Drug Discovery in Liver Disease Using Kinome Profiling

Abstract

The liver is one of the most important organs, playing critical roles in maintaining biochemical homeostasis. Accordingly, disease of the liver is often debilitating and responsible for untold human misery. As biochemical nexus, with kinases being master regulators of cellular biochemistry, targeting kinase enzymes is an obvious avenue for treating liver disease. Development of such therapy, however, is hampered by the technical difficulty of obtaining comprehensive insight into hepatic kinase activity, a problem further compounded by the often unique aspects of hepatic kinase activities, which makes extrapolations from other systems difficult. This consideration prompted us to review the current state of the art with respect to kinome profiling approaches towards the hepatic kinome. We observe that currently four different approaches are available, all showing significant promise. Hence we postulate that insight into the hepatic kinome will quickly increase, leading to rational kinase-targeted therapy for different liver diseases.

Keywords: hepatitis; hepatocellular carcinoma; hepatocytes; kinases; kinome; liver; peptide arrays.

Figure 7

Common signaling pathways in liver disease (primarily HCC) and the kinase inhibitors aimed at these pathways. Receptor tyrosine kinase (RTK) inhibitors are the main group of kinase inhibitors currently used in clinical practice. Most RTKs activate common downstream pathways: the JAK/STAT pathway, the mitogenic Ras/Raf/MEK/ERK pathway, and the PI3K/PKB survival pathway. These pathways show considerable overlap, with Src activating several of their members, and the guanine nucleotide exchange factor SOS being a target for both the adaptor protein Grb (activated via RTKs) and the oncogene Bcr-Abl (Figure based on [69,123]).

Figure 1

Kinobead-based profiling constitutes a chemical proteomics approach that entails the profiling of the interaction of small molecules with kinases. Following capture by immobilized kinase inhibitors the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) or comparable technology.

Figure 2

Kinase activity assay for kinome profiling (KAYAK). Synthetic substrate peptides are pooled and incubated with cell lysate. After quenching, isotope-labeled phosphopeptides of identical sequence to substrate peptides are added in a gnostic concentration to allow absolute quantification. Following enrichment of phosphorylated substrate peptides by immobilized metal-ion affinity chromatography results are obtained by analysis through LC–MS. Pairs of light and heavy peptides differ in mass and can be quantified by calculating the ratio.

Figure 3

Phospho-kinase antibody array. Using specific capture antibodies spotted onto nitrocellulose membranes, the proteome profiler antibody array binds selected kinases when these membranes are incubated with experimental samples. A horseradish-peroxidase-coupled detection antibody, which recognizes phosphorylated tyrosine is added. Kinases are subsequently visualized by chemiluminescent detection reagents.

Figure 4

Peptide array-based profiling. Spatially addressed peptide substrates are immobilized and incubated with biological samples. Kinase activity is detected by incorporation of radioactivity into the substrates.

Figure 5

Kinetic kinase target sequence arrays. The microarray assay for kinase activity profiling is based on measuring peptide phosphorylation by protein kinases. The peptide sequences (13 amino acids long) harbor phosphorylation sites. Fluorescently labeled anti-phospho antibodies are used to detect phosphorylation activity of kinases present in the sample.

Figure 6

Analysis of native proteins for kinome profiling vs. denatured proteins for Western blot analysis. Kinome profiling can be performed on tissue samples or stimulated/unstimulated cells or cell lines. Placing cells on ice is important to fix the state of phosphorylation and enzymatic activity at the timepoint of interest. While for Western blot analysis samples are reduced and denatured by addition of (for instance) Laemmli buffer and boiling of the samples, proteins generally need to remain in their native state for kinome analysis. Mild lysis in the presence of protease inhibitors is recommended, and samples need to be kept cold at all preparatory steps. In order to reactivate resident enzymes, samples are heated to 37 °C for kinome applications depending on enzymatic activity.