Chi K. Leung

An Ultra High-Throughput, Whole-Animal Screen for Small Molecule Modulators of a Specific Genetic Pathway in Caenorhabditis elegans

High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of ∼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.

Unique structure and regulation of the nematode detoxification gene regulator, SKN-1: implications to understanding and controlling drug resistance

Nematodes parasitize an alarming number of people and agricultural animals globally and cause debilitating morbidity and mortality. Anthelmintics have been the primary tools used to control parasitic nematodes for the past several decades, but drug resistance is becoming a major obstacle. Xenobiotic detoxification pathways defend against drugs and other foreign chemicals in diverse organisms, and evidence is accumulating that they play a role in mediating resistance to anthelmintics in nematodes. Related antioxidation pathways may also provide filarial parasites with protection against host free-radical–mediated immune responses. Upstream regulatory pathways have received almost no attention in nematode parasites, despite their potential to coregulate multiple detoxification and antioxidation genes. The nuclear eurythroid 2–related factor 2 (NRF2) transcription factor mediates inducible detoxification and antioxidation defenses in mammals, and recent studies have demonstrated that it promotes multidrug resistance in some human tumors. Recent studies in the free-living model nematode, Caenorhabditis elegans, have defined the homologous transcription factor, SKN-1, as a master regulator of detoxification and antioxidation genes. Despite similar functions, SKN-1 and NRF2 have important differences in structure and regulatory pathways. Protein alignment and phylogenetic analyses indicate that these differences are shared among many nematodes, making SKN-1 a candidate for specifically targeting nematode detoxification and antioxidation.

Phylogenetic, expression, and functional analyses of anoctamin homologs in Caenorhabditis elegans

Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.

Direct Interaction between the WD40 Repeat Protein WDR-23 and SKN-1/Nrf Inhibits Binding to Target DNA

ABSTRACT SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a β-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress.

A Negative-Feedback Loop between the Detoxification/Antioxidant Response Factor SKN-1 and Its Repressor WDR-23 Matches Organism Needs with Environmental Conditions

ABSTRACT Negative-feedback loops between transcription factors and repressors in responses to xenobiotics, oxidants, heat, hypoxia, DNA damage, and infection have been described. Although common, the function of feedback is largely unstudied. Here, we define a negative-feedback loop between the Caenorhabditis elegans detoxification/antioxidant response factor SKN-1/Nrf and its repressor wdr-23 and investigate its function in vivo. Although SKN-1 promotes stress resistance and longevity, we find that tight regulation by WDR-23 is essential for growth and reproduction. By disabling SKN-1 transactivation of wdr-23, we reveal that feedback is required to set the balance between growth/reproduction and stress resistance/longevity. We also find that feedback is required to set the sensitivity of a core SKN-1 target gene to an electrophile. Interestingly, the effect of feedback on target gene induction is greatly reduced when the stress response is strongly activated, presumably to ensure maximum activation of cytoprotective genes during potentially fatal conditions. Our work provides a framework for understanding the function of negative feedback in inducible stress responses and demonstrates that manipulation of feedback alone can shift the balance of competing animal processes toward cell protection, health, and longevity.

Discovery of ML358, a Selective Small Molecule Inhibitor of the SKN-1 Pathway Involved in Drug Detoxification and Resistance in Nematodes.

Nematodes parasitize ∼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report 17 (ML358), a first in class small molecule inhibitor of the SKN-1 pathway. Compound 17 resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure-activity studies. Compound 17 is a potent (IC50 = 0.24 μM, Emax = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode C. elegans to oxidants and anthelmintics. Compound 17 is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to C. elegans (LC50 > 64 μM) and Fa2N-4 immortalized human hepatocytes (LC50 > 5.0 μM). In addition, 17 exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, 17 is a valuable probe to study regulation and function of SKN-1 in vivo. By selective targeting of the SKN-1 pathway, 17 could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics.

SKN-1/Nrf, a new unfolded protein response factor?

