Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance.

Small molecules are difficult to directly detect using commercially available surface plasmon resonance (SPR) instruments. This is because low molecular weight compounds do not have sufficient mass to cause a measurable change in refractive index. Refractive index is sensitive, however, to other properties besides the mass of the analyte. Recently the detection of substantial conformational changes for immobilized proteins using SPR has been reported. However, this property has not yet been exploited for the detection of low molecular weight ligand binding to immobilized protein receptors. Here we demonstrate that ligand-induced conformational changes can be used to monitor the binding of small molecules to immobilized maltose-binding protein and tissue transglutaminase. Ligand binding to a receptor that decreases in hydrodynamic radius yielded a net decrease in refractive index. A net positive change in refractive index was observed for a receptor that increases in hydrodynamic radius. Refractive index changes could not be explained by addition of analyte molecular mass to the surface. These SPR responses were a result of specific receptor-ligand interactions, as judged by the reversibility of the response and the similarities between the SPR-determined equilibrium dissociation constants and reported dissociation constants. Additionally, this technique proved to be effective at detecting specific ligands from a panel of small molecules. This SPR method required no alterations in widely used and commercially available instrumentation yet allowed direct detection of very small molecules such as calcium ions (40 Da). Use of receptor conformation to detect low molecular weight analytes has potential applications in the high-throughput screening of small molecule drug libraries and the development of biosensors.

Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays.

We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment.

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.

Real-time, sequence-specific detection of nucleic acids during strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data.

Spectroscopic studies of single-stranded DNA ligands and oxazole yellow dyes.

Interactions between short single-stranded DNA ligands and fluorescent DNA indicator dyes were used to investigate binding selectivity of the ligands. Conformational differences among four DNA ligands of different sequence and structure, including two that form a G-quartet and two that do not, were confirmed by circular dichroism spectroscopy. Their interactions with indicator dyes YO-pro-1 iodide (YO) and YOYO-1 iodide (YOYO) were probed using measurements of dye absorbance; induced circular dichroism; and fluorescence spectra, anisotropy, and lifetime. Equilibrium binding constants and stoichiometry were determined as well. Results indicate significant differences among the dye interactions and binding stoichiometries of the four ligands. One of the G-quartet forming ligands, a 20-mer of sequence 5'-GGTTTTGGTTTTGGTTTTGG-3', shows distinctly different interactions from the other three ligands, all of which are 15-mers. These studies illustrate the importance of sequence and conformation in determining the binding interactions of short single-stranded DNA.

Temperature and quenching studies of fluorescence polarization detection of DNA hybridization.

The effects of temperature and collisional quenching on fluorescence polarization detection of DNA hybridization were studied using measurements of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in length, were used to attach fluorescein label to the 5' end of the 33-base oligomers. Perrin plots showed that the effective rotating volume decreases with increasing tether length and approximately doubles upon hybridization. Hybridization increases the association between the tethered dye and the DNA for the shorter tethers but displaces the fluorescein on the 12C tether from the DNA, forcing it into greater contact with the bulk solution. The 6C tether appears to promote sequence-specific interaction between fluorescein label and the oligomer, which causes unexpectedly high anisotropy at higher temperatures and increased protection from collisional quenching. In all cases, there appear to exist several possible conformations for the tethered fluorescein. As temperature is increased, these conformations tend to collapse into a single, average or preferred, conformation. The results demonstrate the importance of the selection of tether, dye, and DNA probe in designing a polarization strategy for detection of DNA hybridization, particularly with respect to tether length and DNA probe sequence.

Transferred nuclear Overhauser enhancement experiments show that the monoclonal antibody strep 9 selects a local minimum conformation of a Streptococcus group A trisaccharide-hapten.

