Peptide microarrays in protein kinase and nuclear receptor research

Introduction

Kinases and nuclear hormone receptors are important targets in the development of new therapeutics. High-throughput peptide microarrays can be used to find a compound, which specifically acts on kinases or hormone receptors. In this paper a new technology is described which uses peptide microarrays for monitoring the kinetic behaviour of purified kinases, kinase in cell lysates and nuclear hormone receptors.

Kinases and hormone receptors belong to families of proteins, which comprise hundreds or tens of members, respectively. Both are involved in signal transduction where they are part of the cell’s machinery, orchestrating the mechanisms of transforming an incoming signal into a cellular response. Complex signalling pathways can be triggered involving many protein members. A non-specific reaction of a drug compound with a non-target protein could lead to undesired side effects. The main suspects are proteins from the same family. Proteomic technologies with sufficient throughput can help drug discovery and development by enabling the study of effects on multiple members of a protein target family at once.

This paper describes peptide microarrays based on methodology similar to that used in classical DNA microarrays, but with significant features like kinetic read-out [1,2,3]. Its application is not limited to the detection of protein abundance; it also allows the study and monitoring of protein functions. The monitoring of proteins’ enzymatic activities (kinases) as well as protein interactions (nuclear receptors) will be discussed.

Three-dimensional

Figure 1. The 3-Dimensional flow-through PamChip® array technology
A) Electron microscope image showing the porous structure of the anopore sheets, the solid support in which the peptides are immobilized. B) Each peptide position is called a spot. C) Up to 400 peptide spots are present per microarray. D) A chip comprises 4 or 96 peptide arrays. E) Flow-through fluidics using 25 uL volume sample. F) Kinetics: during the incubation multiple fluorescent microscopic images are taken

Most microarray technologies use a 2-dimensional surface as a solid support for immobilization of detector molecules. However the technology presented here, referred to as PamChip® technology [1,2,3], is based on the porous, 3-dimensional, anopore, an aluminium oxide-based material (figure 1). Up to 400 peptides can be present per array and chips with 4,12 or 96 arrays are available. Together with the fully integrated and automated PamStation™ instruments which pump, wash and capture images, a number of features are available which are not possible with the commonly used two-dimensional systems [4,5,6]. For example, due to the continuous pumping of sample up and down through the microarray, diffusion is not rate limiting. In addition, reaction kinetics can be monitored in real time.

Applications in kinase research
The peptide microarray technology described above has been applied to kinase research for measuring activity of recombinant and purified kinases and also measuring kinase activities in cells. Kinases direct the propagation of cellular signals by transferring a phosphate group from ATP to a particular protein substrate. The part of the sequence being phosphorylated is called a phosphosite. Incubation of an array comprising different phosphosite peptides with either purified kinases or with lysates from cells, results in phosphorylation of these peptides. The phosphorylated peptides are recognized by a fluorescently labeled antibody, which is detected by the integrated CCD based optical system (see Figure 1). In this way kinetic read-outs of phosphorylation reactions are obtained.

Purified kinases

Figure 2. The protein kinase assay. A peptide array comprising 144 different phosphosite peptides is incubated with purified kinases (Csk or Yes shown) or a cell lysate comprising multiple kinase activities (not shown). Time curves of signals plotted versus time are generated for each of the 144 peptides

In Figure 2 an example is shown of an experiment in which the kinase assay was used to investigate the peptide substrate preference of two different purified kinases: Csk kinase and Yes kinase. These kinases were incubated on an array comprising 144 peptide substrates. The incubation mixture contained cofactors like magnesium and ATP. The images generated show different profiles for the two kinases and also show that the signals obtained are ATP dependant. Furthermore, by monitoring the fluorescent signals, it can be seen that the activity of the kinase can be inhibited using a kinase inhibitor (data not shown). Either the identity of the peptide most efficiently phosphorylated or the consensus sequence based on the peptide sequences giving the highest signals is the basis for subsequent experiments. These can vary from the development of a kinase activity assay to the identification of a downstream protein substrate.

