Tackling Influenza A virus by M2 ion channel blockers: Latest progress and limitations.

Influenza outbreaks cause pandemics in millions of people. The treatment of influenza remains a challenge due to significant genetic polymorphism in the influenza virus. Also, developing vaccines to protect against seasonal and pandemic influenza infections is constantly impeded. Thus, antibiotics are the only first line of defense against antigenically distinct strains or new subtypes of influenza viruses. Among several anti-influenza targets, the M2 protein of the influenza virus performs several activities. M2 protein is an ion channel that permits proton conductance through the virion envelope and the deacidification of the Golgi apparatus. Both these functions are critical for viral replication. Thus, targeting the M2 protein of the influenza virus is an essential target. Rimantadine and amantadine are two well-known drugs that act on the M2 protein. However, these drugs acquired resistance to influenza and thus are not recommended to treat influenza infections. This review discusses an overview of anti-influenza therapy, M2 ion channel functions, and its working principle. It also discusses the M2 structure and its role, and the change in the structure leads to mutant variants of influenza A virus. We also shed light on the recently identified compounds acting against wild-type and mutated M2 proteins of influenza virus A. These scaffolds could be an alternative to M2 inhibitors and be developed as antibiotics for treating influenza infections.

Specific N-glycans regulate an extracellular adhesion complex during somatosensory dendrite patterning.

N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate an extracellular protein complex involved in the patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans, regulates the activity of the Menorin adhesion complex without obviously affecting the protein stability and localization of its components. AMAN-2 functions cell-autonomously to allow for decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with the correct set of high-mannose/hybrid/paucimannose N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate DMA-1/LRR-TM receptor function, which, together with three other extracellular proteins, forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex, suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.

Differential effects of D-cycloserine and amantadine on motor behavior and D2/3 receptor binding in the nigrostriatal and mesolimbic system of the adult rat.

D-cycloserine (DCS) and amantadine (AMA) act as partial NMDA receptor (R) agonist and antagonist, respectively. In the present study, we compared the effects of DCS and AMA on dopamine D2/3R binding in the brain of adult rats in relation to motor behavior. D2/3R binding was determined with small animal SPECT in baseline and after challenge with DCS (20 mg/kg) or AMA (40 mg/kg) with [123I]IBZM as radioligand. Immediately post-challenge, motor/exploratory behavior was assessed for 30 min in an open field. The regional binding potentials (ratios of the specifically bound compartments to the cerebellar reference region) were computed in baseline and post-challenge. DCS increased D2/3R binding in nucleus accumbens, substantia nigra/ventral tegmental area, thalamus, frontal, motor and parietal cortex as well as anterodorsal and posterior hippocampus, whereas AMA decreased D2/3R binding in nucleus accumbens, caudateputamen and thalamus. After DCS, ambulation and head-shoulder motility were decreased, while sitting was increased compared to vehicle and AMA. Moreover, DCS increased rearing relative to AMA. The regional elevations of D2/3R binding after DCS reflect a reduction of available dopamine throughout the mesolimbocortical system. In contrast, the reductions of D2/3R binding after AMA indicate increased dopamine in nucleus accumbens, caudateputamen and thalamus. Findings imply that, after DCS, nigrostriatal and mesolimbic dopamine levels are directly related to motor/exploratory activity, whereas an inverse relationship may be inferred for AMA.

Environmental enrichment and amantadine confer individual but nonadditive enhancements in motor and spatial learning after controlled cortical impact injury.