Cell function requires simultaneous regulation of numerous processes, often under variable conditions. Several inducible pathways have been defined as being responsible for maintaining homeostasis under the threat of a particular stress such as heat, oxidizing conditions, exposure to pathogens, or loss of proteostasis. The challenge now is to understand how and why these pathways interact in basal, stress, and pathological states. The Caenorhabditis elegans inducible transcription factor SKN-1, a homolog of mammalian Nrf proteins, has been defined as the transcription factor that responds to oxidative stress. In this issue of PLOS Genetics, Glover-Cutter et al. [1] challenge this paradigm by showing that SKN-1 directly regulates the genes of core regulators and effectors of the endoplasmic reticulum (ER) unfolded protein response (UPR) and that the UPR plays a role in activation of the antioxidant/detoxification response. ER homeostasis requires coordination of protein translation, folding, and covalent modification; availability of energy and substrates; and maintenance of a redox environment that is suitable for disulfide bond formation. Disruption of ER homeostasis can cause accumulation of misfolded proteins in the ER lumen, referred to as “ER stress,” which can impair cell function and eventually trigger cell death [2]–[4]. The UPR is a eukaryotic signaling program that responds to ER stress by inhibiting protein translation, inducing protein folding chaperones, and directing the degradation of misfolded proteins [2]–[4] (Figure 1). Redox homeostasis in animal cells is controlled in part by a family of transcription factors represented by SKN-1 in C. elegans and Nrf1, Nrf2, and Nrf3 in mammals (Figure 1). SKN-1 and Nrf2 have well-established roles in promoting redox homeostasis and small molecule detoxification, and SKN-1 has been shown to promote longevity [5]–[8]. Figure 1 Summary of interactions between the endoplasmic reticulum (ER) unfolded protein response (UPR) and antioxidant/detoxification transcription factor SKN-1 in C. elegans. Glover-Cutter et al. [1] conducted an extensive series of genetic and molecular experiments to investigate regulatory interactions between SKN-1 and the UPR; many of their findings are summarized in Figure 1. They showed that SKN-1 regulates numerous genes involved in ER function during ER stress that are not typically activated by SKN-1 during oxidative stress. These include protein chaperones and homologs of the following core UPR components: BiP (an unfolded protein sensor), PERK (a protein kinase), IRE1 (a protein kinase and mRNA endonuclease), and the transcription factors XBP1, ATF4, and ATF6. During ER stress, SKN-1 protein was shown to associate with loci for homologs of ATF4, ATF6, XBP1, and IRE1, indicating that regulation of core UPR genes by SKN-1 is likely to be direct. How is SKN-1 activated by ER stress? The authors observed elevated skn-1 mRNA and protein levels during ER stress [1]. Processing of protein disulfide bonds in the ER can elevate reactive oxygen species (ROS) [4], [9], a well-established stimulus for SKN-1 that could simply activate it secondarily. However, the authors demonstrated induction of skn-1 mRNA by a strong reducing agent and by silencing of the worm ER oxidoreductase, conditions that cause ER stress and decrease ROS [1]. Furthermore, the C. elegans homologs of XBP1 and ATF6, and SKN-1 itself, all associated with the skn-1 locus during ER stress and were found to play a role in induction of skn-1 mRNA [1]. Therefore, activation of SKN-1 during ER stress appears to be at least partly transcriptional via UPR transcription factors. If SKN-1 is required for the UPR, then could the UPR also be required for the antioxidant/detoxification response? The answer may be yes. During oxidative stress, core components of the UPR were required for induction of skn-1 mRNA and some SKN-1 target genes. It is of additional interest that activation of p38 MAPK by phosphorylation, which activates SKN-1 under conditions of oxidative stress, also required components of the UPR [10]. Important regulatory and functional interactions between the UPR and Nrf2 were previously known for mammalian cells [4], [11]–[13]. Nrf2 is phosphorylated and activated by PERK during ER stress and promotes cell survival by maintaining redox homeostasis together with ATF4 [12]–[13]. Nrf2 also activates expression of proteasome subunits and may support degradation of misfolded proteins during ER stress [11]. So what is different about the current findings? Transcriptional regulation of core UPR transcription factors and downstream effectors by SKN-1 is a far more central function in the ER stress response than has previously been reported for SKN-1/Nrf family members. Details of the molecular interactions may not all be conserved, but the findings for SKN-1 raise the possibility that Nrf1, Nrf2, or Nrf3 may be centrally integrated into the mammalian UPR. As with any new findings, important new questions follow. SKN-1 was shown to contribute to survival of ER stress in vivo [1]. Determining the relative importance of SKN-1–mediated UPR gene regulation versus redox homeostasis would be challenging, but is needed to understand the function of these newly identified regulatory interactions. Other than PERK phosphorylation of Nrf2 [12]–[13], little is known about post-translational regulation of SKN-1/Nrf proteins during ER stress. Evidence was provided for association of a long SKN-1 variant with the ER [1], and Nrf1 and Nrf3 each have a predicted transmembrane domain and have been reported to be associated with the ER membrane [14]–[16]. In unstressed cells, ATF6 is a membrane protein tethered to the ER by BiP [3]. During ER stress, ATF6 undergoes cleavage to its active transcription factor form in the golgi [3]. Further work is needed to determine if SKN-1/Nrf proteins at the ER have a similar fate. Coordination between SKN-1/Nrf proteins and the UPR has been evolutionarily conserved and, therefore, may be fundamentally important to cell homeostasis. Direct regulation of the UPR implies that the ER may be able to prepare for protein damage under harsh conditions detected by SKN-1/Nrf family members. It may also ensure that redox homeostasis in the cytosol is compatible with redox-dependent protein processing in the ER. The activity of SKN-1 also responds to changes in the nucleolus [17], the proteasomes [18], nutrient signaling [5]–[6], and protein translation [19]. Therefore, an extremely complex network of signals likely converges on SKN-1 to ensure that redox status and detoxification activity are compatible with a number of cellular processes. Deciphering this signaling network will provide mechanistic insights into how a single transcription factor is influenced by multiple signals. As we continue to refine our understanding of cellular stress responses and their roles in disease and aging, it will be increasingly important to investigate how different pathways coordinate responses to optimize homeostasis, avoid incompatibilities, and mitigate competition for common substrates.

