Probe Ligand Research Articles

Structure Activity Relationships of Nuclear Receptor, GPCR and Kinase Modulators Revealed with Differential HDX

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has emerged as a powerful technology for analysis of protein conformational dynamics and ligand interactions. The regulation of transcriptional output by nuclear receptors (NRs) is driven by alterations in the conformational ensemble of the receptor upon ligand binding and previously we have shown that HDX can be used to determine a novel mechanism of ligand activation of PPARγ, detailed analysis of binding modes of ligands within the ligand binding pocket of two ER isoforms, and how ERα ligands can be classified and correlated to their pharmacology based on receptor HDX signatures. More recently, we have applied HDX to probe the conformational dynamics of intact full length nuclear receptor complexes upon interaction with DNA and coactivator proteins. These studies have demonstrated that DNA binding alters conformational dynamics of the nuclear receptor heterodimer in regions remote of the DBD. These alterations in conformational selection appear to be important for coactivator binding to the heterodimer suggesting that DNA acts as an allosteric ligand. In addition to work on NRs, the lab recently demonstrated the use of HDX for probing ligand interaction with G protein coupled receptor (GPCRs) and kinases. We have extended these studies to probe differential receptor perturbation upon interacting with functionally selective ligands, and with kinases probed both ligand and co-regulatory protein interactions. Results from these studies will be presented.

Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes

Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A2A Adenosine Receptor

Molecular modeling of agonist binding to the human A(2A) adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A(2A)AR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A(2A)AR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles.

Trimethylsilyl-Substituted Hydroxycyclopentadienyl Ruthenium Hydrides as Benchmarks to Probe Ligand and Metal Effects on the Reactivity of Shvo Type Complexes.

The bis(trimethylsilyl)-substituted hydroxycyclopentadienyl ruthenium hydride [2,5-(SiMe(3))(2)-3,4-(CH(2)OCH(2))(η(5)-C(4)COH)]Ru(CO)(2)H (10) is an efficient catalyst for hydrogenation of aldehydes and ketones. Because 10 transfers hydrogen rapidly to aldehydes and ketones and because it does not form an inactive bridging hydride during reaction, hydrogenation of aldehydes and ketones can be performed at room temperature under relatively low hydrogen pressure (3 atm); this is a significant improvement compared with previously developed Shvo type catalysts. Kinetic and (2)H NMR spectroscopic studies of the stoichiometric reduction of aldehydes and ketones by 10 established a two-step process for the hydrogen transfer: (1) rapid and reversible hydrogen bond formation between OH of 10 and the oxygen of the aldehyde or ketone, (2) followed by slow transfer of both proton and hydride from 10 to the aldehyde or ketone. The stoichiometric and catalytic activities of complex 10 are compared to those of other Shvo type ruthenium hydrides and related iron hydrides.

Chemoselective Surface Immobilization of Proteins through a Cleavable Peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

Controlling Conformational Flexibility of an O2-Binding H-NOX Domain

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.

Determinants of the Heme–CO Vibrational Modes in the H-NOX Family

The Heme Nitric oxide/OXygen binding (H-NOX) family of proteins have important functions in gaseous ligand signaling in organisms from bacteria to humans, including nitric oxide (NO) sensing in mammals, and provide a model system for probing ligand selectivity in hemoproteins. A unique vibrational feature that is ubiquitous throughout the H-NOX family is the presence of a high C-O stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe-CO and C-O stretching frequencies were probed in the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) using resonance Raman (RR) spectroscopy. Four classes of heme pocket mutants were generated to assess the changes in stretching frequency: (i) the distal H-bonding network, (ii) the proximal histidine ligand, (iii) modulation of the heme conformation via Ile-5 and Pro-115, and (iv) the conserved Tyr-Ser-Arg (YxSxR) motif. These mutations revealed important electrostatic interactions that dampen the back-donation of the Fe(II) d(π) electrons into the CO π* orbitals. The most significant change occurred upon disruption of the H-bonds between the strictly conserved YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations. One conformer was structurally similar to Tt H-NOX WT, whereas the other displayed a decrease in ν(C-O) of up to ∼70 cm(-1) relative to the WT protein, with minimal changes in ν(Fe-CO). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high ν(C-O) by decreasing the Fe d(π) → CO π* back-donation and suggest that the dominant mechanism by which this family modulates the Fe(II)-CO bond likely involves the YxSxR motif.

