October 15, 2006 (Vol. 26, No. 18)

Radioactive and Fluorescent Methods Draw Considerable Interest

Kinases play a key role in many cellular signaling pathways and in several diseases, such as cancer and inflammation. Kinase inhibition assays comprise about 20–30% of current high-throughput screening drug discovery programs. Some of the most current technologies were discussed at the recent Society for Biomolecular Sciences conference in Seattle.

Technologies to address kinase assays typically fall into three categories: radioactive, fluorescent, and general methodologies. Radioactive assays are time-tested, use radio-labeled phosphates to detect kinase phosphorylation, and are sensitive and specific. Fluorescent kinase assays are based on time-resolved fluorescence (TF), fluorescence polarization (FP), or time-resolved fluorescent resonant energy transfer (TR-FRET).

FRET is an event that occurs when donor and acceptor fluorescent molecules get into close proximity, whereupon the energy normally emitted from the donor is transferred in a radiationless event to excite the acceptor. This in turn leads to light observed at the acceptor’s emission wavelength. When the donor is a long-lifetime fluorophor, such as a lanthanide complex, the fluorescence resulting from a FRET event can be read in a time-resolved mode.

TR-FRET allows one to circumvent inhibitor compound and background fluorescence interference. Fluorescent assays use specific antibodies to detect kinase phosphorylation, or chelates, that can bind phosphorylated residues. General methodologies include ATP-consumption assays, assessing ADP production, or using miniaturized electrophoresis devices that separate the kinase-phosphorylated moieties.


“A good technology for kinase assays should ideally address the following—Is it compatible with a customer’s screening needs, and in particular, with specific assay requirements (e.g. ATP concentration)? Is the enzyme consumption for assays economical? Is it compatible with natural protein and peptide substrates?” said Francois Degorce, Ph.D., head of HTRF (homogeneous time-resolved fluorescence) marketing and business development of Cisbio (www.htrf.com).

Cisbio presented a tutorial on kinases and discussed the company’s HTRF® KinEASE™ technology for serine/threonine kinase activity assays. “HTRF KinEASE is an antibody-based fluorescent kinase assay system that uses a proprietary antiphosphoserine-specific monoclonal antibody that recognizes a conserved epitope in the serine/threonine kinase family and a choice of three biotinylated substrates.

“The conserved epitope gets phosphorylated by the exogenously added kinase and detected by the specific antibody in the presence of streptavidin, respectively conjugated to Europium Cryptate and XL665. Our HTRF KinEASE kits have been validated on more than 104 serine/threonine kinases and provide additional benefits, such as low enzyme consumption, high sensitivity, activity over a large range of ATP concentration, short assay development time, and amenability to assay miniaturization,” explained Dr. Degorce.

Nonantibody-based Technology

Antibody-based kinase assays, however, have there own challenges, noted Annegret Boge, Ph.D., director for reagent and assay R&D at Molecular Devices (www.moleculardevices.com). “There was a need in the market for a homogenous, reliable, and nonantibody-based technology for detecting kinase activity.

“Classic kinase assays typically use radioactive isotopes or highly specific antibodies. While antibodies exist for detecting activity of tyrosine kinases, good antibodies for phospho-serine and -threonine epitopes have limited breadth of application. This makes it necessary to invest considerable resources into antibody generation during assay development for serine/threonine kinases. Even then antibodies as biological products often have cost and stability issues, as well as lot-to-lot variabilities.”

Dr. Boge described the company’s IMAP® (immobilized metal assay of phosphorylation) platform as a nonradioactive homogenous assay technology that is centered around the specific and high-affinity interaction of phosphorylated molecules with trivalent metals immobilized on nanoparticles. The phosphorylation can be on serine, threonine, or tyrosine, making IMAP a generic platform to assess kinase activity without the need for specific antibodies.

Fluorescently (Fam or TAMRA)-labeled peptides serve as substrates for the kinase reaction. The IMAP binding system stops the kinase reaction and specifically binds the phosphorylated substrates. Phosphorylation and subsequent binding of the substrate can be detected either by FP or TR-FRET. The terbium complex (instead of europium lanthanide) used for the IMAP platform is very stable even under the low pH and high ionic strength employed in IMAP.

Dr. Boge discussed data that demonstrated the validity of this platform for screening sphingoshine kinase (a lipid kinase) inhibitors. Sphingoshine kinase is often upregulated in cancer cells and acts as a signaling molecule that promotes the characteristic out-of-control cell proliferation and differentiation in cancerous tumors.

Combining GFP and TR-FRET

Kurt Vogel, Ph.D., technical area manager for biochemistry at Invitrogen (www.invitrogen.com), unveiled the company’s TR-FRET-based GFP-fusion protein substrates for use in biochemical and cell-based kinase assays. SBS was the launching pad for Invitrogen’s cellular LanthaScreen™ format.

“A vast majority of kinase assays use synthetic and unnatural peptides as substrates for kinases. We already offer the TR-FRET LanthaScreen peptide-based kinase assay format, where the phosphorylated peptide substrate is detected by terbium-labeled antibody that recognizes the phosphorylated site. With LanthaScreen ‘Whole Protein’ kinase substrates, protein substrates are fused to a green fluorescent protein (GFP) protein. The phosphorylated protein is detected by a terbium-labeled antibody that recognizes the phosphorylated entity.

