May 15, 2008 (Vol. 28, No. 10)

Combining Chemistry and Instrumentation to Make the Most of microHTS and HTS Formats

Protein kinases comprise the largest family of mammalian enzymes, totaling at least 500. These ubiquitous enzymes play key roles in cellular regulation by catalyzing the reversible phosphorylation of more than 10,000 proteins.

Dysfunctional intracellular signaling through protein kinases is associated with about 400 human diseases, most notably cancers. As a result of the clinical success of the p210 Bcr-Abl inhibitor (Novartis’ Gleevec) for treatment of chronic myelogenous leukemia, pharma companies have applied massive resources to finding the next blockbuster kinase activity-modulating drug. And, as kinase-focused drug discovery enterprises expand, so does the need for discovery tools that automate and scale them.

At the Society for Biomolecular Screening’s (SBS) annual meeting held last month, companies collaborated in workshops, combining their technologies to address formidable challenges in kinase profiling and to facilitate selection of appropriate assay options.

In a workshop entitled “Assay Optimization and Kinase Profiling in micro HTS Format,” Labcyte (, Deerac Fluidics (, Promega (, Corning Life Sciences (, and BMG Labtech ( teamed up to present a complete platform approach for kinase-assay development.

Using Labcyte’s ultralow volume-test technology, Corning Life Sciences’ specially developed dual assay plates, luminescent kinase-assay technology from Promega, Deerac Fluidics’ reagent-dispensing technology, and BMG Labtech’s PHERAstar luminescent-detection instrumentation, these companies demonstrated the complementarity and adaptability of their diverse technologies for the development of extremely low-volume, protein-kinase assays.

Test Compound and Reagent Dispensing

According to Elaine Heron, Ph.D., Labcyte’s CEO, successful kinase screening campaigns in any format require precise liquid handling, robust assays, and sensitive detection methods combined to produce accurate results.

“The dirty little secret about compound dispensing in assays is that when performing transfers using a plastic pipette and an intermediate dilution, five to ten percent of the compounds in a given library are often lost,” Dr. Heron reported. She further explained that Labcyte’s technology addresses this specific problem by very precisely dispensing low volumes onto assay plates.

“Our system also allows people to save money by reducing the size of assays with better results,” Dr. Heron added. Labcyte’s Acoustic Droplet Ejection (ADE) technology, which is embodied in its Echo 555 liquid handler, enables dispensing of test compounds in extremely low volumes for screening and compound titration applications.

ADE works by transmitting sound waves through microplate wells containing test compounds. The pressure generated by a focused acoustic wave creates upswelling at the fluid surface, thereby causing a droplet to fly upward from the liquid surface. These droplets are then captured by the assay plate, which is suspended upside down over the source plate.

For the Echo systems, the size of the droplet is 2.5 nL; higher droplet volumes are attained by rapidly ejecting multiple droplets. Since there is no contact between the ejection mechanism and the sample, the technology is particularly suited to biological assays in which precise transfers are critical and cross-contamination interferes with accurate results.

According to Labcyte, its contact-free approach means that no tips or washing are needed, and compounds are not lost by adsorption onto plastic surfaces. The ability to accurately transfer 2.5 nL droplets allows miniaturization of assays to 384-, 1,536-, and 3,456-well formats economically, and with less solvent and plastic waste, Dr. Heron said.

In January, Labcyte acquired Deerac Fluids, a provider of liquid-handling systems that enable dispensing of a wide variety of liquids in 0.05–50 µL volumes. According to Dr. Heron, Deerac’s systems “expand the range of low-volume liquid-handling systems we are able to offer our customers.”

Deerac’s technology consists of a feed-back-controlled, air-displacement technology originally developed at Trinity College, Dublin. For the microHTS-format kinase-assay applications described at the workshop, Labcyte’s ADE-based dispensers were used to add test compounds to the assays and the Deerac products to add reagents.

Corning Life Sciences worked closely with Labcyte to develop the compound-assay plates that allow ADE-based dispensing with Echo systems. For these assay configurations, the plates containing the wells in which the assays are performed are upside down over the right-side up Echo source plates. “The 1,536-well flat-bottomed plates are made of an acrylic olefin copolymer that is acoustically clear, meaning that an acoustic signal can transfer the Echo system signal clearly,” explained Kim Titus, business development manager for Corning Life Sciences.

These source plates, she added, were developed with a well-geometry design that allows accurate transfer to the center of the assay plate wells, reducing the chance that test drugs will “jump into wells where you don’t want them.”