Environmental enrichment (EE) and amantadine (AMT) enhance motor and cognitive outcome after experimental traumatic brain injury (TBI). However, there are no data on the effects of combining these two therapies. Hence, the aim of the current study was to combine EE and AMT after TBI to determine if their net effect further enhances motor and cognitive performance. Anesthetized adult male rats received either a cortical impact of moderate severity or sham injury and then were randomly assigned to EE or standard (STD) housing and once daily administration of AMT (20 mg/kg; i.p.) or saline vehicle (VEH, 1 mL/kg; i.p.) beginning 24 h after injury for 19 days. Motor and cognitive function were assessed on post-surgical days 1-5 and 14-19, respectively. Cortical lesion volume was quantified on day 21. There were no statistical differences among the sham groups regardless of therapy, so the data were pooled. EE, AMT, and their combination (EE + AMT) improved beam-balance, but only EE and EE + AMT enhanced beam-walking. All three treatment paradigms improved spatial learning and memory relative to the VEH-treated STD controls (p < 0.05). No differences were revealed between the EE groups, regardless of treatment, but both were better than the AMT-treated STD group on beam-walking and spatial learning (p < 0.05). Both EE groups equally reduced cortical lesion volume relative to the STD-housed AMT and VEH groups (p < 0.05). The results indicate that although beneficial on their own, EE + AMT do not provide additional benefits after TBI. It is important to note that the lack of additive effects using the current treatment and behavioral protocols does not detract from the benefits of each individual therapy. The findings provide insight for future combination studies.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the ß-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the ß-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment. Herein, we introduce a bioorthogonal assembly strategy based on a synthetic host-guest system featuring Cucurbit[7]uril (CB[7]) and adamantylamine (ADA). We demonstrate that highly selective and ultrastable host-guest interaction between CB[7] and ADA provides a noncovalent mechanism for assembling labeling agents, such as fluorophores and DNA, in cells and tissues for bioorthogonal imaging of molecular targets. Additionally, by combining with covalent reaction, we show that this CB[7]-ADA based noncovalent interaction enables simultaneous bioorthogonal labeling and multiplexed imaging in cells as well as tissue sections. Finally, we show that interaction between CB[7] and ADA fulfills the demands of specificity and stability that is required for assembling molecules in the complexities of a living cell. We demonstrate this by sensitive detection of metastatic cancer-associated cell surface protein marker as well as by showing the distribution and dynamics of F-actin in living cells.

Extending the scope of amantadine drug by incorporation of phenolic azo Schiff bases as potent selective inhibitors of carbonic anhydrase II, drug-likeness and binding analysis.

A series of Amantadine-based azo Schiff base dyes 6a-6e have been synthesized and characterized by 1 H NMR and 13 C NMR and evaluated for their in vitro carbonic anhydrase II inhibition activity and antioxidant activity. All of the synthesized showed excellent carbonic inhibition. Compound 6b was found to be the most potent derivative in the series, and the IC50 of 6b was found to be 0.0849 ± 0.00245 µm (standard Acetazolamide IC50  = 0.9975 ± 0.049 µm). The binding interactions of the most active analogs were confirmed through molecular docking studies. Docking studies showed 6b is interacting by making two hydrogen bonds w at His93 and Ser1 residues, respectively. All compounds showed a good drug score and followed Lipinski's rule. In summary, our studies have shown that these amantadine-derived phenolic azo Schiff base derivatives are a new class of carbonic anhydrase II inhibitors.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite's survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite's hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein.

The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.

Interpreting Thermodynamic Profiles of Aminoadamantane Compounds Inhibiting the M2 Proton Channel of Influenza A by Free Energy Calculations.

The development of novel anti-influenza drugs is of great importance because of the capability of influenza viruses to occasionally cross interspecies barriers and to rapidly mutate. One class of anti-influenza agents, aminoadamantanes, including the drugs amantadine and rimantadine now widely abandoned due to virus resistance, bind to and block the pore of the transmembrane domain of the M2 proton channel (M2TM) of influenza A. Here, we present one of the still rare studies that interprets thermodynamic profiles from isothermal titration calorimetry (ITC) experiments in terms of individual energy contributions to binding, calculated by the computationally inexpensive implicit solvent/implicit membrane molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach, for aminoadamantane compounds binding to M2TM of the avian "Weybridge" strain. For all eight pairs of aminoadamantane compounds considered, the trend of the predicted relative binding free energies and their individual components, effective binding energies and changes in the configurational entropy, agrees with experimental measures (ΔΔG, ΔΔH, TΔΔS) in 88, 88, and 50% of the cases. In addition, information yielded by the MM-PBSA approach about determinants of binding goes beyond that available in component quantities (ΔH, ΔS) from ITC measurements. We demonstrate how one can make use of such information to link thermodynamic profiles from ITC with structural causes on the ligand side and, ultimately, to guide decision making in lead optimization in a prospective manner, which results in an aminoadamantane derivative with improved binding affinity against M2TM(Weybridge).

