Blocking Peptides Research Articles

In vitro digestive stability of complexes between gliadin and synthetic blocking peptides

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions.

Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes

Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.

Development of a competition ELISA to assess stability and measure ShK‐186 in plasma samples

ShK-186, a potent (IC50 70 pM) and selective peptide blocker of Kv1.3, is scheduled to begin human phase 1 trials in 2011 as a therapeutic for autoimmune disease. Kv1.3 selectivity is due to a negatively charged phosphotyrosine (pTyr) at the N-terminus. Here, we describe the development of a competition ELISA (cELISA) to measure ShK-186 concentrations in plasma samples. Six monoclonal antibodies (mAbs) were raised and specificities were determined against a panel of ShK analogs. Four mAbs reacted with the pTyr moiety (e.g. P2F12), and two reacted only to the peptide-body. A cELISA was developed with P2F12 to detect the intact fully phosphorylated form. Standard curves generated with varying concentrations of ShK-186 in plasma were reproducible (n=9), and QC samples were measured within 9% precision. Alpha-1 acid glycoprotein, a plasma protein known to bind cationic peptides, sequestered ShK-186 in a time-dependent manner. High salt reversed this effect, suggesting a charge-charge interaction. Dephosphorylation, detected by loss of P2F12 reactivity and confirmed by LC-MS, was evident in 1 hour in rat and human plasma, and was anti-coagulant dependent. In patch-clamp experiments, the dephosphorylated peptide (ShK-198) blocked Kv1.3 (IC50 4.3 pM) and Kv1.1 (IC50 3 pM) with greater potency but lower selectivity than ShK-186. PK studies in rats are underway. Supported by NIH (RO1NS48252) and SBIR (1R43A1085691-01)

Comparative analysis of inactivated-state block of N-type (Cav2.2) calcium channels

The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays. The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology. N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable. The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.

Pulmonary Surfactant Protein A Enhances Endolysosomal Trafficking in Alveolar Macrophages through Regulation of Rab7

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.

Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

BackgroundThe characterization and cellular localization of transcription factors like NF-κB requires the use of antibodies for western blots and immunohistochemistry. However, if target protein levels are low and the antibodies not well characterized, false positive data can result. In studies of NF-κB activity in the CNS, antibodies detecting NF-κB proteins have been used to support the finding that NF-κB is constitutively active in neurons, and activity levels are further increased by neurotoxic treatments, glutamate stimulation, or elevated synaptic activity. The specificity of the antibodies used was analyzed in this study.MethodsSelectivity and nonselectivity of commonly used commercial and non-commercial p50 and p65 antibodies were demonstrated in western blot assays conducted in tissues from mutant gene knockout mice lacking the target proteins.ResultsA few antibodies for p50 and p65 each mark a single band at the appropriate molecular weight in gels containing proteins from wildtype tissue, and this band is absent in proteins from knockout tissues. Several antibodies mark proteins that are present in knockout tissues, indicating that they are nonspecific. These include antibodies raised against the peptide sequence containing the nuclear localization signals of p65 (MAB3026; Chemicon) and p50 (sc-114; Santa Cruz). Some antibodies that recognize target proteins at the correct molecular weight still fail in western blot analysis because they also mark additional proteins and inconsistently so. We show that the criterion for validation by use of blocking peptides can still fail the test of specificity, as demonstrated for several antibodies raised against p65 phosphorylated at serine 276. Finally, even antibodies that show specificity in western blots produce nonspecific neuronal staining by immunohistochemistry.ConclusionsWe note that many of the findings in the literature about neuronal NF-κB are based on data garnered with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes

Nucleotides and nucleosides play important roles by maintaining brain homeostasis, and their extracellular concentrations are mainly regulated by ectonucleotidases and nucleoside transporters expressed by astrocytes. Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, of which the molecular mechanism has not been fully elucidated. Recently, exogenous NAD(+) was reported to enter astrocytes via the P2X7 receptor (P2X7R)-associated channel/pore. In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), showed time- and temperature-dependency, and was significantly enhanced on addition of apyrase, and was reduced by 8-Br-cADPR and ARL67156, inhibitors of CD38 and ectoapyrase, respectively, and P2X7R knockdown, suggesting that the detected uptake of [(14)C]NAD(+) resulted from [(14)C]adenosine acting as a metabolite of [(14)C]NAD(+). Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN-62, oxATP, and siRNA transfection resulted in a decrease of [(3)H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker (10)panx. Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.

