Antibody-based fluorescence polarization assay to screen combinatorial libraries for sweet taste compounds.

Technological advances in instrumentation, chemical synthesis methods, molecular biology and biochemistry have fueled the recent growth in high throughput screening. Assays are available in a vast range for formats, including fluorescence, luminescence, absorbance, and scintillation detection. Antibodies represent a powerful tool for novel compound discovery and their utility in this regard should not be underestimated. We have designed a fluorescence polarization immunoassay for the identification of novel sweeteners. The assay is based on monoclonal antibodies that bind superpotent sweet taste compounds and libraries of suitable test compounds can be rapidly screened using these antibodies as "artificial taste receptors."

Antibody-ligand interactions: computational modeling and correlation with biophysical measurements.

Several new aspects of computer-assisted molecular modeling strategies and biophysical techniques, such as fluorescence spectroscopy, circular dichroism, and absorption spectroscopy, have proved useful in the analysis and description of antibody-ligand interactions. The molecular features involved in determining the specificity of antibody-ligand interactions, such as electrostatics (e.g. partial charges, salt bridges, p-cation motifs), hydrogen-bonds, polarization, hydrophobic interactions, hydration and solvation effects, entropy, and kinetics can be identified using a battery of biophysical techniques. An understanding of these parameters is essential to our use of antibodies as tools in high throughput screening of chemical libraries for the discovery of novel compounds.

Dimeric beta-turn peptidomimetics as ligands for the neurotrophin receptor TrkC.

Twelve dimeric peptidomimetics 1 were prepared via a divergent-convergent strategy. These peptidomimetics incorporated the same amino acids as i +1 and i + 2 residues in key beta-turns of the neurotrophin NT-3. Cytosensor microphysiometry was used to gauge the effects of the dimers 1 on cells that overexpress the NT-3 receptor, TrkC. Increases in extracellular acidification rates were observed for some monomers 3, but the active dimers gave greater effects.

Cellular responses of NG108-15 and SK-N-MC lines to sweet and bitter tastants as measured by extracellular acidification rates.

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of detecting small changes in cellular metabolism in response to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). By measuring the ECAR we were able to detect responses of tissue culture cell lines to a variety of sweet- and bitter-tasting compounds. We examined in detail the responses of the NG108-15 (mouse neuroblastoma x rat glioma hybrid) and SK-N-MC (human neuroepithelioma) cell lines. We determined that NG108-15 cells were consistently very responsive to several potent sweeteners and bitter compounds, such as sodium saccharin, guanidino- sweeteners, denatonium benzoate, quinine, and ranitidine. These compounds could evoke changes in cellular metabolism (measured as ECAR) that were rapid in onset, saturable with respect to ligand concentration, and sensitive to several inhibitors of G-protein-coupled receptor signaling pathways. In sharp contrast, the neuroepithelioma SK-N-MC did not respond to any of the sweet or bitter compounds. Rapid changes in ECAR were easily detectable in both cell lines with the calcium ionophore A23187. Bradykinin elicited changes in the ECAR only in the NG108-15 cell line, which is known to express the B2 receptor. The changes in ECAR of the NG108-15 cell line in response to sweet and bitter taste compounds suggest these cells may expresses a receptor(s) specific for small sapid molecules.

Ultrastructural effects of silicic acid on primary lung fibroblasts in tissue culture.

Transmission and scanning electron microscopic examination of primary lung fibroblasts exposed in tissue culture to polymeric silicic acid (PSA) revealed profound cellular changes in the cell surface membranes, resulting in rapid endocytosis of affected membranes and formation of multivesicular bodies. Exposure to monomeric silicic acid did not appear to exhibit any immediate adverse effects. Appearance of numerous cytoplasmic vacuoles within 1 h of PSA exposure was easily visible by light microscopy. Electron microscopy revealed that PSA exposure caused formation of an 'osmiophilic' cell surface membrane. Numerous osmiophilic cytoplasmic blebs on the surface and subsequent endocytotic vesicles appeared to collapse and aggregate into multivesicular bodies. This study provides ultrastructural evidence of the direct interaction between lung fibroblasts and polymeric silicic acid, which has a dramatic effect the surface membrane, its subsequent internalization and cytoplasmic processing. This interaction could be one of the key steps in the damaging effects of silica containing dust.

CDK4 expression and activity are required for cytokine responsiveness in T cells.

