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 towards the development of drugs for treating SCI and related neurological pathologies.

cJun promotes CNS axon growth

“…The previous observation, along with the fact that JUN has been implicated in peripheral nerve regeneration, led us to further test JUN’s role in CNS axon growth, using an organotypic cortical slice model (Blackmore, et al, 2012; Al-Ali, et al, 2013). In this model PN5 cortical slices are “matured” for 1 week in vitro, after which they are injured by transection, and transected halves are allowed to fuse (Figure 3A and Methods).…”

In vitro models of axon regeneration

“…An overexpression screen using embryonic hippocampal neurons was used to identify KLF4, a member of the KLF family of transcription factors, as a developmental regulator of axon growth 25 . Small molecule screens with these neurons have also identified kinase inhibitors that were later shown to promote regeneration of axons in cortical slice cultures and of CST axons in vivo 43,86 . Overexpression, biochemical, and computational studies have found that many of the kinases that regulate neurite outgrowth in other CNS neurons also regulate neurite outgrowth in hippocampal neurons 42,43 .…”

“…With tools like genetic fate mapping and viral vectors, it is possible to observe growing axons without classical antero- or retrograde tracers 86,192 . In recent years, cortical slice cultures have been used to study axon regrowth following manipulations of regeneration associated transcription factors 193 .…”

“…In recent years, cortical slice cultures have been used to study axon regrowth following manipulations of regeneration associated transcription factors 193 . They have also been used to study how kinase inhibitors affect axon growth 86 . Recently, spinal cord slice cultures were used to study axon growth and remyelination, as well as astrocyte migratory and growth-supportive behavior 194–196 .…”

The opportunities of mining historical and collective data in drug discovery

“…Advances in high-content imaging technologies make it possible to characterize large numbers of compounds by the morphological changes that they induce in cells, as captured by automated microscopy. Descriptive features such as cell or organelle size, shape and intensity are extracted by image processing software and give rise to phenotypic profiles [36,37] that have successfully been used to classify small molecules by their mechanistic class, define the role of a particular drug target, if multiple structurally diverse ligands produced the same phenotype, and identify compounds with unintended cellular activities within a chemical series [38][39][40][41][42].…”