A Small Synthetic Cripto Blocking Peptide Improves Neural Induction, Dopaminergic Differentiation, and Functional Integration of Mouse Embryonic Stem Cells in a Rat Model of Parkinson's Disease

Cripto is a glycosylphosphatidylinositol-anchored coreceptor that binds Nodal and the activin type I (ALK)-4 receptor, and is involved in cardiac differentiation of mouse embryonic stem cells (mESCs). Interestingly, genetic ablation of cripto results in increased neuralization and midbrain dopaminergic (DA) differentiation of mESCs, as well as improved DA cell replacement therapy (CRT) in a model of Parkinson's disease (PD). In this study, we developed a Cripto specific blocking tool that would mimic the deletion of cripto, but could be easily applied to embryonic stem cell (ESC) lines without the need of genetic manipulation. We thus screened a combinatorial peptide library and identified a tetrameric tripeptide, Cripto blocking peptide (BP), which prevents Cripto/ALK-4 receptor interaction and interferes with Cripto signaling. Cripto BP treatment favored neuroectoderm formation and promoted midbrain DA neuron differentiation of mESCs in vitro and in vivo. Remarkably, Cripto BP-treated ESCs, when transplanted into the striatum of PD rats, enhanced functional recovery and reduced tumor formation, mimicking the effect of genetic ablation of cripto. We therefore suggest that specific blockers such as Cripto BP may be used to improve the differentiation of ESC-derived DA neurons in vitro and their engraftment in vivo, bringing us closer towards an application of ESCs in CRT.

Integrin αvβ5 is a primary receptor for adenovirus in CAR-negative cells

BackgroundViruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection.ResultsIn this study we report that cells which express little to no CAR can be efficiently transduced by adenovirus. Using competition experiments between whole virus and soluble viral fiber protein or integrin blocking peptides, we show virus binding is not dependent on fiber binding to cells but rather on penton base binding cellular integrins. Further, we find that binding to low CAR expressing cells is inhibited specifically by a blocking antibody to integrin αvβ5, demonstrating that in these cells integrin αvβ5 and not CAR is required for adenovirus attachment. The binding mediated by integrin αvβ5 is extremely high affinity, in the picomolar range.ConclusionsOur data further challenges the model of adenovirus infection in which binding to primary receptor CAR is required in order for subsequent interactions between adenovirus and integrins to initiate viral entry. In low CAR cells, binding occurs through integrin αvβ5, a receptor previously thought to be used exclusively in internalization. We show for the first time that integrin αvβ5 can be used as an alternate binding receptor.

Control of transient, resurgent, and persistent current by open-channel block by Na channel β4 in cultured cerebellar granule neurons

Voltage-gated Na channels in several classes of neurons, including cells of the cerebellum, are subject to an open-channel block and unblock by an endogenous protein. The Na(V)beta4 (Scn4b) subunit is a candidate blocking protein because a free peptide from its cytoplasmic tail, the beta4 peptide, can block open Na channels and induce resurgent current as channels unblock upon repolarization. In heterologous expression systems, however, Na(V)beta4 fails to produce resurgent current. We therefore tested the necessity of this subunit in generating resurgent current, as well as its influence on Na channel gating and action potential firing, by studying cultured cerebellar granule neurons treated with siRNA targeted against Scn4b. Knockdown of Scn4b, confirmed with quantitative RT-PCR, led to five electrophysiological phenotypes: a loss of resurgent current, a reduction of persistent current, a hyperpolarized half-inactivation voltage of transient current, a higher rheobase, and a decrease in repetitive firing. All disruptions of Na currents and firing were rescued by the beta4 peptide. The simplest interpretation is that Na(V)beta4 itself blocks Na channels of granule cells, making this subunit the first blocking protein that is responsible for resurgent current. The results also demonstrate that a known open-channel blocking peptide not only permits a rapid recovery from nonconducting states upon repolarization from positive voltages but also increases Na channel availability at negative potentials by antagonizing fast inactivation. Thus, Na(V)beta4 expression determines multiple aspects of Na channel gating, thereby regulating excitability in cultured cerebellar granule cells.

Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels

More than a hundred conotoxins are known today and from them, only seven conopeptides have been identified to target voltage-gated potassium channels (Kv). Conotoxin sr11a belongs to the I(2)-superfamily which is characterized by four disulfide bridges and provokes muscle stiffness when injected intracranially in mice. The aim of this work was to test the biological activity of sr11a on recombinant voltage-gated Kv1 potassium channels expressed in Xenopus laevis oocytes. Peptide sr11a was purified by high-performance liquid chromatography from the venom of the vermivorous Conus spurius. We found that peptide sr11a inhibits the delayed rectifiers Kv1.2 and Kv1.6 but had not effect on the slowly inactivating Kv1.3 channel. The functional dyad composed of a basic Lys and a hydrophobic amino acid residue is a crucial structural element, regarding the binding properties and blocking activities of more than a hundred K(+) channel toxins. Peptide sr11a does not contain Lys residues and then, it lacks the functional dyad. Molecular modeling of peptide sr11a reveals the presence of exposed basic residues of Arg and suggests that Arg17 and Arg29 are important on its biological activity.

Protease-Activated Receptor 2 Blocking Peptide Counteracts Endotoxin-Induced Inflammation and Coagulation and Ameliorates Renal Fibrin Deposition in a Rat Model of Acute Renal Failure

Protease-Activated Receptor 2 Blocking Peptide Counteracts Endotoxin-Induced Inflammation and Coagulation and Ameliorates Renal Fibrin Deposition in a Rat Model of Acute Renal Failure

Design of new high-affinity peptide ligands for human leukocyte antigen-DQ2 using a positional scanning peptide library

Human leukocyte antigen (HLA)-DQ2 (DQA1 × 0501/DQB1 × 0201) is associated with several immune disorders, including celiac disease, which is caused by an inappropriate T-cell response to gluten. Interference with peptide presentation by HLA-DQ2, for example, by the use of peptide blockers, is a possible treatment strategy for such HLA-associated disorders. A successful implementation of this strategy will depend on the identification of ligands that bind much better to HLA-DQ2 than the disease related epitopes. We have used a positional scanning nonapeptide library to determine the optimal amino acids for each position of the HLA-DQ2 binding frame. By combining the optimal residues in each position, we were able to design high affinity binders to HLA-DQ2. Interestingly, the decapeptide with highest affinity was composed of the most favorable residues in each position. This sequence bound 50-fold better than the immunodominant gluten epitope DQ2-α-I-gliadin, which makes it an interesting lead compound for the development of blockers. For some natural HLA-DQ2 ligands, the correlation between measured and predicted affinities was poorer, but notably these peptides did not have optimal amino acids at all positions. Our approach represents a straightforward strategy for developing high-affinity binders to HLA class II molecules.

Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O

An immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine. In the strip, the expressed protein of VP1, the main protective antigen of FMDV, labeled with colloidal gold was used as the detector, the staphylococcal protein A (SPA) and swine anti-foot-and-mouth disease virus (FMDV) antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. 296 swine serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial liquid-phage blocking ELISA (LPB ELISA) kit and peptide ELISA kit. The strip was shown to be of high specificity and sensitivity. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents nor equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.