Stimulation of lymphocytes through the Ag receptor can lead to cytokine responsiveness or unresponsiveness. We examined the importance of cyclin-dependent kinase (CDK)4 to establish and maintain IL-2 responsiveness in human T cells. Our results show that a herbimycin A- and staurosporine-sensitive phase of CDK4 expression and activity preceded the acquisition of IL-2-responsiveness in mitogen-stimulated peripheral blood T cells. Intriguingly, CDK4 expression and activity were demonstrable in purified unstimulated peripheral blood T cells from approximately 30% (5/16) of healthy individuals examined for this study. These T cells proliferated in response to IL-2 without additional mitogens, and both the expression and activity of CDK4 and the ability to respond to cytokines were resistant to herbimycin A and staurosporine. The pattern of CDK4 expression and response to IL-2 in this subset of individuals resembled that seen in the human IL-2-dependent Kit-225 T cell line. However, in contrast to normal T cells, Kit-225 cells were rendered unresponsive to IL-2 by stimulation through the Ag receptor. In these cells, PHA, anti-CD3, or PMA induced marked reductions of CDK4 expression and activity that paralleled IL-2 unresponsiveness, and these effects were not reversible by IL-2. Furthermore, IL-2-dependent proliferation could be similarly inhibited in Kit-225 cells by overexpression of the CDK inhibitors p16/Ink4-a or p21/Waf-1a or by overexpression of a kinase-inactive CDK4 mutant. The data indicate that CDK4 expression and activity are necessary to induce and maintain cytokine responsiveness in T cells, suggesting that CDK4 is important to link T cell signaling pathways to the machinery that controls cell cycle progression.

The three-dimensional structure of a complex of a murine Fab (NC10. 14) with a potent sweetener (NC174): an illustration of structural diversity in antigen recognition by immunoglobulins.

The three-dimensional structure of a complex of an Fab from a murine IgG2b(lambda) antibody (NC10.14) with a high potency sweet tasting hap- ten, N-(p-cyanophenyl)-N'-(diphenylmethyl)-N"-(carboxymethyl)guan idine (NC174), has been determined to 2.6 A resolution by X-ray crystallography. This complex crystallized in the triclinic space group P1, with two molecules in the asymmetric unit. In contrast to a companion monoclonal antibody (NC6.8) with a kappa-type light chain and similar high affinity for the NC174 ligand, the NC10.14 antibody possessed a large and deep antigen combining site bounded primarily by the third complementarity-determining regions (CDR3s) of the light and heavy chains. CDR3 of the heavy chain dominated the site and its crown protruded into the external solvent as a type 1' beta-turn. NC174 was nested against HCDR3 and was held in place by two tryptophan side-chains (L91 and L96) from LCDR3. The diphenyl rings were accommodated on an upper tier of the binding pocket that is largely hydrophobic. At the floor of the site, a positively charged arginine side-chain (H95) stabilized the orientation of the electronegative cyano group of the hapten. The negative charge on the acetate group was partially neutralized by a hydrogen bond with the phenolic hydroxyl group of tyrosine H58. Comparisons of the modes of binding of NC174 to the NC6.8 and NC10.14 antibodies illustrate the enormous structural and mechanistic diversity manifest by immune responses.

Vasoactive amine responses in murine cerebrovascular endothelial cells as measured by extracellular acidification rates.

The Cytosensortrade mark microphysiometer device is capable detecting cellular responses to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). Using microphysiometry, we were able to determine that cerebovascular endothelial cells derived from SJL/J mice were more sensitive to serotonin (maximal ECAR response at 100 nM), whereas BALB/c-derived cerebrovascular endothelial cells (CVE) were relatively insensitive (maximal ECAR response at 30 microM). Serotonin (5HT)(1) and 5HT(2) receptor antagonists inhibited the serotonin-mediated increases in ECAR. The cells' responses to histamine in both strains were similar (maximal ECAR required 100 microM of histamine). H(1) and H(3) receptor subtype antagonists specifically inhibited the histamine responses. Bradykinin also revealed sensitivity differences in that maximal ECAR changes for CVE from SJL/J mice could be observed with 1 microM, as compared to the ECAR responses from BALB/c mice CVE, which required 10 microM. Exposure to bradykinin antagonists revealed that the response was due to the stimulation of B(2) receptors. These microphysiometry results reveal that the cerebrovascular endothelial cells of SJL/J mice tend to be more sensitive to vasoactive substances than those of BALB/c mice.

Analysis of correlated motion in antibody combining sites from molecular dynamics simulations.

The crystal structures of the NC6.8-antisweet taste ligand complex and the uncomplexed antibody structures display significant differences in the conformations of residues in the combining site. A molecular dynamics method was employed to understand the flexibility and correlated motion of key combining site residues in the uncomplexed antibody. The simulations reveal that residues that show conformational differences between the complex and uncomplexed structures display strong dynamical correlations. Extensive analysis of the dynamics trajectory using time correlation methods is presented.

Analysis of diversity of nucleotide and amino acid distributions in the VD and DJ joining regions in Ig heavy chains.

