Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the alpha1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel alpha1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.T HE predicted advantages and disadvantages of hapotide site, and their level of dominance can be measured loidy and diploidy, like those of recombination (Kondirectly. drashov 1993), hinge on the ability of populations (a)Two factors are preeminent in determining the effect to cope with deleterious mutation (Kondrashov and of ploidy on the rate of adaptation (Orr and Otto 1994): Crow 1991) and (b) to adapt to new environments (Orr the waiting time for mutations to appear and the fixation and Otto 1994). Our goal in this study was to make a time required for mutations to spread to high frequency direct comparison of ploidy states by measuring the in a population in response to directional selection. Under relative abilities of isogenic haploids and diploids of the conditions of finite population size where the waiting yeast Saccharomyces cerevisiae to adapt to the presence of time for beneficial mutations is the rate-limiting step in the antifungal drug fluconazole (FLC). We followed the adaptation, diploids should, at first glance, have the faster initial, rather than the long-term, responses of haploid rate of adaptation. This is because diploids have twice the and diploid populations to pinpoint the relati...

No abstract

Sequencing of plasmid-enriched samples revealed multiple genetic contexts for the resistance genes detected and highlighted the potential role of small plasmids in the movement of antibiotic resistance genes. The findings are informative for disease management in food animals, but also manure management and antibiotic therapy in human medicine for improved antibiotic stewardship.