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 µM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity.

Why bound amantadine fails to inhibit proton conductance according to simulations of the drug-resistant influenza A M2 (S31N).

The mechanisms responsible for drug resistance in the Asn31 variant of the M2 protein of influenza A are not well understood. Molecular dynamics simulations were performed on wild-type (Ser31) and S31N influenza A M2 in the homotetramer configuration. After evaluation of 13 published M2 structures, a solid-state NMR structure with amantadine bound was selected for simulations, an S31N mutant structure was developed and equilibrated, and the native and mutant structures were used to determine the binding behavior of amantadine and the dynamics of water in the two channels. Amantadine is stable in the plugging region of wild-type M2, with the adamantane in contact with the Val27 side chains, while amantadine in S31N M2 has more variable movement and orientation, and spontaneously moves lower into the central cavity of the channel. Free energy profiles from umbrella sampling support this observation. In this configuration, water surrounds the drug and can easily transport protons past it, so the drug binds without blocking proton transport in the S31N M2 channel.

Novel benzopolycyclic amines with NMDA receptor antagonist activity.

A new series of benzopolycyclic amines active as NMDA receptor antagonists were synthesized. Most of them exhibited increased activity compared with related analogues previously published. All the tested compounds were more potent than clinically approved amantadine and one of them displayed a lower IC50 value than memantine, an anti-Alzheimer's approved drug.

Three-dimensional structure and interaction studies of hepatitis C virus p7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine by solution nuclear magnetic resonance.

Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug-protein and protein-protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.

Host-guest interaction of adamantine with a ß-cyclodextrin-functionalized AuPd bimetallic nanoprobe for ultrasensitive electrochemical immunoassay of small molecules.

A modular labeling strategy was presented for electrochemical immunoassay via supramolecular host-guest interaction between ß-cyclodextrin (ß-CD) and adamantine (ADA). An ADA-labeled antibody (ADA-Ab) was synthesized via amidation, and the number of ADA moieties loaded on a single antibody was calculated to be ~7. The ß-CD-functionalized gold-palladium bimetallic nanoparticles (AuPd-CD) were synthesized in aqueous solution via metal-S chemistry and characterized with transmission electron microscopy and X-ray photoelectron spectra. After the ADA-Ab was bound to the antigen-modified electrode surface with a competitive immunoreaction, AuPd-CD as a signal tag was immobilized onto the immunosensor by a host-guest interaction, leading to a large loading of AuPd nanoparticles. The highly efficient electrocatalysis by AuPd nanoparticles for NaBH4 oxidation produced an ultrasensitive response to chloramphenicol as a model of a small molecule antigen. The immunoassay method showed a wide linear range from 50 pg/mL to 50 µg/mL and a detection limit of 4.6 pg/mL. The specific recognition of antigen by antibody resulted in good selectivity for the proposed method. The host-guest interaction strategy provided a universal labeling approach for the ultrasensitive detection of small molecule targets.

Key binding interactions for memantine in the NMDA receptor.

Memantine (Namenda) is prescribed as a treatment for moderate to severe Alzheimer's Disease. Memantine functions by blocking the NMDA receptor, but the key binding interactions between drug and receptor are not fully elucidated. To determine key binding interactions of memantine, we made side-by-side comparisons of IC(50) for memantine and amantadine, a structurally related drug, in the GluN1/GluN2B NMDA receptor. We identified hydrophobic binding pockets for the two methyl groups on memantine formed by the residues A645 and A644 on the third transmembrane helices of GluN1 and GluN2B, respectively. Moreover, we found that while adding two methyl groups to amantadine to produce memantine greatly improves affinity, adding a third methyl group to produce the symmetrical trimethylamantadine diminished affinity. Our results provide a better understanding of chemical-scale interactions between memantine and the NMDA channel, which will potentially benefit the development of new drugs for neurodegenerative diseases involving NMDA receptors.