Nucleotide fill-in between the germ line V, D and J genes in the H3 loop of immunoglobulins contributes to the diversity of the antibody repertoire. This fill-in process is mediated by terminal deoxynucleotidyl transferase (TdT), which has been widely believed to insert nucleotides in a random fashion. Using a database of 2443 immunoglobulin sequences, we identified the regions of nucleotide fill-in between the V-D and D-J gene regions. We translated the fill-in nucleotides and measured the diversity within the two regions both at the nucleotide and amino acid level. We found that the nucleotide and amino acid distributions that resulted from nucleotide fill-in were in fact not random. Examination of the synonymous substitution rates of nucleotides revealed evidence suggesting that TdT plays a less significant role in generating antibody diversity than previously thought. We observed preferences for polar residues, which are more likely to encourage interaction with ligand than non-polar residues and are often found in loop regions in general. We also observed a preference for the insertion of smaller residues versus larger residues of similar biochemical properties, aiding in loop flexibility. We interpret these findings to reflect the significant influence of biochemical (i.e. folding) constraints and/or binding affinity constraints (at the cellular/selectional level) on the sequence diversity in the H3 region. These constraints act as a filter on the randomness generated by nucleotide addition by TdT, as well as other diversity generating processes such as recombination of VDJ gene segments and somatic mutation. The results of this study suggest that the antibody repertoire might be reduced from what is traditionally believed.

Conservation of cys-cys trp structural triads and their geometry in the protein domains of immunoglobulin superfamily members.

In almost all members of the immunoglobulin superfamily (IgSF) for which an experimental structure has been determined, a triad (C-CW) consisting of two cysteine residues that form a disulfide bond and a neighboring tryptophan can be found in the core of the protein fold. We analyzed the geometry of these C-CW triads among a database of 60 Fab crystal structures and found it to be remarkably conserved. We identified C-CW triads of a similar configuration in other members of the IgSF such as T cell receptor (TCR), major histocompatibility complex antigens (MHC), cell surface antigens CD4 and CD8, and cell-adhesion molecules. We used this C-CW pattern to search a database of non-IgSF proteins, and identified several proteins that contain a disulfide bridge associated with a tryptophan in a similar configuration. Examination of the distances and orientations between triads found in adjacent domains in Fab fragments and TCR also reveal a high degree of conservation, which reflects the invariance of the inter-chain domain packing. This high degree of conservation of the geometry of the C-CW triad in IgSF structures suggests that the Trp may contribute significantly to the stability of the disulfide bond. Knowledge of these geometric parameters may prove useful in the construction and validation of theoretical models of Ig, TCR, and other IgSF members.

Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy.

Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E. coli using the pET-11a system. The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent.

Ligand binding by antibody IgE Lb4: assessment of binding site preferences using microcalorimetry, docking, and free energy simulations.

Antibody IgE Lb4 interacts favorably with a large number of different compounds. To improve the current understanding of the structural basis of this vast cross-reactivity, the binding of three dinitrophenyl (DNP) amino acids (DNP-alanine, DNP-glycine, and DNP-serine) is investigated in detail by means of docking and molecular dynamics free energy simulations. Experimental binding energies obtained by isothermal titration microcalorimetry are used to judge the results of the computational studies. For all three ligands, the docking procedure proposes two plausible subsites within the binding region formed by the antibody CDR loops. By subsequent molecular dynamics simulations and calculations of relative free energies of binding, one of these subsites, a tyrosine-surrounded pocket, is revealed as the preferred point of complexation. For this subsite, results consistent with experimental observations are obtained; DNP-glycine is found to bind better than DNP-serine, and this, in turn, is found to bind better than DNP-alanine. The suggested binding mode makes it possible to explain both the moderate binding affinity and the differences in binding energy among the three ligands.

Crystal structure of monoclonal 6B5 Fab complexed with phencyclidine.

The crystal structure of monoclonal antibody (mAb) 6B5 Fab fragment complexed with 1-(1-phenylcyclohexyl)piperidine (PCP or phencyclidine) was determined at 2.2-A resolution. 6B5 was originally produced from a mouse immunized with a phencyclidine analogue hapten 5-[N-(1'phenylcyclohexyl)amino]pentanoic acid conjugated to bovine serum albumin. This mAb was selected for further study because of its high affinity (Kd = 2 x 10(-9) M/liter) for PCP and usefulness in reversing PCP-induced central nervous system toxicity in laboratory animals. The dominant feature of the 6B5 Fab.PCP complex is the deep binding site and hydrophobic nature of the interaction. The ligand binding pocket of 6B5 Fab has numerous aromatic side chains, as compared with other known Fab structures. The most notable feature of the binding site is a Trp at position 97H (H-chain), and the side chain of this residue appears to act as a hydrophobic umbrella on the ligand in the antigen binding pocket. There are only two other known Fabs found with a Trp at the 97H position in complementarity determining region (CDR) H3, but they do not play a major role in the interaction with their respective antigens; in both Fab TE33 and R6.5 the Trp 97H side chain is positioned away from the bound antigen. Comparison of the CDR residues of 6B5 with other Fab structures with similar CDR sizes and amino acid compositions reveals a number of important patterns of residue substitutions that appear to be critical for specific PCP ligand interactions.

