Modulating a model membrane of sphingomyelin by a tricyclic antidepressant drug.

Tricyclic medicine such as amitriptyline (AMT) hydrochloride, initially developed to treat depression, is also used to treat neuropathic pain, anxiety disorder, and migraines. The mechanism of functioning of this type of drugs is ambiguous. Understanding the mechanism is important for designing new drug molecules with higher pharmacological efficiency. Hence, in the present study, biophysical approaches have been taken to shed light on their interactions with a model cellular membrane of brain sphingomyelin in the form of monolayer and multi-lamellar vesicles. The surface pressure-area isotherm infers the partitioning of a drug molecule into the lipid monolayer at the air water interface, providing a higher surface area per molecule and reducing the in-plane elasticity. Further, the surface electrostatic potential of the lipid monolayer is found to increase due to the insertion of drug molecule. The interfacial rheology revealed a reduction of the in-plane viscoelasticity of the lipid film, which, depends on the adsorption of the drug molecule onto the film. Small-angle X-ray scattering (SAXS) measurements on multilamellar vesicles (MLVs) have revealed that the AMT molecules partition into the hydrophobic core of the lipid membrane, modifying the organization of lipids in the membrane. The modified physical state of less rigid membrane and the transformed electrostatics of the membrane could influence its interaction with synaptic vesicles and neurotransmitters making higher availability of the neurotransmitters in the synaptic cleft.

Graphene-Based Nanomaterials and Their Interactions with Lipid Membranes.

Graphene-based nanomaterials (GNMs) have captured increasing attention in the recent advancement of materials science and nanotechnology owing to their excellent physicochemical properties. Despite having unquestionable advances, the application of GNMs in biological and medical sciences is still limited due to the lack of knowledge and precise control over their interaction with the biological milieu. The cellular membrane is the first barrier with which GNMs interact before entering a cell. Therefore, understanding how they interact with cell membranes is important from the perspective of safe use in biological and biomedical fields. In this review, we systematically summarize the recent efforts in predicting the interactions between GNMs and model cellular membranes. This review provides insights into how GNMs interact with lipid membranes and self-assemble in and around them. Both the computational simulations and experimental observations are summarized. The interactions are classified depending on the physicochemical properties (structure, chemistry, and orientation) of GNMs and various model membranes. The thermodynamic parameters, structural details, and supramolecular forces are listed to understand the interactions which would help circumvent potential risks and provide guidance for safe use in the future. At the end of this review, future prospective and emerging challenges in this research field are discussed.

Ionic Liquid-Induced Assembly of DNA at Air-Water Interface.

DNA nanotechnology is the future of many products in the pharmaceutical and cosmetic industries. Self-assembly of this negatively charged biopolymer at surfaces and interfaces is an essential step to elaborate its field of applications. In this study, the ionic liquid (IL) monolayer-assisted self-assembly of DNA macromolecules at the air-water interface has been closely monitored by employing various quantitative techniques, namely, surface pressure-area (π-A) isotherms, surface potential, interfacial rheology, and X-ray reflectivity (XRR). The π-A isotherms reveal that the IL 1,3-didecyl 3-methyl imidazolium chloride induces DNA self-assembly at the interface, leading to a thick viscoelastic film. The interfacial rheology exhibits a notable rise in the viscoelastic modulus as the surface pressure increases. The values of storage and loss moduli measured as a function of strain frequency suggest a relaxation frequency that depends on the length of the macromolecule. The XRR measurements indicate a considerable increase in DNA layer thickness at the elevated surface pressures depending on the number of base pairs of the DNA. The results are considered in terms of the electrostatic and hydrophobic interactions, allowing a quantitative conclusion about the arrangement of DNA strands underneath the monolayer of the ILs at the air-water interface.

Effects of ionic liquids on biomembranes: A review on recent biophysical studies.

Ionic liquids (ILs) have been emerged as a versatile class of compounds that can be easily tuned to achieve desirable properties for various applications. The ability of ILs to interact with biomembranes has attracted significant interest, as they have been shown to modulate membrane properties in ways that may have implications for various biological processes. This review provides an overview of recent studies that have investigated the interaction between ILs and biomembranes. We discuss the effects of ILs on the physical and chemical properties of biomembranes, including changes in membrane fluidity, permeability, and stability. We also explore the mechanisms underlying the interaction of ILs with biomembranes, such as electrostatic interactions, hydrogen bonding, and van der Waals forces. Additionally, we discuss the future prospects of this field.

