Expression of Serine/Threonine Protein-Kinases and Related Factors in Normal Monkey and Human Retinas: The Mechanistic Understanding of a CDK2 Inhibitor Induced Retinal Toxicity

Abstract

Protein-kinase inhibitors are among the most advanced compounds in development using the new drug discovery paradigm of developing smallmolecule drugs against specific molecular targets in cancer. After treatment with a cyclin dependent kinase CDK2 inhibitor in monkey, histopathological analysis of the eye showed specific cellular damage in the photoreceptor layer. Since this CDK2 inhibitor showed activity also on other CDKs, in order to investigate the mechanism of toxicity of this compound, we isolated cones and rods from the retina of normal monkey and humans by Laser Capture Microdissection. Using Real-Time PCR we first measured the expression of cyclin dependent protein-kinases (CDK)1, 2, 4, 5, Glycogen synthase kinase 3β (GSK3β) and microtubule associated protein TAU. We additionally verified the presence of these proteins in monkey eye sections by immuno-histochemistry and immunofluorescence analysis and afterwards quantified GSK3β, phospho-GSK3β and TAU by Reverse Phase Protein Microarrays. With this work we demonstrate how complementary gene expression and protein-based technologies constitute a powerful tool for the understanding of the molecular mechanism of a CDK2 inhibitor induced toxicity. Moreover, this investigative approach is helpful to better understand and characterize the mechanism of species-specific toxicities and further support a rational, molecular mechanism-based safety assessment in humans.

Microtubule-associated protein tau in bovine retinal photoreceptor rod outer segments: Comparison with brain tau

“…Thus, it can be further speculated that, similarly to the development of human brain diseases in human retina, tau-mediated bovine brain diseases may also be developed in bovine retina. Second, Cdk5, one of the protein kinases responsible for the hyperphosphorylation of human brain tau, is present in bovine retina including ROS [31, 32]. Third, large quantities of bovine preparations are available.…”

“…The retina, a part of the visual system, contains both tau [29, 30] and protein kinases responsible for brain tau hyperphosphorylation [31, 32]. Recent studies suggest that abnormal tau may play a role in some retinal degenerative diseases [33–41].…”

ATP‐Noncompetitive Inhibitors of CDK–Cyclin Complexes

“…[22] For example, flavopiridol provokes the appearance of the hyperacute tumor lysis syndrome which determines the dose limitation of the drug, [23] and the histopathological analysis of monkey-treated eyes showed specific cellular damage in the photoreceptor layer. [24] Additionally in combination with docetaxel, flavopiridol induces neutropenia in patients with metastatic breast cancer. Moreover, the antitumor activity of these compounds in clinical trials has been modest.…”

Evanescent wave fluorescence biosensors: Advances of the last decade

“…RPPAs are distinct from standard antibody- or DNA-type arrays in that instead of using an array of different “capture” molecules immobilized in a planar array to interrogate a sample mixture of different proteins, RPPAs comprise spots of immobilized protein lysates, which are then subsequently probed with different antibodies, enzymes, or ligands. Zeptosens’ waveguide-based microarrays have been used for biomarker discovery (Assadi et al 2013; Weissenstein et al 2006), following protein expression and modification over time (Pirnia et al 2009), profiling enzymatic activities and signaling pathways (Van Oostrum et al 2009; Voshol et al 2009), and drug discovery and testing (Saturno et al 2007; Tegnebratt et al 2014) using tissues and lysates supplied by each research group and appropriate for each application.…”