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed to investigate the bound conformation of the trisaccharide repeating unit of the Streptococcus Group A cell-wall polysaccharide. Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyran osyl)- alpha-L-rhamnopyranoside [C(A')B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational preferences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous solution, thus displaying relatively flexible behavior. The NOE HNAc-H2A', which was not detected in previous work, accounts for a conformation at the beta-(1-->3) linkage with a phi angle of approximately 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was further complicated by spin diffusion. With the use of transferred rotating-frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that all of the observed long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together with repulsive constraints, derived from the absence of TRROE effects, were used as input parameters in simulated annealing and molecular mechanics calculations to determine the bound conformation of the trisaccharide. Complexation by the antibody results in the selection of one defined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the alpha-(1-->2) linkage when compared to the global minimum conformation. The results infer that the bound conformation of the Streptococcus Group A cell-wall polysaccharide is different from its previously proposed solution structure (Kreis et al., 1995).

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.

N-modified analogues of cocaine: synthesis and inhibition of binding to the cocaine receptor.

Cocaine methiodide (2), N-norcocaine (1b), N-benzyl-N-norcocaine (1c), and N-nor-N-acetylcocaine (1d) were synthesized and evaluated for their ability to inhibit binding of [3H]-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester (WIN 35,428) to the cocaine receptor. The study showed that removal of the N-methyl group to give 1b, or replacement with the larger N-benzyl group to give 1c, has a relatively small effect on binding potency. In contrast, replacement of the N-methyl group by the acetyl moiety to give 1d, or the addition of a methyl group to give 2, reduces affinity for the receptor by a large factor. In order to gain preliminary information concerning the importance of the nitrogen location on the tropane ring system, the receptor binding affinity of 8-methyl-8-azabicyclo[3.2.1]octan-3 beta-ol benzoate (5, beta-tropacocaine) was compared to that of the isomeric 6-methyl-6-azabicyclo[3.2.1]octan-3 beta-ol benzoate (4d). The fact that both compounds have similar binding affinities for the cocaine receptor suggests that 3 beta-(benzoyloxy)-6-methyl-6-azabicyclo[3.2.1] octane-2-carboxylic acid methyl ester, which is isomeric with cocaine, may possess binding potency similar to cocaine.

Synthesis, molecular modeling studies, and muscarinic receptor activity of azaprophen analogues.

Synthesis, radioligand binding, and pharmacologic activities of a series of muscarinic receptor ligands including and related to azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3 alpha-ol 2,2-diphenylpropionate, 1) have been measured to determine activity and selectivity for muscarinic receptor subtypes. Pharmacologic affinities of antagonists were determined as pA2 values for antagonism of methacholine-induced tension responses in guinea pig ileum. Binding affinities were measured by competition against [3H]QNB binding in guinea pig ileum, rat heart and brain, and m1- or m3-transfected Chinese hamster ovary (CHO) cells. The efficacies of muscarinic agonists in brain were determined by the ratio of binding affinities against [3H]QNB or [3H]NMS and [3H]oxotremorine-M ([3H]Oxo-M). Nine muscarinic antagonists, including azaprophen, did not discriminate significantly between the subtypes of muscarinic receptors. KI values for receptor binding for azaprophen (1) were between 8.81 x 10(-11) and 4.72 x 10(-10) M in ileum, heart, brain, and m1- or m3-transfected CHO cells. The alpha- and beta-benzilate esters 5 and 6 are as potent as azaprophen, and diphenylacetate esters 3 and 4 and N-(6)-benzyl alpha-isomer 7 are less potent than azaprophen. Significant stereoselectivity was exhibited with (+)-azaprophen being approximately 200 times more potent than the (-)-enantiomers and the 3 beta-ol isomer 2 being ca. 50 times less potent than azaprophen in all systems. A molecular modeling-molecular mechanics study was conducted to account for the difference. Putative muscarinic agonists (analogues and isomers of 6-methyl-6-azabicyclo[3.2.1]octan-3-ol acetate) did not discriminate muscarinic receptor subtypes with KI values between 2.77 x 10(-6) and 4.33 x 10(-5) M without significant stereoselectivity in the systems examined. The most active analogue was (1R,3R,5S)-6-[1(R)-phenylethyl]-6-azabicyclo[3.2.1]octan-3 alpha-ol acetate. However, efficacies of these putative agonists were in general very low.