“With the cellular LanthaScreen format, Invitrogen has taken it one step further. The GFP fusion-protein substrate can be stably or transiently expressed in a cell, stimulated in a physiologically relevant context, and its phosphorylation state determined by subsequent cell lysis. This combination of GFP and TR-FRET allows the readout to be done in a standard fluorescent plate reader.

“The LanthaScreen whole protein kinase substrates enable routine analysis of difficult kinases in a standard biochemical assay and are adaptable to a cell-based format,” explained Dr. Vogel. He presented data that validated this technology in TNF-a stimulated phosphorylation of IKBµ, as well as inhibition of IKBµ phosphorylation in HEK293 cells.

Gain-of-Signal Assays for Kinases

“With growing interest in evaluating unactive kinases as putative targets, the market is looking for approaches where screening technology is a nonactivity-based approach,” said Sailaja Kuchibhatla, senior vice president of business development at DiscoveRx (www.discoverx.com). “HitHunter™ kinase-binding assays are preferred when both kinase substrate and detection antibody are unknown and kinase activation is not necessary. It is the only high-throughput screening technology currently available for unactive and low-activity kinases.”

DiscoveRx presented two posters, the ADP Hunter Plus and the HitHunter™-b-galactosidase enzyme fragment complementation for kinase binding assays. The ADP Hunter Plus is a nonradioactive, nonantibody approach that measures ADP generation (gain-of-function) in the context of ATP depletion instead of just measuring ATP depletion (loss-of-signal).

“Unlike ATP-depletion assays, ADP-production assays can be performed with whole protein or peptide substrates. ADP output is measured by subsequent ADP-mediated fluorescent signal. The assay conditions have a high tolerance for DTT and are not hampered by background or compound fluorescence,” explained Kuchibhatla.

HitHunter kinase binding assays are based on b-galactosidase-based enzyme fragment complementation. This technology is based on two inactive fragments (large fragment EA and peptide fragment ED) of b-galactosidase enzyme that become active when combined in solution. For kinase binding assays, the ED fragment is coupled to ligands that compete with kinase inhibitors for binding to kinase of interest.

Seth Cohen, Ph.D., director of application sciences at Caliper Life Sciences (www.caliperls.com), presented a tutorial on “Labchip technology from assay development to mechanism of action.” He discussed the use and validation of Caliper’s LabChip® 3000 Drug Discovery System with mobility-shift kinase assays.

“A key feature of the LabChip 3000 system is it allows one to look at enzyme rates in a real-time mode, as one can analyze samples while the kinase reaction is in progress. The LabChip 3000 uses Caliper’s sipper chips for real-time automated sampling.

“Combining real-time kinetics analysis using the LC3000 with robotic automation and design of experimentation software, a wide variety of assay conditions enable rapid kinase assay development. One can, for example, run 370 kinase reaction conditions varying buffer type, pH, detergents, and salts in a single-plate experiment.

“The ability to combine robotics in assay execution along with automated data analysis results in an efficient and noniterative assay-development process,” said Dr. Cohen. Many firms have implemented LabChip technology, notably Pfizer, which is doing compound profiling of kinase inhibitors using the mobility-shift format.

Kinase Inhibitor Assays

Qing-Fen Gan, Ph.D., principal research scientist at Roche (www.roche.com), presented a poster titled, “Mn2+ Decreases the Observed Potency of Kinase Inhibitors.” Roche has one compound in Phase II for rheumatoid arthritis. Dr. Gan’s poster focused on the importance of carefully evaluating reaction components in kinase assays. She presented and discussed results from a kinase inhibitor study that demonstrated that added Mn2+ might decrease assay sensitivity to compounds.

“Mn2+ is sometimes added to kinase activity assays in addition to or instead of Mg2+. It is particularly useful to improve the activity of low-activity kinase enzymes. However, when we used a commercial Mn2+-containing kinase assay to test the activity of about fifty inhibitor compounds, we found that their IC50 potencies were lower with Mn2+ than in the absence of Mn2+. Our results reinforce the need for careful assay validation,” explained Dr. Gan.

Cellular kinase Solutions

Lisa Martel, Ph.D., scientist II at MDS Pharma Services (www.mdsps.com), discussed the company’s “Cellular Kinase Solutions” at the recent CHI show. MDS Pharma provides a comprehensive line of kinase assay services to address the in vitro and in vivo aspects of drug discovery. Dr. Martel discussed two key offerings, FastKinase™ Profiling for rapid kinase specificity and selectivity profiling and the PathTrak™ assays for predicting kinase efficacy and selectivity in the native cellular environment.

“The ability to generate highly potent and selective compounds is a key challenge faced by drug discovery programs. Selectivity is a particular challenge when targeting kinases, because many compounds are designed to bind in the highly homologous ATP-binding domain.

“PathTrak detects phosphorylation states of natural proteins that are already present in a cell without the need for over-expression. It is particularly useful in characterizing potency and selectivity of compounds early on in the drug discovery process. The assay measures changes in phospho-protein levels due to compound-mediated inhibition of upstream kinase activity in signaling pathways. We use pre-validated and well-characterized cell systems, such as primary HUVECs and Jurkats, as well as additional, tumor cell lines, for cellular kinase studies. They work equally well for known kinase or known target, as well as for unknown kinase or unknown targets,” explained Dr. Martel.

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