Regulation of amantadine hydrochloride binding with IIA subdomain of human serum albumin by fatty acid chains.

Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X-ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.

The involvement of a Na⁺- and Cl⁻-dependent transporter in the brain uptake of amantadine and rimantadine.

Despite their structural similarity, the two anti-influenza adamantane compounds amantadine (AMA) and rimantadine (RIM) exhibit strikingly different rates of blood-brain barrier (BBB) transport. However, the molecular mechanisms facilitating the higher rate of in situ BBB transport of RIM, relative to AMA, remain unclear. The aim of this study, therefore, was to determine whether differences in the extent of brain uptake between these two adamantanes also occurred in vivo, and elucidate the potential carrier protein facilitating their BBB transport using immortalized human brain endothelial cells (hCMEC/D3). Following oral administration to Swiss Outbred mice, RIM exhibited 2.4-3.0-fold higher brain-to-plasma exposure compared to AMA, which was not attributable to differences in the degree of plasma protein binding. At concentrations representative of those obtained in vivo, the hCMEC/D3 cell uptake of RIM was 4.5-15.7-fold higher than that of AMA, with Michaelis-Menten constants 6.3 and 238.4 µM, respectively. The hCMEC/D3 cellular uptake of both AMA and RIM was inhibited by various cationic transporter inhibitors (cimetidine, choline, quinine, and tetraethylammonium) and was dependent on extracellular pH, membrane depolarization and Na⁺ and Cl⁻ ions. Such findings indicated the involvement of the neutral and cationic amino acid transporter B°,⁺ (ATB°,⁺) in the uptake of AMA and RIM, which was demonstrated to be expressed (at the protein level) in the hCMEC/D3 cells. Indeed, AMA and RIM appeared to interact with this transporter, as shown by a 53-70% reduction in the hCMEC/D3 uptake of the specific ATB°,⁺ substrate ³H-glycine in their presence. These studies suggest the involvement of ATB°,⁺ in the disposition of these cationic drugs across the BBB, a transporter with the potential to be exploited for targeted drug delivery to the brain.

Confocal laser scanning microscopy (CLSM) based evidence for cell permeation by mono-4-(N-6-deoxy-6-amino-ß-cyclodextrin)-7-nitrobenzofuran (NBD-ß-CyD).

Beta-cyclodextrin (ß-CyD), amantadine and glucose were fluorescently tagged with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD chloride) to afford NBD-ß-CyD, NBD-amantadine and NBD-glucose, respectively. NBD-ß-CyD/amantadine and ß-CyD/NBD-amantadine inclusion complexes were prepared. Fluorescence emission maxima (λ(max) 544nm) and relative fluorescence intensities for NBD-ß-CyD and NBD-ß-CyD/amantadine were virtually identical, precluding the use of emission spectrum shifts for distinguishing free NBD-ß-CyD from the complex. Intracellular accumulation of NBD-ß-CyD was studied in HepG2 and SK-MEL-24 cells using confocal laser scanning microscopy (CLSM). No major differences were observed between uptake of NBD-ß-CyD and NBD-ß-CyD/amantadine. Serum proteins did not perturb uptake, whereas temperature-dependent uptake, indicative of cell entry via diffusion, was observed. Intracellular distribution favoured mitochondria, with less fluorescent material present in cytoplasm and none in cell nuclei. No experimental evidence of NBD-ß-CyD breakdown to NBD-glucose was found upon chromatographic analysis of incubation mixtures, providing additional evidence of intact NBD-ß-CyD entry into these cells. Endocytosis and/or cholesterol-independent membrane modulation are discussed as possible mechanisms for the transmembrane passage of NBD-ß-CyD.