Cholesterol-Controlled Interaction of Ionic Liquids with Model Cellular Membranes.

While ionic liquids (ILs) are considered as prospective ingredients of new antimicrobial agents, it is important to understand the adverse effects of these molecules on human cells. Since cholesterol is the essential component of a human cell membrane, in the present study, the effect of an imidazolium-based IL has been investigated on the model membrane in the presence of cholesterol. The area per sphingomyelin lipid is found to reduce in the presence of the IL, which is quantified by the area-surface pressure isotherm of the lipid monolayer formed at the air-water interface. The effect is considerably diminished in the cholesterol-containing monolayer. Further, the IL is observed to decrease the rigidity of the cholesterol-free monolayer. Interestingly, the presence of cholesterol does not allow any change in this property of the layer at lower surface pressure. However, at a higher surface pressure, the IL increases the elasticity in the cholesterol-induced condensed phase of the lipid layer. The X-ray reflectivity measurement on a stack of cholesterol-free lipid bilayers proved the formation of IL-induced phase-separated domains in the matrix of a pure lipid phase. These domains are found to be formed by interdigitating the chains of the lipids, producing a thinner membrane. Such a phase is less intense in the cholesterol-containing membrane. All of these results indicate that the IL molecules may deform the cholesterol-free membrane of a bacterial cell, but the same may not be harmful to human beings as cholesterol could restrict the insertion in the cellular membrane of a human cell.

Temperature-dependent anomalous viscosity of aqueous solutions of imidazolium-based ionic liquids.

Ionic liquids (ILs) are organic salts with a low melting point compared to inorganic salts. Room temperature ILs are of great importance for their widespread potential industrial applications. The viscosity of aqueous solutions of two imidazolium-based ILs, investigated in the present study, exhibits an anomalous temperature variation. Unlike conventional molecular fluids, the viscosity of 1-methyl-3-octyl imidazolium chloride [OMIM Cl] and 1-methyl-3-decyl imidazolium chloride [DMIM Cl] solutions is found to increase with temperature and then depress. The Small Angle X-ray Scattering (SAXS) data suggest that the lattice parameter of the body-centered cubic lattice formed by the spherical micelles of these ILs, and the morphology of the micelles remain intact over the measured temperature range. The molecular dynamics simulation shows the micelles to be more refined with their integrated structure on increasing the temperature. On further increase of the temperature, the structure is found to be loosened, which is corroborated by the simulation work. The ionic conductivity of these IL solutions shows a trend that is opposite to that of the viscosity. The observed anomalous nature of the viscosity is attributed to the trapped dissociated ions in the network of the micellar aggregates.

Discerning perturbed assembly of lipids in a model membrane in presence of violacein: Effects of membrane hydrophobicity.

For the lack of effective antibiotics towards antibiotic resisting bacteria, it is required to discover new antibiotics and to understand their antimicrobial mechanism. Violacein is a violet pigment found in several gram-negative bacteria possessing antimicrobial properties to gram-positive bacteria. This present article investigates the insertion ability of this molecule into a model membrane composed of zwitterionic phospholipids. Thermodynamic characterization of lipid monolayers in the presence of violacein was carried out using a single lipid layer formed at air-water interface. The molecule inserts into the layer altering the area occupied by each lipid and the in-plane compressibility of the film. This insertion increases with the hydrophobic chain length of the lipid. The perturbed self-assembly of lipids in a bilayer is quantified using a lipid multilayer system applying the X-ray reflectivity technique. The electron density profile from the reflectivity data shows that the molecule inserts into the fluid phase creating a relatively ordered chain conformation. Further, the insertion into the gel phase is observed to increase with the increased thickness of the hydrophobic core of a bilayer.

Thermodynamics and In-Plane Viscoelasticity of Anionic Phospholipid Membranes Modulated by an Ionic Liquid.

