Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.