# Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury

> **NIH NIH R01** · UNIVERSITY OF MIAMI SCHOOL OF MEDICINE · 2020 · $465,464

## Abstract

Title: Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury
Project Summary:
 Spinal cord injury (SCI) patients experience limited functional recovery, owing in part to the paucity of axon
regrowth from injured CNS neurons. Effective treatments are lacking, likely because of multiple factors, intrinsic and
extrinsic, that inhibit axon growth. Thus we require agents that target more than one source of regeneration failure.
 Kinases are ubiquitous signal transducers that regulate most cellular processes, including axon growth. To begin
to identify compounds that positively regulate axon growth, we screened 1600 small-molecule kinase inhibitors (KIs) in
an in vitro CNS neurite outgrowth assay and identified “hit” KIs that reproducibly and strongly promote outgrowth. Due
to homology of catalytic domains, KIs typically inhibit multiple kinases. This makes it difficult to identify the kinase(s)
that mediate a KI's effects on cells. We used information theory and machine learning to analyze the inhibition profiles of
KIs in relation to their effects on neurite outgrowth. This enabled us to identify, and later validate via siRNA knockdown
in primary neurons, multiple kinase targets (i.e. kinases that should be inhibited to promote neurite outgrowth). These
included previously known targets that regulate intrinsic and extrinsic inhibitor factors, in addition to several novel
candidates. Conversely, we identified kinases whose activity is critical for neurite outgrowth, and whose inhibition must
be avoided (anti-targets). We discovered several KIs that inhibit multiple targets and no anti-targets. These KIs strongly
promoted neurite outgrowth in vitro.
 We tested the KI, RO48, that had the largest effect in vitro in two in vivo models. Our preliminary experiments
indicate that RO48 is remarkably effective in vivo. It promoted robust axonal growth of the corticospinal tract (CST) in
three separate models of CST injury (pyramidotomy, funiculotomy, dorsal hemisection), and in the dorsal hemisection
model, improved forelimb function. We propose to build on these remarkable results to test the working hypothesis that
the simultaneous inhibition of RO48's five target kinases (ROCK, PKC, PRKG1, PRKX, and RPS6K) promotes sprouting
and regeneration of CST axons. This will be accomplished using viral vectors to knock down expression of the different
target kinases individually and in combination. We will do knockdown in CST neurons in the cortex. We will assess CST
axon growth at the injury site using light microscopy. We will also perform experiments to determine if RO48-induced
CST axon growth promotes axon sprouting, regeneration, or both, and whether RO48 improves behavioral outcomes such
as grasping and walking after a contusion injury.
 These experiments will 1) validate novel kinases as in vivo targets for future development of SCI therapeutics 2)
determine whether these kinases regulate CST axon sprouting, regeneration, or both, and 3) confirm...

## Key facts

- **NIH application ID:** 9917854
- **Project number:** 5R01NS100531-04
- **Recipient organization:** UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
- **Principal Investigator:** John L Bixby
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $465,464
- **Award type:** 5
- **Project period:** 2017-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9917854

## Citation

> US National Institutes of Health, RePORTER application 9917854, Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury (5R01NS100531-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9917854. Licensed CC0.

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