This article presents the effects of an imidazolium-based ionic liquid (IL) on the thermodynamics and in-plane viscoelastic properties of model membranes of anionic phospholipids. The negative Zeta potential of multilamellar vesicles of 14 carbon lipid 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) is observed to reduce due to the presence of few mole % of an IL 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]). The effect was found to be stronger on enhancing the chain length of the lipid. The surface pressure-area isotherms of lipid monolayer formed at air-water interface are modified by the IL reducing the effective area per molecule. Further, the equilibrium elasticity of the film is altered depending upon the thermodynamic phase of the lipids. While the presence of the IL in the DMPG lipid makes it ordered in the gel phase by reducing the entropy, the effect is opposite in the fluid phase. The in-plane viscoelastic parameters of the lipid film is quantified by dilation rheology using the oscillatory barriers of a Langmuir trough. Even though the low chain lipid DMPG does not show any effect of IL on its storage and loss moduli, the longer chain lipids exhibit a prominent effect in the liquid extended (LE) phase. Further, the dynamic response of the lipid film is found to be distinctly different in the liquid condensed (LC) phase from that of the LE phase.

1,3 Dialkylated Imidazolium Ionic Liquid Causes Interdigitated Domains in a Phospholipid Membrane.

Amphiphilic imidazolium-based ionic liquids (ILs) have proven their efficacy in altering the membrane integrity and dynamics. The present article investigates the phase-separated domains in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane induced by 1,3 dialkylated imidazolium IL. Isotherm measurements on DPPC monolayers formed at the air-water interface have shown a decrease in the mean molecular area with the addition of this IL. The positive value of the excess Gibbs free energy of mixing indicates an unfavorable mixing of the IL into the lipid. This leads to IL-induced phase-separated domains in the multilayer of the lipid confirmed by the occurrence of two sets of equidistance peaks in the X-ray reflectivity data. The electron density profile along the surface normal obtained by the swelling method shows the bilayer thickness of the newly formed IL-rich phase to be substantially lower (∼34 Å) than the DPPC phase (∼45.8 Å). This IL-rich phase has been confirmed to be interdigitated, showing an enhanced electron density in the tail region due to the overlapping hydrocarbon chains. Differential scanning calorimetry measurements showed that the incorporation of IL enhances the fluidity of the lipid bilayer. Therefore, the study indicates the formation of an interdigitated phase with a lower order compared to the gel phase in the DPPC membrane supplemented with the IL.

Bioinspired micro/nano structured aluminum with multifaceted applications.

Inspired by many biological systems such as lotus leaves, insect wings and rose petals, great attention has been devoted to the study and fabrication of artificial superhydrophobic surfaces with multiple functionalities. In the present study, a simple and ecological synthesis route has been employed for large scale fabrication of self-assembled, sustainable nanostructures on unprocessed and micro imprinted aluminum surfaces named 'Nano' and 'Hierarchy'. The processed samples show extreme wettability ranging from superhydrophilicity to superhydrophobicity depending on post-processing conditions. The densely packed ellipsoidal nanostructures exhibited superhydrophobicity with excellent water, bacterial and dust repellency when modified by low surface energy material 1H,1H,2H,2H-perfluorooctyltriethoxysilane (FOTES), characterized by a static contact angle of 163 ± 1° and contact angle hysteresis (CAH) ~3°. These coated surfaces show significant corrosion resistance with current density of 6 nA/cm2 which is 40 times lower than unprocessed counterpart and retain chemical stability after prolonged immersion in corrosive media. These surfaces show excellent self-cleaning ability with significantly low water consumption (< 0.1 µl/mm2-mg) and prevent biofouling which ensures its applicability in biological environment and marine components. The nanostructured superhydrophilic aluminum shows maximum antibacterial activity due to disruption of cell membrane. This work can offer a simple strategy to large scale fabrication of multifunctional biomimetic metallic surfaces.

Partitioning of a Hybrid Lipid in Domains of Saturated and Unsaturated Lipids in a Model Cellular Membrane.

