Pharmacopeia Inc.

Pharmacopeia Drug Discovery, Inc.

Amira, Inc.

Jonathan J Burbaum

James Inglese

Stephen Skwish

Rima Kaddurah-Daouk

Daniel Chelsky

James W Lillie

Timothy Pryor

John J Baldwin

Ge Li

Lawrence Wayne Dillard

Bassam Damaj

John C Reader

Gregory L Kirk

Theodore S Widlanski

Wenguang Zeng

Maria Webb

Craig J Forsyth

J Fraser Glickman

Thomas Dy Chung

Kenneth C Appell

Philippe Samama

Ke Yang

Ilana L Stroke

A method for determining the level of tyrosine kinase activity in a biological sample is disclosed. The method employs an anti-phosphotyrosine antibody as both the capture agent and the detecting agent. The detecting antibody is labeled with a fluorescent label, for instance, Cy5, Cy5.5 or Cy7 or a lanthanide ion, such as europium, as the signal generating entity. The method is particularly well suited to high throughput screening, for example, for compounds which modulate tyrosine kinase activity.

Fluorescent capture assay for kinase activity employing an anti-phosphotyrosine antibody as both the capture agent and detecting agent

Techniques for high throughput parallel separation, filtration and plate-to-plate transfer are described. Cells, proteins, chemical compounds and the like are being developed in multi-well, small volume well plates, such as 1536-well plates having wells of a volume on the order of 1 &mgr;l. A mechanism for clamping such plates together so that the wells will be aligned allows rapid separation or transfer by simply centrifuging the assembly. A membrane may be clamped between the plates. Alternatively, a membrane may replace one of the plates. Centrifuge dependent and independent designs are described.

Methods and apparatus for high throughput plate to plate or plate to membrane transfer

The present invention encompasses polypeptides that comprise a chemokine receptor binding sequence and are useful in determining the affinity of a compound for a chemokine receptor. Substitution of one of the amino acids of the C-terminal region of the polypeptide with a cysteine enables the polypeptide to be detectably labelled without loss of receptor binding activity and without the problems inherent in radioiodine labelling. Methods for use of the polypeptides in competitive binding assays are also disclosed.

Site-specific bioconjugates of chemokines and their use in assays

Caged enzyme substrates as probes for detecting reporter enzyme activity in cell-based assays are disclosed. Caged substrates for reporter gene assays are shown as compounds of Formula I or Formula II: ##STR1## wherein Z is a luminescent functionality; G is a labile group, cleavable by enzymatic action; and K is a photolytically cleavable caging group; wherein m and n are independently 0 or 1, but m and n both can not be zero. Test kits for determining reporter enzyme activity using compounds of Formula I or Formula II and photolytic cleavage to produce a compound of Formula III:

Caged enzyme substrates as probes for reporter enzyme activity

Cell culture chambers and incubators are operated to grow cells under a wide variety of conditions. Where those conditions result in too rapid evaporation of the well media for low volume wells such as those found in 1536-well plates, a subchamber is provided to increase the local humidity so that media evaporation is controlled to an acceptable level. A local liquid source is provided. That liquid may be water or an isotonic solution matched to the well media for a particular application.

Cell culture chamber for multiple well plates

A homogeneous high throughput assay is described which screens compounds for enzyme inhibition, or receptor or other target binding. Inhibition (or binding) by the library compounds causes a change in the amount of an optically detectable label that is bound to suspendable cells or solid supports. The amounts of label bound to individual cells or solid supports are microscopically determined, and compared with the amount of label that is not bound to individual cells or solid supports. The degree of inhibition or binding is determined using this data. Confocal microscopy, and subsequent data analysis, allow the assay to be carried out without any separation step, and provide for high throughput screening of very small assay volumes using very small amounts of test compound.

High-throughput assay

A lawn assay is described for determining compounds that affect enzyme activity or that bind to target molecules. Compounds to be screened are cleaved, and diffused from solid supports into a colloidal matrix. Enzymatic catalysis or binding to target molecules by the compounds is carried out in the matrix. Active compounds are found by monitoring a photometrically detectable change in a substrate, coenzyme, or cofactor involved in the enzymatic reaction, or in a labeled ligand bound to the target molecule, that produces a zone of activity associated with the compounds.

Lawn assay for compounds that affect enzyme activity or bind to target

Combinatorial libraries are disclosed which are represented by Formula I:

Process for preparing intermediates for a combinatorial

A method of inhibiting growth, transformation and/or metastasis of mammalian cells, particularly epithelial cells, in which activity of at least one enzyme, which participates in purine metabolism or regulation of nucleotide levels or the relative ratios of their phosphorylated states, is elevated. In particular, a method of inhibiting transformation, growth and/or metastasis of mammalian cells in which a DNA tumor virus, a DNA tumor virus factor or other factor which has an equivalent effect on cells has acted.

Method of inhibiting transformation of cells in which purine metabolic

The present invention relates to the use of analogs of creatine, such as cyclocreatine, as antiviral agents. Analogs of creatine can be used as antiviral agents against a variety of viruses, particularly DNA viruses, such as Herpes viruses (e.g., HSV-1, HSV-2, cytomegaloviruses, Varicella-Zoster virus) and adenovirus. The invention further relates to creatine analogs including four classes of creatine analogs selected as candidate antiviral compounds: (1) creatine analogs that can be phosphorylated by creatine kinase but differ in their phosphoryl group transfer potential, (2) bisubstrate inhibitors of creatine kinase comprising covalently linked structural analogs of adenosine triphosphate (ATP) and creatine, (3) creatine analogs which can act as irreversible inhibitors of creatine kinase, and (4) N-phosphorocreatine analogs bearing non-transferable moieties which mimic the N-phosphoryl group.

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Cranbury, NJ, United States of America

Jonathan J Burbaum