Biophysics of claudin proteins in tight junction architecture: Three decades of progress.

Tight junctions are cell-cell adhesion complexes that act as gatekeepers of the paracellular space. Formed by several transmembrane proteins, the claudin family performs the primary gate-keeping function. The claudin proteins form charge and size-selective diffusion barriers to maintain homeostasis across endothelial and epithelial tissue. Of the 27 known claudins in mammals, some are known to seal the paracellular space, while others provide selective permeability. The differences in permeability arise due to the varying expression levels of claudins in each tissue. The tight junctions are observed as strands in freeze-fracture electron monographs; however, at the molecular level, tight junction strands form when multiple claudin proteins assemble laterally (cis assembly) within a cell and head-on (trans assembly) with claudins of the adjacent cell in a zipper-like architecture, closing the gap between the neighboring cells. The disruption of tight junctions caused by changing claudin expression levels or mutations can lead to diseases. Therefore, knowledge of the molecular architecture of the tight junctions and how that is tied to tissue-specific function is critical for fighting diseases. Here, we review the current understanding of the tight junctions accrued over the last three decades from experimental and computational biophysics perspectives.

Lipidation alters the phase-separation of resilin-like polypeptides.

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

Supramolecular Peptoid Structure Strengthens Complexation with Polyacrylic Acid Microgels.

We have studied the complexation between cationic antimicrobials and polyanionic microgels to create self-defensive surfaces that responsively resist bacterial colonization. An essential property is the stable sequestration of the loaded (complexed) antimicrobial within the microgel under a physiological ionic strength. Here, we assess the complexation strength between poly(acrylic acid) [PAA] microgels and a series of cationic peptoids that display supramolecular structures ranging from an oligomeric monomer to a tetramer. We follow changes in loaded microgel diameter with increasing [Na+] as a measure of the counterion doping level. Consistent with prior findings on colistin/PAA complexation, we find that a monomeric peptoid is fully released at ionic strengths well below physiological conditions, despite its +5 charge. In contrast, progressively higher degrees of peptoid supramolecular structure display progressively greater resistance to salting out, which we attribute to the greater entropic stability associated with the complexation of multimeric peptoid bundles.

An Affordable Topography-Based Protocol for Assigning a Residue's Character on a Hydropathy (PARCH) Scale.

The hydropathy of proteins or quantitative assessment of protein-water interactions has been a topic of interest for decades. Most hydropathy scales use a residue-based or atom-based approach to assign fixed numerical values to the 20 amino acids and categorize them as hydrophilic, hydroneutral, or hydrophobic. These scales overlook the protein's nanoscale topography, such as bumps, crevices, cavities, clefts, pockets, and channels, in calculating the hydropathy of the residues. Some recent studies have included protein topography in determining hydrophobic patches on protein surfaces, but these methods do not provide a hydropathy scale. To overcome the limitations in the existing methods, we have developed a Protocol for Assigning a Residue's Character on the Hydropathy (PARCH) scale that adopts a holistic approach to assigning the hydropathy of a residue. The parch scale evaluates the collective response of the water molecules in the protein's first hydration shell to increasing temperatures. We performed the parch analysis of a set of well-studied proteins that include the following─enzymes, immune proteins, and integral membrane proteins, as well as fungal and virus capsid proteins. Since the parch scale evaluates every residue based on its location, a residue may have very different parch values inside a crevice versus a surface bump. Thus, a residue can have a range of parch values (or hydropathies) dictated by the local geometry. The parch scale calculations are computationally inexpensive and can compare hydropathies of different proteins. The parch analysis can affordably and reliably aid in designing nanostructured surfaces, identifying hydrophilic and hydrophobic patches, and drug discovery.

Self-Defensive Antimicrobial Shape Memory Polyurethanes with Honey-Based Compounds.

Infection treatment plays a crucial role in aiding the body in wound healing. To that end, we developed a library of antimicrobial polymers based on segmented shape memory polyurethanes with nondrug-based antimicrobials (i.e., honey-based phenolic acids (PAs)) using both chemical and physical incorporation approaches. The antimicrobial shape memory polymers (SMPs) have high transition temperatures (>55 °C) to enable maintenance of temporary, programmed shapes in physiological conditions unless a specific external stimulus is present. Polymers showed tunable mechanical and shape memory properties by changing the ratio, chemistry, and incorporation method of PAs. Cytocompatible (∼100% cell viability) synthesized polymers inhibited growth rates of Staphylococcus aureus (∼100% with physically incorporated PAs and >80% with chemically incorporated PAs) and Escherichia coli (∼100% for samples with cinnamic acid (physical and chemical)). Crystal violet assays showed that all formulations inhibit biofilm formation in surrounding solutions, and chemically incorporated samples showed surface antibiofilm properties with S. aureus. Molecular dynamics simulations confirm that PAs have higher levels of interactions with S. aureus cell membranes than E. coli. Long-term antimicrobial properties were measured after storage of the sample in aqueous conditions; the polymers retained their antimicrobial properties against E. coli after up to 20 days. As a proof of concept, magnetic particles were incorporated into the polymer to trigger user-defined shape recovery by applying an external magnetic field. Shape recovery disrupted preformed S. aureus biofilms on polymer surfaces. This antimicrobial biomaterial platform could enable user- or environmentally controlled shape change and/or antimicrobial release to enhance infection treatment efforts.

