Identification of a family of species-selective complex I inhibitors as potential anthelmintics.

Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.

Identification of enzymes that have helminth-specific active sites and are required for Rhodoquinone-dependent metabolism as targets for new anthelmintics.

Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets in the anaerobic metabolic pathways STHs need to survive in their mammalian host. These require rhodoquinone (RQ), an electron carrier used by STHs and not their hosts. We identified 25 genes predicted to act in RQ-dependent metabolism including sensing hypoxia and RQ synthesis and found 9 are required. Since all 9 have mammalian orthologues, we used comparative genomics and structural modeling to identify those with active sites that differ between host and parasite. Together, we found 4 genes that are required for RQ-dependent metabolism and have different active sites. Finding these high confidence targets can open up in silico screens to identify species selective inhibitors of these enzymes as new anthelmintics.

Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers.

CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1, whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.

CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing.

SF3B1 is the most frequently mutated splicing factor in cancer. Mutations in SF3B1 likely confer clonal advantages to cancer cells but they may also confer vulnerabilities that can be therapeutically targeted. SF3B1 cancer mutations can be maintained in homozygosis in C. elegans, allowing synthetic lethal screens with a homogeneous population of animals. These mutations cause alternative splicing (AS) defects in C. elegans, as it occurs in SF3B1-mutated human cells. In a screen, we identified RNAi of U2 snRNP components that cause synthetic lethality with sftb-1/SF3B1 mutations. We also detected synthetic interactions between sftb-1 mutants and cancer-related mutations in uaf-2/U2AF1 or rsp-4/SRSF2, demonstrating that this model can identify interactions between mutations that are mutually exclusive in human tumors. Finally, we have edited an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Thus, we have established a multicellular model for SF3B1 mutations amenable for high-throughput genetic and chemical screens.

Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.

CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.

Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin.

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.This article has an associated First Person interview with the first author of the paper.

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis.

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.