Transport of Alzheimer's associated amyloid-ß catalyzed by P-glycoprotein.

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-ß (Aß) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aß. We observed transport of Aß40 and Aß42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aß42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aß42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

Optimizing Targeted Inhibitors of P-Glycoprotein Using Computational and Structure-Guided Approaches.

Overexpression of ABC transporters like P-glycoprotein (P-gp) has been correlated with resistances in cancer chemotherapy. Intensive efforts to identify P-gp inhibitors for use in combination therapy have not led to clinically approved inhibitors to date. Here, we describe computational approaches combined with structure-based design to improve the characteristics of a P-gp inhibitor previously identified by us. This hit compound represents a novel class of P-gp inhibitors that specifically targets and inhibits P-gp ATP hydrolysis while not being transported by the pump. We describe here a new program for virtual chemical synthesis and computational assessment, ChemGen, to produce hit compound variants with improved binding characteristics. The chemical syntheses of several variants, efficacy in reversing multidrug resistance in cell culture, and biochemical assessment of the inhibition mechanism are described. The usefulness of the computational predictions of binding characteristics of the inhibitor variants is discussed and compared to more traditional structure-based approaches.

Prolonged inhibition of P-glycoprotein after exposure to chemotherapeutics increases cell mortality in multidrug resistant cultured cancer cells.

One common reason for cancer chemotherapy failure is increased drug efflux catalyzed by membrane transporters with broad pump substrate specificities, which leads to resistances to a wide range of chemically unrelated drugs. This multidrug resistance (MDR) phenomenon results in failed therapies and poor patient prognoses. A common cause of MDR is over-expression of the P-glycoprotein (ABCB1/P-gp) transporter. We report here on an MDR modulator that is a small molecule inhibitor of P-glycoprotein, but is not a pump substrate for P-gp and we show for the first time that extended exposure of an MDR prostate cancer cell line to the inhibitor following treatment with chemotherapeutics and inhibitor resulted in trapping of the chemotherapeutics within the cancerous cells. This trapping led to decreased cell viability, survival, and motility, and increased indicators of apoptosis in the cancerous cells. In contrast, extended exposure of non-Pgp-overexpressing cells to the inhibitor during and after similar chemotherapy treatments did not lead to decreased cell viability and survival, indicating that toxicity of the chemotherapeutic was not increased by the inhibitor. Increases in efficacy in treating MDR cancer cells without increasing toxicity to normal cells by such extended inhibitor treatment might translate to increased clinical efficacy of chemotherapies if suitable inhibitors can be developed.

Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced mortality of multidrug resistant tumor cells.

Overexpression of ATP-binding cassette (ABC) transporters is often linked to multidrug resistance (MDR) in cancer chemotherapies. P-glycoprotein (P-gp) is one of the best studied drug transporters associated with MDR. There are currently no approved drugs available for clinical use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein. Using computational studies, we previously identified several compounds that inhibit P-gp by targeting its nucleotide binding domain and avoiding its drug binding domains. Several of these compounds showed successful MDR reversal when tested on a drug resistant prostate cancer cell line. Using conventional two-dimensional cell culture of MDR ovarian and prostate cancer cells and three dimensional prostate cancer microtumor spheroids, we demonstrated here that co-administration with chemotherapeutics significantly decreased cell viability and survival as well as cell motility. The P-gp inhibitors were not observed to be toxic on their own. The inhibitors increased cellular retention of chemotherapeutics and reporter compounds known to be transport substrates of P-gp. We also showed that these compounds are not transport substrates of P-gp and that two of the three inhibit P-gp, but not the closely related ABC transporter, ABCG2/BCRP. The results presented suggest that these P-gp inhibitors may be promising leads for future drug development.

Exhaled nitric oxide and vascular endothelial growth factor as predictors of cold symptoms after stress.

OBJECTIVE: Prior research has demonstrated that psychosocial stress is associated with respiratory infections. Immunologic, endocrine, and cardiovascular predictors of such infections have been explored with varying success. We therefore sought to study the unexplored role of airway mucosal immunity factors, nitric oxide (NO) and vascular endothelial growth factor (VEGF). NO is secreted by airway epithelial cells as part of the first line of defense against bacteria, viruses, and fungi. VEGF is expressed by mast cells in respiratory infections and recruits immune cells to infected sites, but in excess lead to vulnerability of the airway epithelium. METHODS: In this proof-of-concept study we measured exhaled NO, exhaled breath condensate (EBC) VEGF, salivary VEGF, and salivary cortisol in 36 students undergoing final academic examinations at three occasions: a low-stress baseline during the term, an early phase of finals, and a late phase of finals. Participants also reported on cold symptoms at these time points and approximately 5 and 10days after their last academic examination. RESULTS: Higher baseline NO was associated with fewer cold symptoms after stress, whereas higher baseline VEGF in EBC and saliva were associated with more cold symptoms after stress. Perceived stress at baseline as well as salivary VEGF and cortisol late in the finals also contributed to the prediction of later cold symptoms. CONCLUSION: Basal levels of NO and VEGF may inform about mucosal immunocompetence and add to preventative treatments against airway infections from periods of stress in daily life.

