Surface Plasmon Resonance Screening to Identify Active and Selective Adenosine Receptor Binding Fragments.

Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.

The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography.

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca(2)(+)-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca(2+)-S100B (1.5-A resolution) and S100B-Ca(2)(+)-TRTK-12 (2.0-A resolution) determined here indicate that the S100B-Ca(2+)-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca(2+)-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca(2)(+)-p53 peptide complex, TRTK-12 binding to Ca(2+)-S100B was found to increase the protein's Ca(2)(+)-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca(2+) ligands and/or improved the Ca(2+) coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca(2+)-TRTK-12 and S100B-Ca(2+) were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca(2+)-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR (15)N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca(2+)-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca(2+)-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.

Platelet kainate receptor signaling promotes thrombosis by stimulating cyclooxygenase activation.

RATIONALE: Glutamate is a major signaling molecule that binds to glutamate receptors including the ionotropic glutamate receptors; kainate (KA) receptor (KAR), the N-methyl-d-aspartate receptor, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Each is well characterized in the central nervous system, but glutamate has important signaling roles in peripheral tissues as well, including a role in regulating platelet function. OBJECTIVE: Our previous work has demonstrated that glutamate is released by platelets in high concentrations within a developing thrombus and increases platelet activation and thrombosis. We now show that platelets express a functional KAR that drives increased agonist induced platelet activation. METHODS AND RESULTS: KAR induced increase in platelet activation is in part the result of activation of platelet cyclooxygenase in a mitogen-activated protein kinase-dependent manner. Platelets derived from KAR subunit knockout mice (GluR6(-/-)) are resistant to KA effects and have a prolonged time to thrombosis in vivo. Importantly, we have also identified polymorphisms in KAR subunits that are associated with phenotypic changes in platelet function in a large group of whites and blacks. CONCLUSIONS: Our data demonstrate that glutamate regulation of platelet activation is in part cyclooxygenase-dependent and suggest that the KAR is a novel antithrombotic target.

Sodium cholate aggregation and chiral recognition of the probe molecule (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate (BNDHP) observed by 1H and 31P NMR spectroscopy.

Bile salt micelles can be employed as a pseudostationary phase in micellar electrokinetic capillary chromatography (MEKC) separations of chiral analytes. To improve MEKC separations of chiral analytes, a molecular level understanding of micelle aggregation in the presence of analyte is needed. Here, aggregation of sodium cholate has been observed by exploiting the presence of a model analyte molecule. The 31P and 1H nuclear magnetic resonance spectroscopy (NMR) chemical shifts of (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate ((R,S)-BNDHP), a model analyte in chiral MEKC separations, are demonstrated to be very sensitive to the aggregation state of the bile salt sodium cholate. In addition to probing micellar aggregation, the NMR spectral resolution of enantiomeric species is also stronglycorrelated with chiral separations in MEKC. In this work, the aggregation of sodium cholate in basic solutions (pH 12) has been observed over the concentration range 0-100 mM. The primary critical micelle concentration (cmc) was found to be 14 +/- 1 mM for basic solutions of sodium cholate. In addition, a primitive aggregate is clearly observed to form at 7 +/- 1 mM sodium cholate. The data also show pseudo-cmc behavior for secondary aggregation observed in the regime of 50-60 mM cholate. Finally, the H5-H7 edge of BNDHP is shown to be sensitive to chirally selective interactions with primary cholate micelles.

Platelet factor 4 mediates inflammation in experimental cerebral malaria.

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM.

In vivo platelet-endothelial cell interactions in response to major histocompatibility complex alloantibody.

Platelets recruit leukocytes and mediate interactions between leukocytes and endothelial cells. Most studies examining this important platelet immune function have focused on the development of atherosclerosis, but similar mechanisms may contribute to acute and chronic vascular lesions in transplants. Platelets have been described as markers of transplant rejection, but little investigation has critically examined a role for platelets in transplant vasculopathy and, in particular, alloantibody-mediated transplant rejection. We now demonstrate using a skin transplant model that alloantibody indirectly induces platelet activation and rolling in vivo. Repeated IgG2a alloantibody injections result in sustained platelet-endothelial interactions and vascular pathology, including von Willebrand factor release, small platelet thrombi, and complement deposition. Maintenance of continued platelet-endothelial interactions are dependent on complement activation. Furthermore, we demonstrate that platelets recruit leukocytes to sites of alloantibody deposition and sustain leukocyte-endothelial cell interactions in vivo. Taken together, our model demonstrates an important role for platelets in alloantibody induced transplant rejection.