Combined Computational-Biochemical Approach Offers an Accelerated Path to Membrane Protein Solubilization.

Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here, we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank-ordered for their proposed ability to solubilize GOAT. The simulations were performed at all-atom resolution for a combined simulation time of 24 µs. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization, supporting downstream functional and structural studies.

Claudin-23 reshapes epithelial tight junction architecture to regulate barrier function.

Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.

Lipidation Alters the Structure and Hydration of Myristoylated Intrinsically Disordered Proteins.

Lipidated proteins are an emerging class of hybrid biomaterials that can integrate the functional capabilities of proteins into precisely engineered nano-biomaterials with potential applications in biotechnology, nanoscience, and biomedical engineering. For instance, fatty-acid-modified elastin-like polypeptides (FAMEs) combine the hierarchical assembly of lipids with the thermoresponsive character of elastin-like polypeptides (ELPs) to form nanocarriers with emergent temperature-dependent structural (shape or size) characteristics. Here, we report the biophysical underpinnings of thermoresponsive behavior of FAMEs using computational nanoscopy, spectroscopy, scattering, and microscopy. This integrated approach revealed that temperature and molecular syntax alter the structure, contact, and hydration of lipid, lipidation site, and protein, aligning with the changes in the nanomorphology of FAMEs. These findings enable a better understanding of the biophysical consequence of lipidation in biology and the rational design of the biomaterials and therapeutics that rival the exquisite hierarchy and capabilities of biological systems.

Self-Defensive Antimicrobial Surfaces Using Polymyxin-Loaded Poly(styrene sulfonate) Microgels.

Self-defensive antimicrobial surfaces are of interest because they can inhibit bacterial colonization while minimizing unnecessary antimicrobial release in the absence of a bacterial challenge. One self-defensive approach uses self-assembly to first deposit a submonolayer coating of polyelectrolyte microgels and subsequently load those microgels by complexation with small-molecule antimicrobials. The microgel/antimicrobial complexation strength is a key parameter that controls the ability of the antimicrobial both to remain sequestered within the microgels when exposed to medium and to release in response to a bacterial challenge. Here we study the relative complexation strengths of two FDA-approved cationic antibiotics─colistin (polymyxin E) and polymyxin B─with microgels of poly(styrene sulfonate) (PSS). These polymyxins are similar cyclic polypeptides with +5 charge at pH 7.4. However, polymyxin B substitutes an aromatic ring for a dimethyl moiety in colistin, and this aromaticity can influence complexation via π and hydrophobic interactions. Coarse-grained molecular dynamics shows that the free-energy change associated with polymyxin B/PSS complexation is more negative than that of colistin/PSS complexation. Experimentally, in situ optical microscopy of microgel deswelling shows that both antibiotics load quickly from low-ionic-strength phosphate buffer. The enhanced polymyxin B/PSS complexation strength is then manifested by subsequent exposure to flowing antibiotic-free buffer with varying NaCl concentration. Microgels loaded with polymyxin B remain stably deswollen to higher salt concentrations than do colistin/PSS microgels. Importantly, exposing loaded microgels to E. coli in nutrient-free-flowing phosphate buffer shows that bacteria are killed by physical contact with the loaded microgels consistent with the contact-transfer mechanism of self-defensiveness. In vitro culture experiments show that these same surfaces, nevertheless, support the adhesion, spreading and proliferation of human fetal osteoblasts. These findings suggest a pathway to create a self-defensive antimicrobial surface effective under physiological conditions based on the nonmetabolic bacteria-triggered release of FDA-approved antibiotics.

Unique structural features of claudin-5 and claudin-15 lead to functionally distinct tight junction strand architecture.