Lighting the Way to Kinase Inhibitors

Promega’s bioluminescent systems provided the kinase chemistry for the micro-HTS-format kinase assays described at the workshop. Promega offers three bioluminescent assay systems, Kinase-Glo®, Kinase-Glo Plus, and Kinase-Glo Max, to identify kinase inhibitors in screening applications, each of which can be used with increasing ATP substrate concentrations.

According to Promega, kinase screening at higher ATP levels increases the likelihood of identifying nonATP binding-site inhibitors. The assays detect the effect of potential inhibitors on kinase activity by quantifying increasing concentrations of the kinase substrate ATP as kinase activity decreases.

Designed for use with multiwell plate formats, the assays are suited to automated microHTS and HTS screening. “Our assays are platform agnostic, allowing us to collaborate with a variety of companies,” noted Neal Cosby, HTS/drug screening marketing manager at Promega. “We can adapt our technology to different customers’ needs and formats.”

The Kinase-Glo assay procedure involves the addition of a single reagent directly to a completed kinase reaction, thereby resulting in the generation of a luminescent signal produced by a recombinant luciferase. The signal correlates with the amount of ATP present and is therefore inversely proportional to kinase activity. The half-life of the luminescent reaction output is greater than five hours and is therefore well suited to HTS applications.

Cosby reported that Kinase-Glo recombinant luciferase-based assays are less likely to be affected by background interference from sample components. Data presented at the workshop demonstrated that scaling down to microHTS formats did not adversely affect assay quality as measured by Z factor analysis.

Flexible Detection Technology

BMG Labtech’s PHERAstar was used to detect both the luminescent signal generated by the Kinase-Glo assay in the microHTS format described above, and the output of a time-resolved fluorescence immunoassay embodied as Invitrogen’s ( Adapta™ Universal Kinase Assay in an HTS format.

BMG and Invitrogen teamed up to host a workshop entitled “Screening for Lipid Kinase Inhibitors on the PHERAstar using Adapta Universal Kinase Assay.” The workshop featured the adaptation of BMG’s multidetection micoplate reader and Invitrogen’s time-resolved fluorescence resonance energy transfer (TR-FRET) technology.

E.J. Dell, Ph.D., an applications scientist at BMG Labtech, commented that lipid kinases pose a particular challenge to kinase assay developers because “they can be sticky, ubiquitously interacting with different proteins and lipids.” This tendency to stick to multiple effectors compromises accurate detection and readout of phosphorylation reactions making them a difficult and expensive kinase target to study.

Invitrogen and BMG Labtech’s solution was to combine PHERAstar’s simultaneous dual emission detection system with the Adapta assay, which Invitrogen licensed from Bellbrook Labs.

According to Dr. Dell, the Adapta assay quantifies kinase activity indirectly in terms of ADP formation, the by-product of a phosphorylation reaction, instead of measuring kinase activity or ATP depletion, thus allowing more accurate measurements of lipid kinases to be achieved.

“Due to the inherent complexity of the PI3 Kinase family, traditional biochemical approaches used with protein kinase assays are not readily transferable to the lipid kinases,” said Robert Horton, Ph.D., senior scientist at Invitrogen. “The sensitivity of the Adapta assay to ADP formation, and its versatility to accommodate a wide gamut of substrates, enables profiling of small molecule inhibitors across the entire PI3 Kinase family with a single assay format.”

Adapta, being a TR-FRET assay, is extremely sensitive to ADP formation. This, according to Dr. Dell, increases the assay’s suitability to detect low-level kinase activity since most of the signal changes occur within the first 10–20% of ATP to ADP conversion. The assay kit can be used for a wide range of ATP concentrations (1–500 mM), eliminating the need for multiple kit purchases, he added.

The assay itself measures ADP formation during the course of a kinase reaction by providing a tracer amount of ADP labeled with a red acceptor fluorophore and an antiADP antibody tagged with a Europium chelate, that when excited in turn can excite the flurophore, causing light to be emitted in the far-red range.

When no ADP is present, the tracer ADP is complexed with the Europium-labeled antibody producing a high TR-FRET signal, measured at 665 nm and 620 nm (emission wavelengths of the red fluorophore and Europium, respectively).

When ADP is formed, it displaces the tracer amount of ADP thereby causing the TR-FRET signal to decrease. Potential lipid kinase inhibitors could be found if a high TR-FRET signal is maintained because there would be no kinase activity and no ADP formation.

According to Dr. Dell, the PHERAstar multidetection microplate reader can simultaneously detect both emissions at once to yield results. “Other instruments need to read the plate twice, decreasing sensitivity and increasing assay time and intra-assay variability in terms of percent CVs and Z prime values.”

Patricia F. Dimond, Ph.D., is a life science consultant. E-mail: [email protected]

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