Glutamate mediates platelet activation through the AMPA receptor.

Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target.

Proton NMR assignments for R,S-1,1'-binaphthol (BN) and R,S-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNDHP) interacting with bile salt micelles.

We report proton chemical shifts for two model chiral analytes that are commonly used in the study of micellar electrokinetic capillary chromatography (MEKC), R,S-1,1'-binaphthol (1, BN) and R,S-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (2, BNDHP), in the absence and presence of monomers and micelles of sodium cholate and sodium deoxycholate. The analytes undergo fast exchange in and out of the micelles, which perturbs the analytes' chemical shifts, and which we use to resolve some resonances that are degenerate at both 300 and 600 MHz. Although BN and BNDHP are simple molecules, the proton assignments are only unambiguously established with the aid of the exchange with micelles, an attractive alternative to other methodologies such as the use of paramagnetic shift reagents which may also cause spectral distortions. We rely also upon 2D-NOE spectra of samples in the presence of micelles to perform these assignments. Recently published assignments, which were based upon 2D-COSY spectroscopy, appear to be in error and are corrected here. Finally, we note that these shifts are information-rich reporters on the nature of the interactions of these model analytes with the micelles.

Accessible NMR experiments studying the hydrodynamics of (15) N-enriched ubiquitin at low fields.

We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available (15) N-enriched human ubiquitin. These experiments take advantage of (15) N direct detection of the NMR signal. The first experiment develops skills in acquiring and interpreting one-dimensional and two-dimensional NMR data of an aqueous protein sample (D. Rovnyak, L. E. Thompson (2005) An accessible two-dimensional solution nuclear magnetic resonance experiment on human ubiquitin, Biochem. Mol. Biol. Educ. 33, 117-122). In the second experiment, discussed here, students examine the overall dynamics of a protein in solution. Students measure spin relaxation times T(1) and T(2) and use these data to estimate a rotational correlation time, τ(c) , and the diameter of ubiquitin using the Stokes-Einstein relation. This is a fast and simple experiment, requiring about 1 h of instrument time, and a valuable opportunity to provide a hands-on experience in studying macromolecular size and dynamics. This experiment is conducted in synchrony with lecture material on hydrodynamics and sedimentation.

Microwave-assisted regioselective addition of P(O)-H bonds to alkenes without added solvent or catalyst.

The addition of P(O)-H bonds to alkenes has been accomplished using microwave irradiation in the absence of added solvent and catalyst. In addition to single addition reactions, tandem hydrophosphinylation reactions with alkynes afforded unsymmetrical species such as phosphine oxide-phosphinates. [reaction: see text]

An accessible two-dimensional solution nuclear magnetic resonance experiment on human ubiquitin*.

Solution-state nuclear magnetic resonance (NMR) is an invaluable tool in structural and molecular biology research, but may be underutilized in undergraduate laboratories because instrumentation for performing structural studies of macromolecules in aqueous solutions is not yet widely available for use in undergraduate laboratories. We have implemented an experiment that is ideal for more commonly available 4.8-7.0 Tesla, double-channel NMR instruments that would not usually be used for biomolecular NMR work. We analyzed a commercially available, (15) N-enriched human ubiquitin sample with a two-dimensional correlation experiment using indirect (1) H evolution and direct (15) N detection, which produced spectra with high resolution on a spectrometer operating at 7.0 Tesla (300 MHz (1) H resonance frequency). The simplicity of the experiment makes it possible to be configured by undergraduate students with minimal supervision from the instructor. Students gain experience in acquiring multidimensional biomolecular NMR experiments, confirm that ubiquitin is stably folded, and observe the correspondence between specific signals and individual amino acids in ubiquitin.