Synthesis and functional characterisation of small molecule activator of p300/CBP lysine acetyltransferase activity: Implications in spinal injury repair.

p300/CBP is a lysine acetyltransferase enzyme that modifies the lysine residue of histones by adding acetyl groups from acetyl-CoA. Dysregulation of p300/CBP KAT activity has been implicated in several neurological disorders. Pharmacological activation of p300/CBP via small-molecule provides a promising strategy to restore acetylation balance and promote neuronal repair. Our laboratory has reported that the p300/CBP-specific small molecule KAT activator TTK21 (N-[4-chloro-3-(trifluoromethyl)phenyl]-2-propoxy-benzamide), when conjugated to a glucose-derived carbon nanosphere (CSP), efficiently crossed the plasma membrane and induced acetylation. Surprisingly, its systemic administration in mice resulted in increased acetylation levels in the frontal cortex and hippocampus. Furthermore, it induced axon regeneration and improved functional recovery after spinal cord injury. Interestingly, oral administration of CSP-TTK21 also induced acetylation and promoted motor recovery in a rat model of spinal cord injury. In this chapter, we illustrate a method for the chemical synthesis of TTK21, glucose-derived carbon nanosphere (CSP), and their conjugate (CSP-TTK21). In addition, we systematically describe the methodologies for the chemical and biological characterization of the KAT activator, including the evaluation of histone acetylation in vitro and in vivo. Lastly, we present detailed experimental procedures to assess the therapeutic efficacy of CSP-TTK21 in models of spinal cord injury. Collectively, this chapter provides comprehensive protocols and practical guidance for the characterization and biological evaluation of p300/CBP activators for spinal cord injury repair.