Small‐Molecule Mechanism of Action Studies in Caenorhabditis elegans. Issue 17 (7th October 2013)

• Record Type: Journal Article
• Authors: Zlotkowski, Katherine; Eliasen, Anders M.; Mitra, Aurpon; Siegel, Dionicio
• Is Part Of: Chembiochem. Volume 14:Issue 17(2013)
• Page Start: 2338
• Page End: 2344
• Publication Date: 2013-10-07
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201300399
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>A general protocol for exogenous small‐molecule pull‐down experiments with <italic>Caenorhabditis elegans</italic> is described; it provides a link between small‐molecule screens in worms and existing mutant and RNAi technologies, thereby enabling organismal mechanism of action studies for the natural product clovanemagnolol. Forward chemical genetic screens followed by mechanism of action studies with <italic>C. elegans</italic>, when coupled with genetic validation of identified targets to reproduce the small molecule's phenotypic effects, provide a unique platform for discovering the biological targets of compounds that affect multicellular processes. First, the use of an immobilized FK506 derivative and soluble competition experiments with optimally prepared soluble <italic>C. elegans</italic> proteome successfully identified interactions with FK506 binding proteins 1 to 6. This approach was used to determine an unknown mechanism of action for clovanemagnolol, a small molecule that promotes axonal branching in both primary neuronal cultures and in vivo in <italic>C. elegans</italic>. Following the synthesis of an appropriately functionalized solid‐phase reagent bearing a clovanemagnolol analogue pull‐down experiments employing soluble competition identified kinesin light chain‐1 (KLC‐1), a protein involved in axonal cargo transport, as a putative target. This was corroborated through the use of mutant worms lacking <italic>klc‐1</italic> and possessing GFP neuronal labeling, reproducing the axonal branching phenotype induced by the small molecule clovanemagnolol.</p> </abstract>