Retraction for Leung et al., Direct Interaction between the WD40 Repeat Protein WDR-23 and SKN-1/Nrf Inhibits Binding to Target DNA

Volume 34, no. 16, p. 3156 –3167, 2014. The results of an investigation initiated by the corresponding author and conducted at the University of Florida found that data in Fig. 6F were falsified by the first author. Given that one of our central conclusions concerning the ability of WDR-23 to block DNA binding by SKN-1 was based on these results, we retract the article. We emphasize that we have been able to confirm that WDR-23 preferentially interacts with the SKN-1c variant (as depicted in Fig. 1), that the top of many WDR-23 blades are required for the interaction with SKN-1 (as depicted in Fig. 3 and 4), and that the N-terminal GLRWRD domain of SKN-1c is required for the interaction (Fig. 3F and 5B to C). We are currently working to properly map the WDR-23 interaction domain within SKN-1c and identity biochemical mechanisms of regulation. We sincerely apologize to the scientific community for any time and effort wasted because of these actions.

Retraction for Leung et al., A Negative-Feedback Loop between the Detoxification/Antioxidant Response Factor SKN-1 and Its Repressor WDR-23 Matches Organism Needs with Environmental Conditions

Volume 33, no. 17, p. 3524 –3537, 2013. The results of an investigation initiated by the corresponding author and conducted at the University of Florida found that data in Fig. 1F and 6A, D, and E were falsified by the first author. The falsified data have two main scientific consequences. First, they compromise our conclusions that skn-1 controls wdr-23 mRNA levels and negative feedback controls detoxification gene mRNA levels in wild-type worms under basal conditions (see the end of the first paragraph in column 2 of p. 3531 and Fig. 6A). Second, they misrepresent differences in gst-4 induction between worms with the wild-type and mutated wdr-23 promoter at two concentrations of acrylamide tested (Fig. 6E and F at 0.875 and 7.0 mM); as a result, there is no longer support for interaction 3 of the model in Fig. 8. The falsifications in Fig. 1F and 6D were inconsequential to the conclusions of the study. To our knowledge, other conclusions are not compromised by these findings of falsification, including the central conclusion of negative feedback via a direct interaction between SKN-1 protein and the wdr-23 promoter, which has been confirmed independently of the first author. Therefore, we partially retract the paper. We sincerely apologize to the scientific community for any time and effort wasted because of these actions.