Chemical biology studies of neuroregenerative small molecules using Caenorhabditis elegans

The debilitating effects of spinal cord injury can be attributed to a lack of regeneration in the central nervous system. Identification of growth-promoting pathways, particularly ones that can be controlled by small molecules, could provide significant advancements in regenerative science and lead to potential treatments for spinal cord injury. The biological investigations of neuroregenerative small molecules, specifically the natural products clovanemagnolol and vinaxanthone, have been expanded to a whole organism context using the nematode Caenorhabditis elegans (C. elegans) as a tool for these studies. A straightforward assay using C. elegans was developed to screen for compounds that promote neuronal outgrowth in vivo. This outgrowth assay was then used to guide the design of chemically edited analogs of clovanemagnolol that maintained biological activity while possessing structures amenable to further modification for mechanism of action studies. Pull-down experiments using affinity reagents synthesized from a neuroactive structural derivative, clovanebisphenol, and the C. elegans proteome combined with mass spectrometry-based protein identification and genetic recapitulation using mutant C. elegans identified the putative protein target of the small molecule as a kinesin light chain, KLC-1. Furthermore, the small molecule-promoted regeneration of injured neurons in vivo was studied using laser microsurgery to cut specific axons in C. elegans followed by treatment with a library of analogs of the growth-promoting natural product vinaxanthone. Enhanced axonal regeneration was observed following small molecule treatment and the results were used to determine the structure-activity relationship of vinaxanthone, which may guide future development of potential drug candidates for the treatment of spinal cord injury.