A. ABSTRACT A significant impediment towards improvement of outcome after spinal cord injury (SCI) is the limited axonal regeneration due to insufficient regrowth of the axonal fibers at the lesion site. Signaling mediated by RhoA GTPases presents a significant impediment to growth. To date, there exists no small-molecule RhoA antagonists that have shown in vivo efficacy. This is attributed to the fact that small GTPases like RhoA do not have druggable pockets, making the development of small molecules that directly bind to them extremely difficult. However, over the years, several proteins with druggable pockets have been discovered to interact with RhoA. We stipulated that small molecules that bind to these druggable targets will promote axonal regeneration and recovery to a similar or greater extent than direct inhibition of RhoA activity. To identify such compounds we pursued a unique approach that consisted of structure-based computational docking of large chemical libraries to druggable targets that interact with RhoA. We tested the top 50-100 compounds for their effect on neurite outgrowth and axonal regeneration. One compound, RLA-47, inhibited in vitro neurite outgrowth and axonal regeneration. The compound also showed substantial axon crossings in an animal model of spinal cord injury. Our central hypothesis is that RLA-47 and its derivatives will promote axonal regeneration and recovery following spinal cord injury. Our preliminary data puts us in a strong position to test our hypothesis. In our first aim, we propose to design and synthesize derivatives of RLA-47 that will be tested in our neurite outgrowth and axonal regeneration in vitro assays. The most promising candidates will be explored in our in vivo hemisection model of spinal cord injury. In our second aim, we propose to develop RLA-47 derivatives with suitable pharmacokinetics and blood- brain permeability that promote axonal regeneration in vivo. We also test the most promising compounds using a more clinically relevant contusion model of spinal cord injury. We will use a standard chemical biology strategy of affinity purification and mass spectrometry to uncover targets of RLA-47 and its derivatives. Along with RNA sequencing, these studies will provide deeper insight into the mechanism of action of compounds. We expect 1-2 derivatives of RLA-47 to exhibit superior pharmacokinetics, brain permeability and efficacy. These compounds will lead to investigational new drug filing that we expect will eventually lead to candidates that can be explored in clinical trials either alone, or in combination with other therapies to promote regeneration and recovery in spinal cord injury patients.

Spinal cord injury is a debilitating condition that afflicts hundreds of thousands of people worldwide. In the United States alone, there are approximately 12,000 new cases that occur each year and about 270,000 people now live with spinal cord injury. The economic cost of spinal cord injury is substantial, estimated at tens of thousands of dollars per patient every year. Treatments that promote the recovery of some lower-extremity motor and sensory activity after a complete paraplegic spinal cord injury could have significant value. Currently, effective treatments are limited. There are relatively few efforts to develop small-molecule therapeutic agents to treat spinal cord injury patients. In this project, we propose to develop small molecules that could lead to therapeutic agents that could be used either alone, or in combination with existing therapies. We expect that small molecules that inhibit a protein associated with the RhoA GTPase will promote axonal regrowth and lead to repair and recovery in spinal cord injury patients.