Understanding the stability, electronic and molecular structure of some copper(III) complexes containing alkyl and non alkyl ligands: Insights from DFT calculations

DFT calculations have been performed for some Cu(III)-alkyl complexes. Complexes 1– 19 were optimized to the square planar ( sq) geometry and observed no imaginary frequencies. Although formally copper adopts d 8 configuration (Cu(III)) in all the complexes, the Natural Population Analysis (NPA) revealed that the copper actually in d 10 (Cu(I)) configuration, Bond order calculation suggested that the Cu(III)–Et trans bond gets more bond order in the presence of poor π-acidic co-ligand (probe ligand). Relatively smaller bond order was calculated for Cu(III)–Me cis bond than Cu(III)–Et trans bond and therefore Cu(III)–Et trans bond is the strongest bond in all the complexes. Calculated less Chemical hardness ( η) of complexes 1– 19 suggested that all these complexes are less stable in nature. Energy Decomposition Analysis (EDA) revealed that the Cu(III)–Et trans bond is relatively more stable than the Cu(III)–Me cis and Cu(III)–L (L = co-ligand/probe ligand) bonds. And also the Cu(III)–alkyl (Cu(III)–Me cis and Cu(III)–Et trans ) bond in complexes 1– 17 is more of ionic in nature. However, Cu(III)–Et trans bond is relatively more ionic than Cu(III)–Me cis bond.

Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins: DETECTION PROBES AND AFFINITY STANDARDS

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants β(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ∼10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.

An Australian tuberous sclerosis cohort: Are surveillance guidelines being met?

This study aims to describe the phenotypic and genotypic characteristics of 45 Australian patients with tuberous sclerosis complex (TSC), to assess risk factors for intellectual disability, to compare patients with TSC1 and TSC2 mutations and to assess adherence to surveillance recommendations. Phenotypic features were recorded in 45 patients who fulfilled established criteria for a diagnosis of definite TSC. All patients underwent TSC1 and TSC2 sequencing and multiplex ligand probe amplification. Features were compared in patients with TSC1 mutations versus TSC2 mutations. Recent surveillance was recorded at the point of first contact. Surveillance adherence was compared in the adult and paediatric cohorts. This cohort consisted of 31 children and 14 adults with definite TSC. The rates of TSC manifestations and TSC1 and TSC2 mutation detection rates were consistent with previous studies. There was a trend towards greater severity for patients with TSC2 mutations compared with their TSC1 counterparts, particularly for autistic spectrum disorder, but this did not reach statistical significance. The presence of seizures was shown to be a risk factor for intellectual disability (P < 0.001). Overall, 12/45 patients (27%) were not undergoing recommended surveillance at the point of first contact. Surveillance guidelines were being followed in 3/31 (11%) children compared with 9/14 (64%) adult patients (P < 0.05). The genotypic and phenotypic characteristics of this TSC cohort were consistent with previous studies. Surveillance rates in adult patients were significantly lower than in paediatric patients. This highlights the need for patients with TSC to undergo a focussed transition into adult services.

FUNCTIONAL PEPTIDE DENDRIMERS AS MAGNETIC RESONANCE IMAGING PROBES

Novel synthetic strategies and methods have been developed for the synthesis of magnetic resonance imaging(MRI) contrasts.DTPA ligand,DOTA ligand and peptide dendrimer-based MRI probes with controllable structure were prepared.The third generation L-lysine dendrimers were synthesized by solution-phase synthesis method and convergent approach,and the primary amine groups on the surface of the dendrimers were protected by different groups.The ratio between Boc group and Cbz groups was 18 ∶ 6.The peptide dendrimers were selectively deprotected and reacted with DTPA or DOTA derivates in the presence of succinic anhydride to yield controllable ‘peripheral’functional dendrimer-based MRI contrast agents.The new compounds were confirmed by NMR,ESI-TOF MS and so on.The peptide dendrimer-based MRI probes possessing highly controlled structures and single molecular weight were tested at 1.5 T magnetic field in a clinical MRI scanner,and a 3-fold increase in T1 relaxivity to 12.53 Gd mmol.L-1.s-1 was noticed comparing to that of the clinical agent Gd-DTPA.The successful approach to synthesize the controllable dendrimer-based agent and the high T1 relaxivity of the agent opened an opportunity for the design of multifunctional dendritic contrasts for MRI.

Direct Evaluation of Ligand-Receptor Interaction by a Novel Imaging Model Based on Fluorescent Silica Nanoparticle as Ligand Probe and Molecular Imprinting Polymer as Artificial Receptor

A novel fluorescent imaging system was established to monitor ligand-receptor interactions. Fluorescent silica nanoparticles (FNPs) conjugated with ligand molecules were employed as ligands and molecular imprinting polymer (MIP) as artificial receptors in this work. Fluorescent silica nano-particles which have controllable and uniform size of 120 nm were synthesized and characterized by scanning electronic microscopy (SEM). The FNPs with excitation and emission peaks at 289 nm and 570 nm provide strong fluorescence intensity, excellent photostability, and easy surface modification. L-phenylalaninamide was employed as a test ligand and MIP template. By use of grafting initiator of 4,4′-azobis (4-cyanovaleric acid), a MIP receptor membrane was firmly grafted on the support layer by layer. Results showed that the specific affinity between artificial MIP receptor and ligand conjugated to the FNPs can be successfully monitored. It also suggests that the designed imaging system is suitable of studying molecular interactions and has a great potential for further application.