Members of the claudin family impart unique paracellular selectivity to tight junctions. However, the structure-function relationship between claudin's strand architecture and the paracellular charge- and size-selectivity is not well-understood. This work examines the molecular assembly of claudin-5, a barrier-forming protein, and claudin-15, a channel-forming protein, to determine their structural and functional properties. We adopt a bottom-up approach starting from claudin monomers to build the molecular architecture of the tight junction strands. First, we investigated the cis assembly of claudin-5 and -15 dimers using the Protein Association Energy Landscape method. Out of the millions of dimer conformations, we narrowed down key cis claudin-5 and -15 dimer conformations that were thermodynamically and kinetically stable. Second, we performed the trans assembly of dimers to identify the tetrameric building blocks that serve as the repeat units for strand formation. Finally, the strand assembly of the tetrameric repeat units showed fundamentally distinct strand architectures. In claudin-5, the cis and trans interactions seal the paracellular space, while in claudin-15, the gaps in the paracellular space lead to pore formation. This detailed study suggests that each member of the claudin family is unique and requires systematic molecular-level analysis for determining the strand architecture.

Joint Profile Characteristics of Long-Latency Transient Evoked and Distortion Otoacoustic Emissions.

PURPOSE: In clinical practice, otoacoustic emissions (OAEs) are interpreted as either "present" or "absent." However, OAEs have the potential to inform about etiology and severity of hearing loss if analyzed in other dimensions. A proposed method uses the nonlinear component of the distortion product OAEs together with stimulus frequency OAEs to construct a joint reflection-distortion profile. The objective of the current study is to determine if joint reflection-distortion profiles can be created using long-latency (LL) components of transient evoked OAEs (TEOAEs) as the reflection-type emission. METHOD: LL TEOAEs and the nonlinear distortion OAEs were measured from adult ears. Individual input-output (I/O) functions were created, and OAE level was normalized by dividing by the stimulus level yielding individual gain functions. Peak strength, compression threshold, and OAE level at compression threshold were derived from individual gain functions to create joint reflection-distortion profiles. RESULTS: TEOAEs with a poststimulus window starting at 6 ms had I/O functions with compression characteristics similar to LL TEOAE components. The model fit the LL gain functions, which had R 2 > .93, significantly better than the nonlinear distortion OAE gain functions, which had R 2 = .596-.99. Interquartile ranges for joint reflection-distortion profiles were larger for compression threshold and OAE level at compression threshold but smaller for peak strength than those previously published. CONCLUSIONS: The gain function fits LL TEOAEs well. Joint reflection-distortion profiles are a promising method that could enhance diagnosis of hearing loss, and use of the LL TEOAE in the profile for peak strength may be important because of narrow interquartile ranges. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.20323593.

Adaptive Recombinant Nanoworms from Genetically Encodable Star Amphiphiles.

Recombinant nanoworms are promising candidates for materials and biomedical applications ranging from the templated synthesis of nanomaterials to multivalent display of bioactive peptides and targeted delivery of theranostic agents. However, molecular design principles to synthesize these assemblies (which are thermodynamically favorable only in a narrow region of the phase diagram) remain unclear. To advance the identification of design principles for the programmable assembly of proteins into well-defined nanoworms and to broaden their stability regimes, we were inspired by the ability of topologically engineered synthetic macromolecules to acess rare mesophases. To test this design principle in biomacromolecular assemblies, we used post-translational modifications (PTMs) to generate lipidated proteins with precise topological and compositional asymmetry. Using an integrated experimental and computational approach, we show that the material properties (thermoresponse and nanoscale assembly) of these hybrid amphiphiles are modulated by their amphiphilic architecture. Importantly, we demonstrate that the judicious choice of amphiphilic architecture can be used to program the assembly of proteins into adaptive nanoworms, which undergo a morphological transition (sphere-to-nanoworms) in response to temperature stimuli.

Persister control by leveraging dormancy associated reduction of antibiotic efflux.

Persistent bacterial infections do not respond to current antibiotic treatments and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 µg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 µg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles' heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.

Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics.

Bacterial colonization of biotic and abiotic surfaces and antibiotic resistance are grand challenges with paramount societal impacts. However, in the face of increasing bacterial resistance to all known antibiotics, efforts to discover new classes of antibiotics have languished, creating an urgent need to accelerate the antibiotic discovery pipeline. A major deterrent in the discovering of new antibiotics is the limited permeability of molecules across the bacterial envelope. Notably, the Gram-negative bacteria have nutrient specific protein channels (or porins) that restrict the permeability of non-essential molecules, including antibiotics. Here, we have developed the Computational Antibiotic Screening Platform (CLASP) for screening of potential drug molecules through the porins. The CLASP takes advantage of coarse grain (CG) resolution, advanced sampling techniques, and a parallel computing environment to maximize its performance. The CLASP yields comprehensive thermodynamic and kinetic output data of a potential drug molecule within a few hours of wall-clock time. Its output includes the potential of mean force profile, energy barrier, the rate constant, and contact analysis of the molecule with the pore-lining residues, and the orientational analysis of the molecule in the porin channel. In our first CLASP application, we report the transport properties of six carbapenem antibiotics-biapenem, doripenem, ertapenem, imipenem, meropenem, and panipenem-through OccD3, a major channel for carbapenem uptake in Pseudomonas aeruginosa. The CLASP is designed to screen small molecule libraries with a fast turnaround time to yield structure-property relationships to discover antibiotics with high permeability. The CLASP will be freely distributed to enable accelerated antibiotic drug discovery.