A novel biomarker associated with distress in humans: calcium-binding protein, spermatid-specific 1 (CABS1).

Calcium-binding protein spermatid-specific 1 (CABS1) is expressed in the human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 in rats. Here, we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Volunteers participated across three studies: 1) weekly baseline measures; 2) a psychosocial speech and mental arithmetic stressor under evaluative threat; and 3) during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; and salivary and nasal inflammatory markers. We identified a CABS1 immunoreactive band at 27 kDa in all participants and additional molecular mass forms in some participants. One week temporal stability of the 27-kDa band was satisfactory (test-retest reliability estimate = 0.62-0.86). Acute stress increased intensity of 18, 27, and 55 kDa bands; 27-kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27-kDa band was also positively associated with VEGF and salivary leukotriene B4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study.

Cationic branched polymers for cellular delivery of negatively charged cargo.

Receptor-independent cellular uptake of small molecule therapeutics is limited by their physical interaction with the negatively charged surface of cellular membranes. Passive diffusion through the hydrophobic membrane bilayer follows this process. Unless specific carriers exist in the biological membrane, such interactions limit therapeutics to those that are hydrophobic with modest positive charge at physiological pH. Small negatively charged molecules are therefore not efficient as therapeutics. To enable delivery of such molecules into eukaryotic cells, cationic branched polymers with tetraalkylammonium pendant groups were synthesized by copolymerization of a functional monomer (glycidyl methacrylate) with degradable and non-degradable divinyl crosslinkers in the presence of an efficient chain transfer agent, CBr4, followed by reaction of the multiple pendant epoxide groups and most of the alkyl bromide chain ends with amines. Cationic branched polymers with covalently attached fluorescent labels entered human cancerous and non-cancerous cells. The non-labeled analogues were able to carry anionic cargo (carboxyfluorescein) into the cells, while no uptake was observed in the absence of the cationic carriers. Most of the polymers were not significantly toxic at the concentrations used. This pilot study showed that cellular uptake of anionic small molecules can be promoted even in the absence of natural uptake mechanisms.

Effects of academic exam stress on nasal leukotriene B4 and vascular endothelial growth factor in asthma and health.

OBJECTIVE: To examine the effect of final exam stress on the concentrations of leukotriene B4 (LTB4) and vascular endothelial growth factor (VEGF) in the upper airways among healthy and asthmatic individuals. METHOD: Nasal samples were collected from 12 individuals with asthma and 23 healthy controls early and late in a final exam period, and during a low-stress period in the semester. We determined LTB4 and VEGF concentrations using Enzyme-Linked Immunoassays. RESULTS: Mixed effects analysis of variance models showed that asthmatic participants with allergies in contrast to healthy individuals experienced a decrease in nasal LTB4 during the final exam period as compared to mid-semester (low stress period). There were no significant changes in nasal VEGF across the observation period. Changes in nasal LTB4 and VEGF were not associated with salivary cortisol, exhaled nitric oxide, or spirometric lung function. CONCLUSIONS: Our results suggest that nasal LTB4 concentrations change in periods of psychological stress for asthmatic individuals with allergies.

In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture.

Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716-726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 µmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances.

Multiple Drug Transport Pathways through Human P-Glycoprotein.

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp.

The effect of academic exam stress on mucosal and cellular airway immune markers among healthy and allergic individuals.

Research suggests that psychological stress can exacerbate allergies, but relatively little is known about the effect of stress on mucosal immune processes central to allergic pathophysiology. In this study, we quantified vascular endothelial growth factor (VEGF), interferon gamma (IFN-γ), and interleukin-4 concentrations in saliva (S) and exhaled breath condensate (EBC) during final exams and at midsemester among 23 healthy and 21 allergic rhinitis individuals. IFN-γs decreased during exams for both groups while VEGF(EBC) increased (and increases in VEGFs were a trend). Elevated negative affect ratings predicted higher VEGF(EBC) in allergic individuals. IFN-γ(EBC) increased in healthy individuals early during exams and then decreased, while allergic individuals showed a decrease in IFN-γ(EBC) throughout final exams. These findings suggest that psychological stress can suppress cellular immune function among allergic individuals while increasing VEGF.

Effects of psychosocial stress on the pattern of salivary protein release.

