored with fluorescence microscopy. We found that a very mild electroporation, undetectable with the classical propidium assay but associated with a transient increase in intracellular Ca2+, can already have a profound effect on excitability close to the electrostimulation threshold, as corroborated by multiscale computational modelling. These results are of great relevance for understanding the effects of pulse delivery on cell excitability observed in context of the rapidly developing cardiac pulsed field ablation as well as other electroporation-based treatments in excitable tissues.

Pitfalls in evaluating permeability experiments with Caco-2/MDCK cell monolayers.

When studying the transport of molecules across biological membranes, intrinsic membrane permeability (P0) is more informative than apparent permeability (Papp), because it eliminates external (setup-specific) factors, provides consistency across experiments and mechanistic insight. It is thus an important building block for modeling the total permeability in any given scenario. However, extracting P0 is often difficult, if not impossible, when the membrane is not the dominant transport resistance. In this work, we set out to analyze Papp values measured with Caco-2/MDCK cell monolayers of 69 literature references. We checked the Papp values for a total of 318 different compounds for the extractability of P0, considering possible limitations by aqueous boundary layers, paracellular transport, recovery issues, active transport, a possible proton flux limitation, and sink conditions. Overall, we were able to extract 77 reliable P0 values, which corresponds to about one quarter of the total compounds analyzed, while about half were limited by the diffusion through the aqueous layers. Compared to an existing data set of P0 values published by Avdeef, our approach resulted in a much higher exclusion of compounds. This is a consequence of stricter compound- and reference-specific exclusion criteria, but also because we considered possible concentration-shift effects due to different pH values in the aqueous layers, an effect only recently described in literature. We thus provide a consistent and reliable set of P0, e.g. as a basis for future modeling.