Functional Reconstitution of a GIRK1-Chimera and its Regulation by the βγ Subunits of G Proteins

The molecular details of ion channel regulation by G proteins remain unknown. A first step in this direction is to define the characteristics of interacting proteins of known structure in isolation and in complex form. The three-dimensional structure of a GIRK1-chimera determined by single particle electron microscopy at 25A is consistent with the crystal structure (Nishida et al., 2007, EMBO J 26:4005-15). We have functionally reconstituted this GIRK1-chimera into a 1:1 ratio of phosphatidylethanolamine to phosphatidylserine planar lipid bilayers. The GIRK1-chimera produces a conductance of approximately 23 pS that shows Mg2+-dependent inwardly rectifying K+ currents and an absolute requirement on the presence of phosphatidylinositol-4,5-bisphosphate for activation. These currents are blocked by PIP2 antibody and poly-lysine applied from the cis but not the trans side. Moreover, the channel shows a high affinity for diC8-PIP2 (EC50 ∼ 7.5 μM). GIRK1-chimera channel currents are blocked by Ba2+ and the GIRK peptide blocker tertiapin when applied from the trans but not the cis side. Interestingly, Gβγ applied from the cis side inhibits GIRK1-chimera currents and shifts phosphoinositide sensitivity by decreasing the apparent affinity to PIP2. This is in contrast to the Gβγ effects on full-length GIRK1∗ channels assayed in Xenopus oocytes or planar lipid bilayers.

Pan-Aurora Kinase Inhibitor Mk-0457 Synergistically Potentiates Apo2L/Trail Cytotoxicity in Multiple Mieloma Cells Sensitive and Resistant to Bortezomib.

Abstract 1837Poster Board I-863Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. Aurora kinase inhibitors have been shown to abrogate proliferation and induce apoptosis in human myeloma cells lines (HMCLs) and primary myeloma cells. In addition previous studies have shown the antimyeloma activity of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) as a single agent or in combination with certain chemotherapeutic agents. The aim of this study was to investigate whether the combined treatment with pan-Aurora kinase inhibitor MK-0457 also known as VX-680(Vertex/Merck) and Apo2L/TRAIL has cytotoxic effects on MM cells. Because we found that MK-0457 (0.2–0.5 μM) partially activated the extrinsic caspase-8 mediated pathway both in HMCLs RPMI 8226 (highly sensitive to Apo2L/TRAIL: median lethal dose (LD50) at 48 hours was 4.9 ng/mL), and bortezomib-resistant 8226/R5 (barely sensitive to Apo2L/TRAIL: LD50 at 48 hours was 90.9 ng/mL) we attempted to examine whether MK-0457 potentiates the Apo2L/TRAIL-mediated apoptosis in HMCLs that have differential sensitivity to Apo2L/TRAIL. We first analyzed the pharmacologic interactions between MK-0457 and Apo2L/TRAIL using a fixed-ratio experimental design and found that the combined treatment resulted in the synergistic induction of apoptosis in both HMCLs (Chou-Talalay method): after 48 hours of treatment the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in MK-0457 plus Apo2L/TRAIL were 0.04 ± 0.05 and 0.03 ± 0.02 in RPMI 8226 and 8226/R5 respectively. Consistent with these results, we found that Aurora-A and -B were expressed at similar levels in RPMI 8226 and 8226/R5 cells and we demonstrated that the functional knock-out of Aurora-A or -B gene expression by small interfering (si)RNAs significantly increased (P< .001 Dunnett test) the TRAIL- induced apoptosis in both HMCLs. To investigate the molecular mechanisms by which MK-0457/Apo2L/TRAIL induced MM cell apoptosis first we compared the effect on caspase activation in RPMI 8226 and 8226/R5. The cells were treated with MK-0457 (0.2–0.5 μM) and/or Apo2L/TRAIL (2.4 ng/mL and 9.6 ng/mL in RPMI 8226 and 8226/R5 respectively) for 24 hours and caspase activation and PARP fragmentation were analyzed by Western blotting: the treatment with MK-0457 strongly potentiated the Apo2L/TRAIL -induced activation of caspase-8, caspase-3 and PARP cleavage in both HMCLs. In addition, a strong activation of caspase-9 was observed in the MK-0457/Apo2L/TRAIL-treated 8226/R5. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in MK-0457/Apo2L/TRAIL -induced apoptosis in RPMI 8226 and 8226/R5: the inhibition of caspase-8 significantly (P< .001 Dunnett test) reduced the MK-0457/Apo2L/TRAIL -induced apoptosis in both cell lines. Moreover, the pancaspase inhibitor Z-VAD-FMK protected MM cells from MK-0457/Apo2L/TRAIL -induced apoptosis, confirming that caspase activity was indispensable in MK-0457/Apo2L/TRAIL -induced apoptosis. Since antiapoptotic Mcl-1 and proapoptotic Bim play a pivotal role in controlling MM cell survival and apoptosis, and Bim, as previously demonstrated, can interfere with the activation of both intrinsic and extrinsic apoptotic pathways in MM cells, we analyzed their expression in MK-0457/Apo2L/TRAIL treated cells. We found that monotreatment with neither MK-0457 nor Apo2L/TRAIL (or their combination) was able to substantially modulate the expression of Mcl-1 or Bim in RPMI 8226; in contrast in HMCL 8226/R5, that showed low sensitivity to Apo2L/TRAIL, the treatment with Apo2L/TRAIL increased the intracellular amount of the anti-apoptotic protein Mcl-1 and MK-0457 reverted Apo2L/TRAIL-induced up-regulation of Mcl-1, thus correlating with the enhanced cytotoxicity of combined treatment. In conclusion, our data indicate that targeting aurora kinase potentiates the apoptotic effect of Apo2L/TRAIL in MM cells with differential sensitivity to Apo2L/TRAIL through the activation of the extrinsic pathway. More important, MK-0457/Apo2L/TRAIL can induce apoptosis in MM cells displaying resistance to bortezomib. Together, these findings suggest that a strategy combining Aurora kinase inhibitors with Apo2L/TRAIL warrants attention in MM. Disclosures:Lunghi:MERCK sharp and Dohme: Research Funding. Bonati:MERCK sharp and Dohme: Research Funding.