The cellular membranes are composed of hundreds of components such as lipids, proteins, and sterols that are chemically and physically distinct from each other. The lipid-lipid and lipid-protein interactions form domains in this membrane, which play vital roles in membrane physiology. The hybrid lipids (HLs) with one saturated and one unsaturated chain can control the shape and size of these domains, ensuring the thermodynamic stability of a membrane. In this study, the thermodynamics of mixing of a HL and its structural effects on the phase separated domains in a model membrane composed of a saturated and an unsaturated lipid have been investigated. The HL is observed to mix into an unsaturated lipid reducing the Gibbs free energy, whereas the mixing is unfavorable in a saturated lipid. The presence of an HL in an unsaturated lipid tends to increase its area fraction, which is reflected in the enhanced correlation length across the bilayers in a multilayered sample. There is a feeble effect on the domain structure of the saturated lipid due to the presence of the HLs at the phase boundary. This study concludes that the HLs preferentially participate in the unsaturated lipid regions compared to that of a saturated lipid.

Self-Assembly of Graphene Oxide Nanoflakes in a Lipid Monolayer at the Air-Water Interface.

The graphene family, especially graphene oxide (GO), has captured increasing prospects in the biomedical field due to its excellent physicochemical properties. Understanding the health and environmental impact of GO is of great importance for guiding future applications. Although their interactions with living organisms are omnipresent, the exact molecular mechanism is yet to be established. The cellular membrane is the first barrier for a foreign molecule to interact before entering into the cell. In the present study, a model system consisting of a lipid monolayer at the air-water interface represents one of the leaflets of this membrane. Surface pressure-area isotherms and advanced synchrotron X-ray scattering techniques have been employed to comprehend the interaction by varying the electrostatics of the membrane. The results depict a strong GO interaction with positively charged phospholipids, weak interaction with zwitterionic lipids, and interestingly negligible interaction with negatively charged lipids. GO flakes induce significant changes in the out-of-plane organization of a positively charged lipid monolayer with a minor influence on in-plane assembly of lipid chains. This interaction is packing-specific, and the influence of GO is much stronger at lower surface pressure. Even though for zwitterionic phospholipids, the GO flakes may partly insert into the lipid chains, the X-ray scattering results indicate that the flakes preferentially lie horizontally underneath the positively charged lipid monolayer. This in-depth structural description may pave new perspectives for the scientific community for the development of GO-based biosensors and biomedical materials.

Interaction of cyclotide Kalata B1 protein with model cellular membranes of varied electrostatics.

A uni-molecular layer of lipids at air-water interface mimicking one of the leaflets of the cellular membrane provides a simple model to understand the interaction of any foreign molecules with the membrane. Here, the interactions of protein Kalata B1 (KB1) of cyclotide family with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), and 1,2-distearoyl-sn-glycero-3-ethylphosphocholine chloride salt (DSEPC) have been investigated. The addition of KB1 induces a change in pressure of the lipid monolayers. The characteristic time of the change in pressure is found to be dependent on the electrostatic nature of the lipid. Even though the protein is weakly surface active, it is capable of modifying the phase behavior and elastic properties of lipid monolayers with differences in their strength and nature making the layers more floppy. The KB1-lipid interaction has been quantified by calculating the excess Gibb's free energy of interaction and the 1-anilino-8-naphthalenesulfonate (ANS) binding studies. The interaction with zwitterionic DPPC and negatively charged DPPG lipids are found to be thermodynamically favorable whereas the protein shows a weaker response to positively charged DSEPC lipid. Therefore, the long ranged electrostatic is the initial driving force for the KB1 to recognize and subsequently attach to a cellular membrane. Thereafter, the hydrophobic region of the protein may penetrate into the hydrophobic core of the membrane via specific amino acid residues.

Discerning perturbed assembly of lipids in a model membrane in presence of violacein.

Violacein is a naturally found pigment that is used by some gram negative bacteria to defend themselves from various gram positive bacteria. As a result, this molecule has caught attention for its potential biomedical applications and has already shown promising outcomes as an antiviral, an antibacterial, and an anti-tumor agent. Understanding the interaction of this molecule with a cellular membrane is an essential step to extend its use in the pharmaceutical paradigm. Here, the interaction of violacein with a lipid monolayer formed at the air-water interface is found to depend on electrostatic nature of lipids. In presence of violacein, the two dimensional (2D) pressure-area isotherms of lipids have exhibited changes in their phase transition pressure and in-plane elasticity. To gain insights into the out-of-plane structural organization of lipids in a membrane, X-ray reflectivity (XRR) study on a solid supported lipid monolayer on a hydrophilic substrate has been performed. It has revealed that the increase in membrane thickness is more pronounced in the zwitterionic and positively charged lipids compared to the negatively charged one. Further, the lipid molecules are observed to decrease their tilt angle made with the normal of lipid membrane along with an alteration in their in-plane ordering. This has been quantified by grazing incidence X-ray diffraction (GIXD) experiments on the multilayer membrane formed in an environment with controlled humidity. The structural reorganization of lipid molecules in presence of violacein can be utilized to provide a detailed mechanism of the interaction of this molecule with cellular membrane.