Various kinase assays have been developed to run on this microarray platform, both from the family of tyrosine kinases as well as serine/threonine kinases. The availability of a panel of different kinase assays enables the screening of compounds, which inhibit kinase activity, on the basis of both potency and selectivity. By focusing on peptides, which are selectively recognized and phosphorylated by two or more kinases of interest, multiplex inhibition assays can also be run. A multiplex assay is a test in which two or more kinases are present per sample. Potency and selectivity of inhibition are measured concomitantly.

Cell based kinase assays
A multiplex assay format is especially of interest in studying compounds’ effects in cells since multiple kinases are present in every cell. Monitoring the effect of kinase inhibitors on the kinase activities present in the cell enables selectivity screening of compounds in cells. Experiments have been performed in which cell lines were treated with different kinase inhibitors (data not shown). For each peptide the percentage of inhibition in comparison to the control has been determined resulting in an inhibition profile. The resulting profiles of selective inhibitors were different from those derived from non-selective inhibitors. Selective compounds hit less kinases and thus less peptide phosphorylations were inhibited. Non-selective compounds hit many kinases and as a result much more peptide phosphorylations were inhibited.

Nuclear hormone receptors
Nuclear hormone receptors are ligand dependent transcription factors that regulate cellular development and metabolism through control of gene expression. Transcriptional activities of these receptors can be regulated by ligand-modulated interaction with co-activator or co-repressor proteins.
The transcription complex comprises multiple interactions between the nuclear receptor and many of these regulating proteins. This complexity is however lost in most in vitro assays, where this system is reduced to an assay in which the nuclear receptor binds to a single peptide representing only one of the multiple activator and repressor proteins.

In Figure 3, an example of an experiment is shown in which a microarray was used comprising nine different peptides containing so-called LxxLL motifs present in the binding site of the co-regulatory proteins responsible for binding to the nuclear hormone receptor. Incubation of this type of peptide array with two different nuclear hormone receptor constructs resulted in clearly different interaction profiles. The differences reflect the preference and selectivity of binding between the nuclear hormone receptors and the LxxLL box containing proteins. These interactions can be modulated by agonist or antagonist compounds present during incubation (data not shown), which can affect the protein-peptide interactions differently. The multiplex nature of the assay allows simultaneous determination of the potency, efficacy and selectivity of such compounds, which is beneficial for finding a selective compound in the drug discovery process [7].

Conclusion
In conclusion, a peptide microarray technology has been developed which extends the toolbox of proteomics techniques and methodologies for the life scientist or drug researcher. Complementary to other technologies this new approach delivers information on enzymatic function of proteins or binding interactions, amongst others. It is suitable for pharmaceutical, academic and diagnostic research and development.

Summary
Protein kinases as well as nuclear hormone receptors are important drug targets. Protein kinases are involved in signal transduction in which a signal is propagated by phosphorylation of tyrosine, serine and threonine residues in target proteins. This paper describes a new peptide microarray assay, which allows the study of these enzymes and their inhibitors in a multiplex way (multiple kinases at once). Up to 400 different target protein-derived peptides are immobilized on a chip. Kinase activity from a sample, incubated on an array, results in peptide dependent phosphorylation detected by fluorescence. One of the unique features of this technology is the kinetic read-out, enabled by the porous nature of the array surface. In a single 96-array experiment several thousands of kinetic phosphorylation curves are generated in parallel. Besides purified kinase samples, activities can be measured in lysates from cell lines or tumor tissue homogenates. This assay format is used to investigate, amongst others, the effect of physiological stimulation of kinase inhibitors on cellular kinase activity profiles.

The same peptide array-based technology is also applicable for studying interactions between nuclear hormone receptors and co-regulatory proteins (activators and repressors). In this assay, peptides derived from the binding sites of multiple different co-regulatory proteins are immobilized on the chip and the interaction with a nuclear receptor is monitored. Furthermore the effect of hormones, agonist and antagonist compounds on these interactions can be studied, including simultaneous measurement of potency, efficacy and selectivity.