Chemical interrogation of the neuronal kinome using a primary cell-based screening assay

Abstract

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention, small molecule protein kinase inhibitors present a promising therapeutic strategy. Here, we report a robust cell-based phenotypic assay, utilizing primary rat hippocampal neurons, for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening, as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened, revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies.

Figure 1. High content analysis of neurons in culture

Neurons in 96 well plates immunostained for βIII-tubulin (cell bodies and neurites), and nuclei (Hoechst). a) DMSO treated control cells, b) traced image of a, c) test compound treated cells, d) traced image of c. The images were automatically analyzed by the software to yield dozens of morphological measurements for each neuron in the well.

Figure 2. Effects of hit compounds on distinct neurite parameters

a) The Pearson correlation coefficient (r²) was calculated for each pair of neurite features using the data obtained for each hit KI at the concentration at which maximum neurite total length (NTL) was observed. b) The effects (% change from control) of KI hits on NTL, NAL, NBA, and NC are shown at the concentrations at which maximal effect on NTL was observed for each compound. Clustering was performed in MATLAB using a Euclidean metric in the KI dimension and a correlation metric in the neurite feature dimension. Activity clustering shows that certain compounds, such as ML-7 and Flt-3, increased multiple aspects of neurite growth, while others, such as VEGF Receptor tyrosine kinase inhibitor II, appeared to have more isolated effects on neurite length.

Figure 3. 12 PKI hits representing different scaffolds and structures

a) Bcr-abl Inhibitor b) Bisindolylmaleimide I c) Compound 56 d) Flt-3 Inhibitor III e) Lck Inhibitor f) Rho Kinase Inhibitor IV g) IKK Inhibitor VII h) Src Kinase Inhibitor I i) Tpl2 Kinase Inhibitor II j) AMPK inhibitor k) ML-7 l) Olomoucine II.

Figure 4. Densitometric analysis of phosphorylation by western blotting

Phosphorylation of indicated markers was analyzed by western blotting in HEK 293T cells treated with 12 different PKI hits for 3 hours. Each colored block represents the change of phosphorylation, where black means no change, red means increased phosphorylation, and green means decreased phosphorylation (values are averaged over three independent experiments). Hierarchical clustering was performed in MatLab using a Euclidean metric. A sample blot is included for total S6 (α-S6) and phospho-S6 (α-pS6): 1) Bcr-abl inhibitor, 2) Bisindolylmaleimide I, 3) Compound 56, 4) Flt-3 inhibitor III, 5) Lck inhibitor, 6) ROCK inhibitor IV, 7) Src Kinase inhibitor I 8) AMPK inhibitor, Compound C, 9) IKK inhibitor VII, 10) ML-7, Hydrochloride, 11) Olomoucine II, 12) Tpl2 Kinase inhibitor II, 13 & 14) DMSO (solvent). Representative western blots for all markers are provided in supplementary figure 3.

Figure 5. ML-7 promotes axon growth in transected cortical slice cultures

a) Bilateral cortical slices connected by the corpus callosum were prepared from P5 rats and treated with viral particles encoding eGFP. After 8 DIV, the hemispheres were separated, paired with unlabeled P5 recipient tissue, and treated with ML-7 (10 μM) or DMSO (control). Axon growth was evaluated 7 days later at a distance of 1 mm from the pairing interface. b) Confocal Z stack of image strips obtained from a slice that received DMSO and a slice that received ML-7. c) Sample of traced image using automated tracing implemented in MatLab (top: confocal image, bottom: grey scale version of confocal image with superimposition of traced axons in red). d) Three replicate slices from two experiments were analyzed. ***P < 0.01, paired t test, mean ± SEM.