# Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury.

> **NIH NIH R01** · UNIVERSITY OF MIAMI SCHOOL OF MEDICINE · 2021 · $16,781

## Abstract

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 whether the substantial stimulation of axon growth induced by treatment
with RO...

## Key facts

- **NIH application ID:** 10393353
- **Project number:** 3R01NS100531-05S1
- **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:** 2021
- **Award amount:** $16,781
- **Award type:** 3
- **Project period:** 2017-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10393353

## Citation

> US National Institutes of Health, RePORTER application 10393353, Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury. (3R01NS100531-05S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10393353. Licensed CC0.

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