Ligand-induced domain movement in an antibody Fab: molecular dynamics studies confirm the unique domain movement observed experimentally for Fab NC6.8 upon complexation and reveal its segmental flexibility.

Two molecular dynamics simulations were carried out for the antibody Fab NC6.8, both with and without the guanidinium sweetener ligand NC174, in order to assess the segmental flexibility as well as the conformational changes upon ligand binding. Trajectory analyses of the simulation of the uncomplexed Fab suggest low-amplitude motions of the Ig domains with respect to each other, most clearly reflected by a periodic alteration of the elbow angle within a range of 11 degrees. Upon insertion of the hapten into the binding site, the quaternary structure of the Fab exhibits considerable rearrangements: the elbow angle changes by almost 30 degrees, the light chain is elongated and the heavy chain becomes more flexed. Comparison with experiment reveals some interesting agreements with X-ray crystallographic results published previously.

A labeled guanidine ligand for studying sweet taste.

A synthesis of a biotinylated-, coumarin-substituted-N,N'-diarylguanidine 1 is reported. This ligand has structural features conducive to studying sweet taste including a fluorescent tag to facilitate spectroscopic studies of binding to protein-receptors for sweet ligands and an anchor for affinity purification.

Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse.

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

Libraries of opiate and anti-opiate peptidomimetics containing 2,3-methanoleucine.

A library of 96 peptides/peptidomimetics was prepared, in which half was based on the YGGFL-NH2 sequence, while the remainder were derivatives of a presumed anti-opiate peptide, YGGFLRF-NH2. Of the 48 compounds in each half of the library, 32 contained a stereoisomer of 2,3-methanoleucine substituted for Leu5. Binding of the YGGFL-NH2 derivatives to the mu- and delta-opioid receptors, and to the anti-beta-endorphin monoclonal antibody (clone 3E7), indicated any change at the Leu5 had little effect on the binding when compared with modifications to the YGGF-sequence. Conversely, cyclo-Leu residues did alter the binding of YGGFLRF-NH2 derivatives when substituted for Leu5. Of these 32 peptidomimetics, three derivatives of 2S,3S-cyclo-Leu had relatively low Ki values for binding to an NPFF receptor. Differences between the outcome of the screens were interpreted in terms of the position of the cyclo-Leu residue in the two sequences.

Local histamine release increases leukocyte rolling in the cerebral microcirculation of the mouse.

Histamine-mediated induction of leukocyte rolling and adhesion in the cerebral microcirculation was examined in two inbred strains of mice (SJL/J and BALB/c). A cranial window was surgically prepared for the visualization of the cerebral microcirculation using intra-vital microscopy. Leukocyte rolling and adhesion to pial venular walls were assessed during off-line video playback analyses. The surgical preparation of the cranial windows was found to trigger 'spontaneous' leukocyte rolling, and this was attributed to disruption of dural mast cells and localized release of vasoactive histamine. This spontaneous leukocyte rolling was observed only in the SJL/J strain of mice, and could be prevented by presurgical treatment with the mast cell stabilizer sodium cromoglycate. BALB/c mice did not show 'spontaneous' leukocyte rolling or adhesion; this strain is known to have low numbers of CNS-associated mast cells. Exogenous histamine, applied topically to the cerebral microcirculation via the cranial window in mice pretreated with sodium cromoglycate, produced significant dose-dependent increases in leukocyte rolling and adhesion to pial venules in SJL/J mice, but not in BALB/c mice. Diphenhydramine (H1 receptor antagonist), but not cimetidine (H2 receptor antagonist), abolished both 'spontaneous' and histamine-induced leukocyte rolling. Anti-P-selectin antibody was found efficiently to block both spontaneous and histamine-induced increases in leukocyte rolling, but not leukocyte adhesion.

Recognition of superpotent sweetener ligands by a library of monoclonal antibodies.

Monoclonal antibodies (mAb) made to the superpotent guanidino sweet tasting ligand, N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidineacetic acid were examined for their molecular recognition specificities using 14 different sweetener analogues in a competitive radioimmunoassay. The effects of variations in pH on ligand binding was also examined by radioimmunoassay. Photoaffinity labelling of the binding site was accomplished using a radiolabelled azido-derivative of the parent ligand, and L-chain or H-chain labelling was easily identified in several different mAb. For two of the mAb examined in this study (NC6.8 and NC10.14), the analogue binding studies are in agreement with the known Fab-ligand crystal structures. Monoclonal antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste ligands from combinatorial chemical libraries.