Incorporation of a Bioactive Reverse-Turn Heterocycle into a Peptide Template Using Solid-Phase Synthesis To Probe Melanocortin Receptor Selectivity and Ligand Conformations by 2D 1H NMR

By use of a solid-phase synthetic approach, a bioactive reverse turn heterocycle was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein-coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nanomolar potency. Biophysical structural analysis [2D (1)H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference peptide and result in a novel template for further structure-function studies.

Fluorescent detection of an anthrax biomarker based on PVA film

Due to the dangerous nature of anthrax, the development of a cost-effective, sensitive and field-portable sensor for the anthrax biomarker--calcium dipicolinate (CaDPA)--is of exceptional significance for both military and civilian use. Herein, a flexible polymer-film-based ratiometric sensor for detecting CaDPA was demonstrated. A reference dye and a probe ligand were covalently immobilized onto the film surface through a highly selective and efficient "click chemistry" reaction. The reference dye, whose fluorescence intensity does not change with varying amounts of CaDPA, offers a non-interfering internal calibration. The ethylenediaminetetraacetic acid (EDTA)-based ligand binds with Eu(III) and serves as the probe. In the absence of CaDPA, the film sensor exhibited almost no red fluorescence because the Eu(III) ions themselves give no emission without sensitization by CaDPA owing to the small molar absorption coefficients of Eu(III) ions. The presence of CaDPA induces a significantly enhanced emission intensity of the sensor, and thereby enables the film as a ratiometric sensor for CaDPA. This sensor can selectively detect CaDPA in water with a detection limit of 100 nM. Moreover, this sensor exhibited strong anti-interfering capability, it can not only be used in milieus that contain various amino acids and some biologically-abundant cations, but can also be usable in some biological fluids such as urine and serum. This test-paper-like film sensor is suitable for portable field analysis and needs no extra protective measures during transport due to its flexibility, and it can easily be separated from the analyte solution after the detection.

Reduction‐Sensitive, Robust Vesicles with a Non‐covalently Modifiable Surface as a Multifunctional Drug‐Delivery Platform

The design and synthesis of a novel reduction-sensitive, robust, and biocompatible vesicle (SSCB[6]VC) are reported, which is self-assembled from an amphiphilic cucurbit[6]uril (CB[6]) derivative that contains disulfide bonds between hexaethylene glycol units and a CB[6] core. The remarkable features of SSCB[6]VC include: 1) facile, non-destructive, non-covalent, and modular surface modification using exceptionally strong host-guest chemistry; 2) high structural stability; 3) facile internalization into targeted cells by receptor-mediated endocytosis, and 4) efficient triggered release of entrapped drugs in a reducing environment such as cytoplasm. Furthermore, a significantly increased cytotoxicity of the anticancer drug doxorubicin to cancer cells is demonstrated using doxorubicin-loaded SSCB[6]VC, the surface of which is decorated with functional moieties such as a folate-spermidine conjugate and fluorescein isothiocyanate-spermidine conjugate as targeting ligand and fluorescence imaging probe, respectively. SSCB[6]VC with such unique features can be used as a highly versatile multifunctional platform for targeted drug delivery, which may find useful applications in cancer therapy. This novel strategy based on supramolecular chemistry and the unique properties of CB[6] can be extended to design smart multifunctional materials for biomedical applications including gene delivery.

Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists

BackgroundQuantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs).ResultsSynthetic strategies for coupling the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine) to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol) (PEG) displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R)-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM) D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 ± 0.15 μM). The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 ± 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence.ConclusionsThis is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.

Determination of Single Molecule erbB1 Homodimer Lifetimes Using Single Quantum Dot Tracking and a Diffusive Hidden Markov Model