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.

Blocking Peptides Decrease Tissue Transglutaminase Processing of Gliadin in Vitro

Tissue transglutaminase (tTG) plays an important role in celiac disease pathology as it catalyzes deamidation and cross-linking of specific gluten peptides and converts them into potent epitopes recognized by intestinal T-cells. We investigated whether synthetic peptides with high affinity to gliadin could alter tTG activity on gliadin and whole gluten digest. The immobilized substrates were incubated with synthetic peptides identified by the phage display technique and a control peptide with no affinity to gliadin. Transglutaminase activity was measured with time resolved fluorescence. The mean tTG activity, compared to that of the control without the peptides, was reduced by 31, 33, and 36% for three selected gliadin-binding peptides, and 30% for the peptide pool (P < or = 0.001-0.004) when gliadin was the substrate. Finally, substrate specificity experiments suggested that avenin was processed in a manner similar that used for gliadin during in vitro assays with tTG. The results showed that the blocking peptides efficiently reduced tTG processing of gliadin in vitro, and this strategy will be further investigated as an alternative therapy for celiac disease.

New drugs for migraine

After the triptans, a calcitonin gene-related peptide blocker (telcagepant) is the first acute medicine that has been developed primarily for treatment of acute migraine. Otherwise, the new drugs have been developed first for other purposes, like anticonvulsants, antihypertensives and antidepressants used for migraine prophylaxis. For acute attacks, a new way to administer a traditional drug like dihydroergotamine is under way, and documentation of efficacy in migraine has been gained for some commonly used painkillers and anti-inflammatory drugs, and for some herbal extracts. Based on insights into the basic pathophysiological mechanisms of the disorder, some drugs have been developed which seem promising in early phase II studies (NOS inhibitors and 5HT1F-receptor agonists). In the future, development and enhancements of existing medicines must be accompanied by increased efforts to develop truly new migraine drugs based on knowledge of the pathophysiology if one wishes to reduce substantially the great burden migraine poses on patients and society.