Ionic Liquid-Induced Phase-Separated Domains in Lipid Multilayers Probed by X-ray Scattering Studies.

A cellular membrane, primarily a lipid bilayer, surrounds the internal components of a biological cell from the external components. This self-assembled bilayer is known to be perturbed by ionic liquids (ILs) causing malfunctioning of a cellular organism. In the present study, surface-sensitive X-ray scattering techniques have been employed to understand this structural perturbation in a lipid multilayer system formed by a zwitterionic phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The ammonium and phosphonium-based ILs with methanesulfonate anions are observed to induce phase-separated domains in the plane of a bilayer. The lamellar X-ray diffraction peaks suggest these domains to correlate across the bilayers in a smectic liquid crystalline phase. This induced IL-rich lamellar phase has a very low lamellar repeat distance, suggesting the formation of an interdigitated bilayer. The IL-poor phase closely related to the pristine lipid phase shows a decrement in the in-plane chain lattice parameters with a reduced tilt angle. The ammonium and phosphonium-based ILs with a relatively bulky anion, p-toluenemethanesulfonate, have shown a similar effect.

Enhanced Microscopic Dynamics of a Liver Lipid Membrane in the Presence of an Ionic Liquid.

Ionic liquids (ILs) are an important class of emerging compounds, owing to their widespread industrial applications in high-performance lubricants for food and cellulose processing, despite their toxicity to living organisms. It is believed that this toxicity is related to their actions on the cellular membrane. Hence, it is vital to understand the interaction of ILs with cell membranes. Here, we report on the effects of an imidazolium-based IL, 1-decyl-3-methylimidazolium tetrafluoroborate (DMIM[BF4]), on the microscopic dynamics of a membrane formed by liver extract lipid, using quasielastic neutron scattering (QENS). The presence of significant quasielastic broadening indicates that stochastic molecular motions of the lipids are active in the system. Two distinct molecular motions, (i) lateral motion of the lipid within the membrane leaflet and (ii) localized internal motions of the lipid, are found to contribute to the QENS broadening. While the lateral motion could be described assuming continuous diffusion, the internal motion is explained on the basis of localized translational diffusion. Incorporation of the IL into the liver lipid membrane is found to enhance the membrane dynamics by accelerating both lateral and internal motions of the lipids. This indicates that the IL induces disorder in the membrane and enhances the fluidity of lipids. This could be explained on the basis of its location in the lipid membrane. Results are compared with various other additives and we provide an indication of a possible correlation between the effects of guest molecules on the dynamics of the membrane and its location within the membrane.

Relating the physical properties of aqueous solutions of ionic liquids with their chemical structures.

The physical properties of an aqueous solution of a macromolecule primarily depend on its chemical structure and the mesoscopic aggregates formed by many of such molecules. Ionic liquids (ILs) are the macromolecules that have caught significant research interests for their enormous industrial and biomedical applications. In the present paper, the physical properties, such as density, viscosity, ionic conductivity of aqueous solutions of various ILs, have been investigated. These properties are found to systematically depend on the shape and size of the anion and the cation along with the solution concentration. The ionic conductivity and viscosity behavior of the solutions do not strictly follow the Walden rule that relates the conductivity to the viscosity of the solution. However, the modified Walden rule could explain the behavior. A simple calculation based on the geometry of a given molecule could shed the light on the observed results.

One-Dimensional Anomalous Diffusion of Gold Nanoparticles in a Polymer Melt.

We investigated the dynamics of polymer-grafted gold nanoparticles loaded into polymer melts using x-ray photon correlation spectroscopy. For low molecular weight host matrix polymer chains, normal isotropic diffusion of the gold nanoparticles is observed. For larger molecular weights, anomalous diffusion of the nanoparticles is observed that can be described by ballistic motion and generalized Lévy walks, similar to those often used to discuss the dynamics of jammed systems. Under certain annealing conditions, the diffusion is one-dimensional and related to the direction of heat flow during annealing and is associated with an dynamic alignment of the host polymer chains. Molecular dynamics simulations of a single gold nanoparticle diffusing in a partially aligned polymer network semiquantitatively reproduce the experimental results to a remarkable degree. The results help to showcase how nanoparticles can under certain circumstances move rapidly in polymer networks.

