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C P Woodbury

Publications and source records attributed to C P Woodbury.

12 recordsLinked to original sources
PubMed1999

Methods of screening combinatorial libraries using immobilized or restrained receptors.

The screening of combinatorial libraries for compounds with high affinity toward drug receptors is currently a major center of attention. We describe methods recently developed for library screening that involve "constrained" receptors (either immobilized onto a surface or restrained to a compartment by some physical means). These include affinity selection chromatography, ultrafiltration assays, the scintillation proximity assay, a variety of interfacial optical techniques (surface plasmon resonance and its relatives, among others), the quartz crystal microbalance, the jet ring cell, and new interferometric assays using porous silicon to immobilize the receptor. We note some trends in assay development involving assays of membrane-bound complexes, and the coupling of two analytical methods to expand the assay resolution.

Chemistry, Pharmaceutical↗
PubMed1998

Pulsed ultrafiltration characterization of binding.

We describe a new method for measuring binding constants, pulsed ultrafiltration. In this technique, a single injection or "pulse" of ligand is passed through a cell containing macromolecules confined by a conventional ultrafiltration membrane. Any binding of the ligand to the macromolecule alters the elution profile of the ligand. We describe this interaction by a set of coupled differential equations whose solution allows us to extract the binding density as a function of free ligand concentration eluting from the cell. A method of comparing elution profile areas which leads to values for both binding affinity and stoichiometry is also presented. We show that the pulsed ultrafiltration method can generate an extensive binding isotherm with a dense set of data points over a wide range of binding densities. We apply the method to several model ligand-macromolecule binding systems to demonstrate the measurement of equilibrium association constants and binding stoichiometry, the accuracy and precision of the method, and temperature dependence of binding. In general, our results agree with those from the literature, and they show that the approach is a fast and flexible method for characterizing ligand-macromolecule binding.

Chromatography, High Pressure Liquid↗
PubMed1997

Screening solution-phase combinatorial libraries using pulsed ultrafiltration/electrospray mass spectrometry.

A method is described whereby a family of homologues is synthesized in a one-pot reaction, without isolation or purification, and the reaction mixture is screened using a competitive binding assay based on pulsed ultrafiltration/electrospray mass spectrometry (PUF/ESMS) to tentatively identify those derivatives having the highest affinity for a target receptor. As a model system to test this approach, a synthetic scheme designed to prepare a series of analogues of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as diastereomeric mixtures, was carried out. Pulsed ultrafiltration screening of the crude reaction mixture against controls without protein detected protonated molecules corresponding to EHNA-type derivatives and three of its linear, alkyl homologues but did not show protonated molecules for an isobutyl or benzylic EHNA derivative, suggesting the latter was inactive. To verify this conclusion, we prepared E/THNA, the linear homologues, and the benzylic derivative (each as a diastereomeric mixture) and bioassayed them for them adenosine deaminase inhibition index ([I]/[S]0.5). The bioassay results for the individually synthesized analogues were in good agreement with that predicted by the observed relative ion enhancement in the PUF experiments. Thus, the PUF protocol might be used as a general method to quickly provide direction to the chemist in search of drug candidates.

Adenine↗
PubMed1997

Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries.

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.

Adenine↗
PubMed1995

Sample multiplexing for greater throughput in HPLC and related methods.

We present a multiplexing scheme that pools samples for greater efficiency in analysis. The method involves arraying the samples in a matrix and then pooling along rows and columns of the array. Sample pools are analyzed instead of individual samples. The content of a given sample is found by the intersection of a row pool with a column pool. Since this simple pooling scheme can lead to ambiguities in assignments when many samples have the same species, we also suggest a supplementary or augmented pooling scheme, involving the use of pools along diagonals across the sample array, to resolve the ambiguities. We demonstrate the pooling scheme, with resolution of some simple ambiguities, using HPLC for the analysis of fluorophore-labeled amino acids.

Amino Acids↗
PubMed1983

On the determination of deoxyribonucleic acid-protein interaction parameters using the nitrocellulose filter-binding assay.

We examine the effects of filter efficiency on DNA-protein binding data obtained by the popular nitrocellulose filter-binding assay. Graphical procedures for determining the efficiency parameter epsilon (for the efficiency of retention of DNA on the filter, per bound protein molecule) are established. Filter efficiency modified formulas for determining thermodynamic binding parameters are derived for four simple prototypes of DNA-protein binding systems. The effects of experimental error on discrimination between models are considered. Finally, we discuss conceptual errors often made in calculating binding stoichiometry from filter-binding experiments and suggest a general protocol for the analysis of filter-binding data.

DNA↗
PubMed1980

DNA site recognition and reduced specificity of the Eco RI endonuclease.

It has been shown previously (Polisky, B., Green, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M., and Boyer, H. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3310-3314; Hsu, M., and Berg, P. (1978) Biochemistry 17, 131-138) that the cleavage sequence specificity of Eco RI endonuclease can be "relaxed" by various means. In this paper this phenomenon is explored in detail, in order to obtain further insight into the nature and selectivity of sequence recognition patterns between proteins and double-stranded nucleic acids. Using conditions of low ionic strength and alkaline pH, we have mapped the positions of potentially cleavable sites in the (completely sequenced) replicative form of the bacteriophage phi X174 genome, and have deduced their sequence. The time course of digestion of phi X174 DNA suggests that double-stranded sequences reading GGATTT, AAATTT, GAATTT, and GAATTA (only "top" strands, written 5' leads to 3', are shown) are cleaved readily under these conditions, while sequences reading CAATTN (N = A, T, G) resist attack. Cleavages at (at least) the more labile sites result in cohesive ends that are religatable. End group analysis of cleaved phi X174 DNA fragments indicates the presence of a 5'-terminal adenine residue on most of the fragments; some fragments may carry a 5'-terminal guanine residue, consistent with the cleavage site sequences suggested above. Addition of Mn2+ to cleavage reactions carried out at moderate salt concentrations and near-neutral pH induces the same pattern of cleavage seen at low ionic strength and alkaline pH. These results are combined with those from other studies, and are interpreted in terms of a model for the site-specific interaction of the Eco RI endonuclease with its substrate, considering both the effects of changes in DNA sequence and of environmental alterations. The resulting model is compared with data developed on similar grounds for Eco RI methylase (see Woodbury, C. P., Downey, R. L., and von Hippel, P. H. (1980) J. Biol. Chem. 255, 11526-11533), and attempts are made to define both common and differing molecular facets of the DNA recognition specificity of these companion (but genetically distinct) enzymes.

Bacteriophage phi X 174↗