Re-marcs on a New Kinase Assay Format

Literature Search and Review240

Anderson SN, Cool BL, Kifle L, Chiou W, Egan DA,Barrett LW, Richardson PL, Frevert EU, Warrior U,Kofron JL, Burns DJ: Microarrayed compoundscreening (mARCS) to identify activators and inhibi-tors of AMP-activated protein kinase. J BiomolScreen 2004;9:112–121.

Abstract: A novel and innovative high-throughputscreening assay was developed to identify both activa-tors and inhibitors of AMP-activated protein kinase(AMPK) using microarrayed compound screening(mARCS) technology. Test compounds were arrayed ata density of 8,640 on a polystyrene sheet, and the en-zyme and peptide substrate were introduced into the as-say by incorporating them into an agarose gel followedby placement of the gels onto the compound sheet.Adenosine triphosphate (ATP) was delivered via amembrane, and the phosphorylated biotinylated sub-strate was captured onto a streptavidin affinity mem-brane (SAM™). For detection, the SAM™ was re-moved, washed, and imaged on a phosphor screenovernight. A library of more than 700,000 compoundswas screened using this format to identify novel acti-vators and inhibitors of AMPK.

Commentary: HTS is an expensive and time-consumingprocess. In efforts to reduce reagent costs and screeningtimes, the trends have been to miniaturize assays andscreen ever more compounds per plate. Conventionally,high throughput assays have been performed in the in-dividual assay wells of a microtiter plate. Typical mi-crotiter plates can have 96, 384, or 1,536 wells, mean-

ing that this number of compounds can be screened in asingle plate-reading process. The “microarrayed com-pound screening” presented in this article is a novel andefficient alternative radioisotopic method for identifyingprotein kinase inhibitors in HTS. The authors demon-strate that the method uses less reagents and can mea-sure more compounds simultaneously than the more con-ventional formats. In the mARCS format, the compoundsare “spotted” and dried at a very high density (8,640 persheet), and are then assayed in a low melting tempera-ture gel (rather than in aqueous solution) that containsthe protein kinase enzyme and peptide substrate to whichthe [ 33P]ATP cofactor is added via a sheet of tissue pa-per. The gel serves the purpose of limiting the diffusionof the compounds such that individual assay wells are nolonger needed. A peptide product is subsequently cap-tured on a streptavidin capture membrane. If radiola-beled phosphate is transferred to the peptide substrateresiding on the card, then one can visualize the transferon a PhosphorImager as a small spot. Compounds thatinhibit the process would result in a smaller or dimin-ished spot on this phosphor image. The system is able toprocess as many as 200,000 compounds per 8-h day andresults in significantly reduced reagent consumption.Other extensions of such applications would lie in theproteases, for example, where a fluorescent readoutcould be used. The system additionally has the advan-tage of not requiring very sophisticated automated liq-uid-dispensing systems or plate readers. The system wasable to identify inhibitors from a collection of 700,000compounds that were validated using conventionalscreening formats (see Fig. 2).

FIG. 2. Novel mARCS format to identify protein kinases. Reprinted from J Biomol Screen © The Society for BiomolecularScreening, 2004.

Enzyme/Substrate Gel

+

Compound card incubation

15 minute Remove

Remove Incubate

Add SAMTM Enzyme/Substrate Gel

Add ATPmembrane

SAMTM membrane

SAMTM membrane

Wash SAMTM

Dry, imageovernight, read onphosphorscreen

120 minutes

Compoundcard

membrane