High-Level Antibiotic Tolerance of a Clinically Isolated Enterococcus faecalis Strain.

Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 µg · ml-1) were 25.4% ± 4.3% and 51.9% ± 4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections.IMPORTANCEEnterococcus faecalis represents a major group of pathogens causing nosocomial infections that are resistant to multiple classes of antibiotics. An important challenge associated with E. faecalis infection is the emergence of multidrug-tolerant strains, which have normal MICs but do not respond to antibiotic treatment. Here, we report a strain of E. faecalis that was isolated from a cystic fibrosis patient and demonstrated high-level tolerance to ampicillin, vancomycin, and tetracycline. Whole-genome sequencing revealed critical substitutions in one of the tetracycline efflux pump repressors that are consistent with the increased tolerance of E. faecalis UM001B to tetracycline. These findings provide new information about bacterial antibiotic tolerance and may help develop more effective therapeutics.

Non-canonical lipoproteins with programmable assembly and architecture.

The substrate promiscuity of an acyltransferase is leveraged to synthesize artificial lipoproteins bearing a non-canonical PTM (ncPTM). The non-canonical functionality of these lipoproteins results in a distinctive hysteretic assembly-absent from the canonical lipoproteins-and is used to prepare hybrid multiblock materials with precise and programmable patterns of amphiphilicity. This study demonstrates the promise of expanding the repertoire of PTMs for the development of nanomaterials with a unique assembly and function.

Rational identification and characterisation of peptide ligands for targeting polysialic acid.

The alpha-2,8-linked form of the polysaccharide polysialic acid (PSA) has widespread implications in physiological and pathological processes, ranging from neurological development to disease progression. Though the high electronegativity and excluded volume of PSA often promotes interference of biomolecular interactions, PSA-binding ligands have important implications for both biological processes and biotechnological applications. As such, the design, identification, and characterisation of novel ligands towards PSA is critical for expanding knowledge of PSA interactions and achieving selective glycan targeting. Here, we report on a rational approach for the identification of alpha-2,8-PSA-binding peptides, involving design from the endogenous ligand Siglec-11 and multi-platform characterisation of peptide binding. Microarray-based examination of peptides revealed charge and sequence characteristics influencing peptide affinity to PSA, and carbohydrate-peptide binding was further quantified with a novel fluorescence anisotropy assay. PSA-binding peptides exhibited specific binding to polymeric SA, as well as different degrees of selective binding in various conditions, including competition with PSA of alternating 2,8/9-linkages and screening with PSA-expressing cells. A computational study of Siglec-11 and Siglec-11-derived peptides offered synergistic insight into ligand binding. These results demonstrate the potential of PSA-binding peptides for selective targeting and highlight the importance of the approaches described herein for the study of carbohydrate interactions.

Interaction of amphiphilic coumarin with DPPC/DPPS lipid bilayer: effects of concentration and alkyl tail length.

In this work, interactions between amphiphilic amino methyl coumarin and dipalmitoyl-sn-glycero-3-phosphocholine/dipalmitoyl-sn-glycero-3-phosphoserine (DPPC/DPPS) lipid bilayer were investigated. A combination of experimental techniques (zeta potential, fluorescence spectroscopy, and differential scanning calorimetry) along with molecular dynamics simulations was employed to examine the influence of alkyl tail length and concentration of the amphiphilic coumarin on the lipid bilayer. Alkyl tails comprising 5(C5), 9(C9), and 12(C12) carbon atoms were conjugated to amino methyl coumarin via a single-step process. The binding and insertion mechanisms of the amphiphilic coumarins were studied in increasing concentrations for short-tailed (C5) and long-tailed (C12) coumarins. The simulation results show that C5 coumarin molecules penetrate the lipid bilayer, but owing to the short alkyl tail, they interact primarily with the lipid head groups resulting in lipid bilayer thinning; however, at high concentrations, the C5 coumarins undergo continuous insertion-ejection from the outer leaflet of the lipid bilayer. In contrast, C12 coumarins interact favorably with the hydrophobic lipid tails and lack the ejection-reinsertion behavior. Instead, the C12 coumarin molecules undergo flip-flops between the outer and inner leaflets of the lipid bilayer. At high concentrations, the high-frequency flip-flops lead to lipid destabilization, causing the lipid bilayer to rupture. The simulation results are in excellent agreement with the toxicity of amphiphilic coumarin activity in cancer cells. The efficacy of amphiphilic coumarins in liposomal lipid bilayers demonstrates the promise of these molecules as a tool in the treatment of cancer.