Previous research suggests that acute stress can increase the release of immune-relevant proteins in saliva. However, no attempts have been made to examine a wider range of salivary proteins in response to stress. In this study, we identified and quantified changes in the pattern of salivary protein release in a 45 min time period following the Trier Social Stress Test (TSST) in 12 asthmatic and 13 healthy participants. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The relative protein amounts were quantified using the Image J software (NIH), and identified and characterized using mass spectroscopy. Negative affect was increased immediately after stress in both groups. The results showed that alpha amylase, cystatin S and light chain IgA were increased after the TSST and significant increases in glutathione S-transferase and prolactin inducible protein were also observed. Asthma patients showed responses similar to healthy controls, but had a tendency toward overall lower alpha amylase levels. Our findings suggest that a variety of proteins relevant to mucosal immunity are elevated following acute psychosocial stress, including glutathione S-transferase and prolactin inducible protein, which had not been characterized in this context before.

Nucleotide binding to the human multidrug resistance protein 3, MRP3.

We used a spin-labeled ATP analog, SL-ATP, to study nucleotide binding to highly purified human multidrug resistance protein 3, MRP3, which had been expressed in the yeast Pichia pastoris. SL-ATP was shown to be a good substrate analog and is hydrolyzed by MRP3 at about 10% of the Vmax for normal ATP. ESR titrations showed that 2 mol of SL-ATP readily bound per mole of MRP3 with a dissociation constant of about 100 microM in the presence of Mg(2+) ions. The binding curve was easily fitted for a hyperbolic binding relationship. SL-ATP also bound readily to MRP3 in the absence of divalent ions and presence of EDTA. The resulting binding curve, however, could not be satisfactorily fitted using the equation for hyperbola. Analysis showed that a good fit was only obtained with the Hill equation using a Hill coefficient of 4 or close to 4. Lower Hill coefficients resulted in lower goodness of the fit. Such cooperative binding may be explained by a dimerization event triggered in the absence of divalent ions and a close communication of nucleotide binding sites of the interacting dimers. These findings may be of great importance for the overall mechanism and regulation of multidrug resistance proteins.

Effects of small molecule modulators on ATP binding to skeletal ryanodine receptor.

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca2+-channel and is strongly affected by intrinsic modulators like Ca2+, Mg2+ and ATP. We showed differential effects on ATP binding in the presence of Ca2+ or Mg2+ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408-9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca2+, suggestive of a stable "open" channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca2+, while in the presence of inhibitory Ca2+, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.

Accommodating discontinuities in dimeric left-handed coiled coils in ATP synthase external stalks.

ATP synthases from coupling membranes are complex rotary motors that convert the energy of proton gradients across coupling membranes into the chemical potential of the beta-gamma anhydride bond of ATP. Proton movement within the ring of c subunits localized in the F(0)-sector drives gamma and epsilon rotation within the F(1)alpha(3)beta(3) catalytic core where substrates are bound and products are released. An external stalk composed of homodimeric subunits b(2) in Escherichia coli or heterodimeric bb' in photosynthetic synthases connects F(0) subunit a with F(1) subunits delta and most likely alpha. The external stalk resists rotation, and is of interest both functionally and structurally. Hypotheses that the external stalk contributes to the overall efficiency of the reaction through elastic coupling of rotational substeps, and that stalks form staggered, right-handed coiled coils, are investigated here. We report on different structures that accommodate heptad discontinuities with either local or global underwinding. Analyses of the knob-and-hole packing of the E. coli b(2) and Synechocystis bb' stalks strongly support the possibility that these proteins can adopt conventional left-handed coiled coils.

De-novo modeling and ESR validation of a cyanobacterial F(o)F(1)-ATP synthase subunit bb' left-handed coiled coil.

The structure and functional role of the dimeric external stalk of F(o)F(1)-ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb' dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b' subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb'-dimer exists mostly as an elongated alpha-helix. The distant C-terminal end of the dimer, which is thought to interact with the delta-subunit, seemed to be disordered in experiments using soluble bb' proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb' structure exists as a left-handed coiled coil.

Structure of the cytosolic part of the subunit b-dimer of Escherichia coli F0F1-ATP synthase.

The structure of the external stalk and its function in the catalytic mechanism of the F(0)F(1)-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F(1) or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F(1) sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b(2) from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.

Subunit b-dimer of the Escherichia coli ATP synthase can form left-handed coiled-coils.

One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures.

Conformational changes in the Escherichia coli ATP synthase b-dimer upon binding to F(1)-ATPase.

Conformational changes within the subunit b-dimer of the E. coli ATP synthase occur upon binding to the F(1) sector. ESR spectra of spin-labeled b at room temperature indicated a pivotal point in the b-structure at residue 62. Spectra of frozen b +/- F(1) and calculated interspin distances suggested that where contact between b (2) and F(1) occurs (above about residue 80), the structure of the dimer changes minimally. Between b-residues 33 and 64 inter-subunit distances in the F(1)-bound b-dimer were found to be too large to accommodate tightly coiled coil packing and therefore suggest a dissociation and disengagement of the dimer upon F(1)-binding. Mechanistic implications of this "bubble" formation in the tether domain of ATP synthase b ( 2 ) are discussed.