Investigation of the buried planar interfaces in multi-layered inverted organic solar cells using x-ray reflectivity and impedance spectroscopy.

The hole and electron extracting interlayers in the organic solar cells (OSCs) play an important role in high performing devices. The present work focuses on an investigation of Zinc oxide/bulk heterojunction (ZnO/BHJ) and BHJ/MoO x (Molybdenum oxide) buried planar interfaces in inverted OSC devices using the optical contrast in various layers along with the electrical measurements. The x-ray reflectivity (XRR) analysis demonstrates the formation of additional intermixing layers at the interfaces of ZnO/BHJ and BHJ/MoO x . Our results indicate infusion of PC71BM into ZnO layer up to ~4 nm which smoothen the ZnO/BHJ interface. In contrast, thermally evaporated MoO x molecules diffuse into PTB7-Th dominant upper layers of BHJ active layer resulting in an intermixed layer at the interface of MoO x /BHJ. The high recombination resistance (~5 kΩ cm2) and electron lifetime (~70 µs), obtained from the impedance spectroscopy (IS), support such vertical segregation of PTB7-Th and PC71BM in the active layer. The OSC devices, processed in ambient condition, exhibit high power conversion efficiency of 6.4%. We consider our results have great significance to understand the structure of buried planar interfaces at interlayers and their correlation with the electrical parameters representing various interfacial mechanisms of OSCs.

Discovery and cellular stress pathway analysis of 1,4-naphthoquinone derivatives with novel, highly potent broad-spectrum anticancer activity.

BACKGROUND: Chemotherapy and targeted therapies have made important strides in cancer treatment yet they often fail and new therapies are still needed. Here, we employed a phenotypic screen to identify and analyze the mechanism of action of novel small molecules that interfere with critical pathways involved in tumor cell growth, using chemoresistant A375 melanoma cells as a model. METHODS: Cell culture studies were performed in ATCC-recommended media. Compounds, and compound libraries were obtained from Boston University or purchased commercially. Effects on A375 cell viability, proliferation and morphology were determined by Celigo Image Cytometer and viability staining. Anticancer activity of the lead compound was tested in a xenograft nude mouse model. Signaling and cell death pathways were analyzed by SDS-PAGE and immunoblotting, and/or fluorescence microscopy. RESULTS: After evaluating 4477 compounds, one hit compound CB533 was identified that caused significant reduction of A375 cell growth. CB533 is an unexplored 1,4-naphthoquinone (NQ) derivative which unlike 1,4-NQ, induced rapid cell death without generating reactive oxygen species (ROS). Structure-activity relationship analysis showed that a pyrrolidine in the 1,4-NQ nucleus in lead compound Pyr-1 yielded optimal activity. CB533 and Pyr-1 had growth-suppressing effects on a large variety of chemotherapy-resistant cancer cell lines in the nano to picomolar range. Pyr-1 also significantly reduced growth of MDA-MB-231 breast cancer cells in nude mice. Pyr-1 rapidly induced activation of major stress pathways and autophagy, which was efficiently blocked by ERK, and somewhat by PI3K inhibitors. CONCLUSION: CB533 and lead Pyr-1 represent novel broad-spectrum, anticancer compounds that are up to 1000-fold more potent than plumbagin, a natural 1,4-NQ with known anticancer activity. Since the growth suppression activities of CB533 and Pyr-1 are unaffected by the chemotherapy resistance of cancer cells, these compounds have promising therapeutic potential. The pyrrolidine in the 3 position of the 1,4-NQ nucleus of Pyr-1 is a critical component of the pharmacophore. Pyr-1-induced cellular stress was mediated by an ERK, and to a lesser extent by an AKT-dependent pathway without involving apoptosis. Our data suggest that Pyr-1 derives its greatly enhanced antitumor activity via mimicking ROS-induced stress signaling without generating ROS, and likely committing cells to autophagy.