Signaling by the erbB family of transmembrane tyrosine kinase receptors controls many cellular pathways, including growth and differentiation, while dysregulation of erbB signaling is a hallmark of many cancers. To understand how receptor interactions regulate signaling, we have developed new multi-color single molecule tracking and analysis techniques. Diffusion and dimerization of erbB1 receptors on live cells were monitored with single particle tracking by using antibody fragments or ligand probes coupled to quantum dots (QDs). As a probe for resting receptors, we used a non-activating, non-competing, monovalent fragment of an anti-erbB1 heavy-chain only antibody (VhH). To track activated receptors, QDs conjugated to EGF ligand were employed. Dimer lifetimes were determined from two-color single particle trajectories using a modified Diffusive Hidden Markov Model. This analytic two-state (bound and free) model uses the two-color QD trajectories to find bound states and quantify the dimerization lifetime using a global estimation over many trajectories. We capture the behavior of erbB1 receptors diffusing on the surface of live A431 cells, including the formation of long-lived dimers between two EGF-QD-erbB1 complexes. Treatment with PD153035, a specific inhibitor of erbB1 tyrosine kinase activity, increases receptor diffusion rate and reduces the lifetime of erbB1 homodimers, suggesting a role for the active kinase domain in formation of stable dimers. These results demonstrate the capabilities of innovative imaging and analysis approaches to measure protein-protein interaction kinetics in real time.

Multivalent ligands of CD22 for targeting of B cells

Multivalent ligands of CD22 for active targeting of B cells. Mary O’Reilly, Wei Hsu Chen, Gladys Completo, Ying Zeng, Satoshi Futakawa and Cory Rillahan and James C. Paulson, Departments of Chemical Physiology and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037The siglecs comprise 13 members of the immunoglobulin superfamily that recognize sialic acid containing glycans, and are differentially expressed on leukocytes and glial cells. The natural ligands of siglecs typically occur on the same cell (in cis) and/or on adjacent cells (in trans). Cis ligands mask the binding of multivalent synthetic sialoside ligands and are thought to regulate the activity of siglecs as modulators of cell signaling. However, synthetic ligands of sufficient avidity can compete with cis ligands, demonstrating a dynamic equilibrium of cis and trans ligand probes. We have explored the relationship between valency, affinity and geometry for achieving avidity sufficient to compete with cis ligands of CD22 (Siglec-2) in situ. A notable achievement is a hetero-bifunctional ligand approach to create multivalent ligands using antibodies as a protein scaffold. The ligand is comprised of a CD22 ligand, 9-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (BPCNeuAc), coupled to an antigen, 4-hydroxy-3-nitrophenylacetic acid (NP) (BPCNeuAc-NP). The BPCNeuAc-NP ligand is able to efficiently assemble complexes of anti-NP antibodies with CD22 on both asialo- and native B cells. Surprisingly, assembly of the tertiary complex occurs with anti-NP IgM (n=10), IgA (n=4) and IgG (n=2). The results suggest that spacing of ligands using an antibody optimizes the contribution of geometry to achieve high avidity with low valency. Other multivalent configurations also show promise for targeting B cells. In particular, liposomes bearing BPCNeuAc ligands bind avidly to B cells and are endocytosed. Thus, BPCNeuAc-lipososomes may prove effective in delivery of therapeutic agents to B cells (Supported by NIH grants GM60938, AI50143 ).

Multivalent ligands of CD22 for targeting of B cells

AbstractMultivalent ligands of CD22 for active targeting of B cells. Mary O’Reilly, Wei Hsu Chen, Gladys Completo, Ying Zeng, Satoshi Futakawa and Cory Rillahan and James C. Paulson, Departments of Chemical Physiology and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037The siglecs comprise 13 members of the immunoglobulin superfamily that recognize sialic acid containing glycans, and are differentially expressed on leukocytes and glial cells. The natural ligands of siglecs typically occur on the same cell (in cis) and/or on adjacent cells (in trans). Cis ligands mask the binding of multivalent synthetic sialoside ligands and are thought to regulate the activity of siglecs as modulators of cell signaling. However, synthetic ligands of sufficient avidity can compete with cis ligands, demonstrating a dynamic equilibrium of cis and trans ligand probes. We have explored the relationship between valency, affinity and geometry for achieving avidity sufficient to compete with cis ligands of CD22 (Siglec-2) in situ. A notable achievement is a hetero-bifunctional ligand approach to create multivalent ligands using antibodies as a protein scaffold. The ligand is comprised of a CD22 ligand, 9-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (BPCNeuAc), coupled to an antigen, 4-hydroxy-3-nitrophenylacetic acid (NP) (BPCNeuAc-NP). The BPCNeuAc-NP ligand is able to efficiently assemble complexes of anti-NP antibodies with CD22 on both asialo- and native B cells. Surprisingly, assembly of the tertiary complex occurs with anti-NP IgM (n=10), IgA (n=4) and IgG (n=2). The results suggest that spacing of ligands using an antibody optimizes the contribution of geometry to achieve high avidity with low valency. Other multivalent configurations also show promise for targeting B cells. In particular, liposomes bearing BPCNeuAc ligands bind avidly to B cells and are endocytosed. Thus, BPCNeuAc-lipososomes may prove effective in delivery of therapeutic agents to B cells (Supported by NIH grants GM60938, AI50143 ).