Mostafa Zamanian, PhD - US grants

Project Summary Parasitic nematodes infect over 1.5 billion people and pose a major challenge to human health and socioeco- nomic development in endemic countries. This tremendous global disease burden is partly curtailed by mass drug administration (MDA) programs that depend on the continued ef?cacy of a small number of anthelmintic drugs. Parasite resistance to anthelmintic chemotherapy is widespread in veterinary medicine and constitutes a signi?cant and emerging threat in human medicine. The early detection of resistance-associated alleles in nematode parasite populations is critical to the goal of slowing anthelmintic resistance and extending drug lifes- pan. This goal is currently hampered by our inadequate understanding of the genetic and molecular mechanisms that underlie anthelmintic resistance in parasitic nematodes. The experimental intractability of human nema- tode parasites necessitates the development of new approaches to discover and validate relevant markers for resistance. Benzimidazoles and avermectins comprise essential classes of broad-spectrum anthelmintics, and human-approved formulations of these drugs are mainstays in the treatment of intestinal and ?larial nematode infections. I have mapped quantitative trait loci (QTL) associated with avermectin and benzimidazole resistance using an innovative high-throughput whole-genome statistical genetics pipeline in the powerful model nematodes Caenorhabditis elegans and Caenorhabditis briggsae. These data reveal a complex multigenic basis for aver- mectin resistance and implicate an exciting and entirely novel small RNA-mediated mechanism for benzimidazole resistance. I propose to narrow these genomic loci to resolve undiscovered protein-coding and RNA genes that robustly and causally contribute to anthelmintic resistance. My central hypothesis is that these model nematode systems can be used to identify new genetic mechanisms and loci that are predictive of anthelmintic resistance in medically important parasitic nematodes. To test whether determinants of anthelmintic resistance are appreciably conserved, putative genetic markers identi?ed in C. elegans will be experimentally validated in the mosquito-borne human ?larial parasite Brugia malayi, an etiological agent of lymphatic ?lariasis, and the soil-transmitted intesti- nal nematode Ascaris suum. Upon completion, this project will provide fundamental new data on novel genetic mechanisms of nematode avermectin and benzimidazole resistance, and produce a set of validated markers that can be used to monitor anthelmintic resistance in human nematode parasites to help ensure the future success of chemotherapy-based helminth control.

PROJECT SUMMARY: Parasitic Schistosoma blood flukes cause the neglected topical disease schistosomiasis. This disease infects >200 million people but is clinically treated by monotherapy with just one broad spectrum drug, praziquantel (PZQ). The vast scope of the disease and the prospect of PZQ resistance highlight the need for alternative therapies. Indeed, PZQ treatment failure has been reported in the field and resistance can be selected in the lab, indicating that standing genetic variation for resistance already exists. However, drug discovery efforts are hampered by our poor understanding of how existing anti-schistosomal drugs work. Many of these older compounds were discovered >40 years ago using in vivo animal screens. The long-term goal is to identify druggable targets / lead compounds to treat schistosomiasis. The overall objective of this application is to resolve the molecular changes in schistosome biology that underpin the efficacy of existing anti-schistosomal compounds, moving beyond superficial descriptors of worm morphology towards quantitative endpoints that can be assayed for new leads. The central hypothesis is that anthelmintics evoke molecular changes that are more productive screening endpoints than superficial phenotypes. The rationale for this project is that current schistosomiasis drug discovery efforts often focus on in vitro assays for changes in movement or morphology, which are poor predictors of efficacy in vivo. Some anti-schistosomal drugs do cause changes in worm movement / morphology, but others are efficacious in vivo with no effect on movement in vitro, and still other drugs impair movement in vitro but are ineffective in vivo. Better predictors of anti- parasitic action are needed to develop more productive assays. We will pursue two specific aims: (1) Profiling the activity of 10 chemically diverse anti-schistosomal drugs using a panel of molecular assays and (2) Identifying transcriptional signatures of anthelmintics with distinct mechanisms of action. The first aim will resolve mechanistic similarities and differences between these 10 anthelmintics by systematically assessing cellular and molecular changes in worms exposed to drug in vivo. These outcomes will serve as endpoints for the development of quantitative assays for future screens. The second aim will compare the transcriptional responses of parasites to each drug, providing an unbiased readout of global changes to schistosome biology and allowing drugs to be binned according to putative mechanisms of action. Insight into the comparative mechanisms of anti-schistosomal compounds will allow us to rationally select alternative drugs in the event of PZQ treatment failure. This information will also inform drug-combination strategies to prevent the emergence of drug resistance. The research proposed in this application is significant because it will provide new assays for future drug discovery efforts that are better predictors of in vivo anti-parasitic efficacy than existing superficial phenotypes of worm movement and morphology. This research is innovative because it is will establish mechanism-based assays to identify PZQ-alternatives to combat and prevent drug resistance.

Project Summary Parasitic nematodes infect over 1.5 billion humans. Control of this poverty-associated global health burden relies almost entirely on the administration of a small number of anthelmintic drugs. The prospects of anthelmintic resistance and the sub-optimal nature of these drugs in many nematode parasites demand new approaches to parasite treatment and control. However, the need to develop new antiparasitic treatment options is hampered by large gaps in our basic knowledge of the nematode biological processes that promote the establishment and maintenance of infection. Excretory-secretory (ES) products released by parasitic nematodes into their host environments are essential for host immune modulation and successful parasitism. Despite the general under- standing that the ES system is a conduit for the release of molecules (proteins and vesicles) that promote parasite survival, we have a poor understanding of the underlying structure and function of the ES apparatus in medically important parasitic nematodes. To address this gap in knowledge, this project will identify regulators of secretory function in Brugia malayi, a mosquito-transmitted ?larial nematode and causative agent of human lymphatic ?lar- iasis (LF). Recent studies in B. malayi, including on the mode of action of ivermectin, support the premise that the ES apparatus is a lucrative and unexploited source of new therapeutic targets. Our overarching hypothesis is that cell-surface receptors localized to the B. malayi ES system directly or indirectly control parasite secretory function, and that they can be targeted to interfere with the release of ES-derived molecules. We will pursue three aims, motivated by preliminary receptor leads and made feasible by innovative methods we have optimized to resolve the transcriptomic state of the B.malayi ES system and to pro?le receptors implicated in parasite se- cretory function. We focus our efforts on G protein-coupled receptors (GPCRs), which are proli?c drug targets and known to be expressed in nematode ES cells and adjacent cell types that may act on the ES system. In Aim 1, we will use innovative spatial transcriptomics approaches to resolve the transcriptome of the B. malayi ES region across intra-host stages and to identify candidate GPCRs that regulate ES function. In Aim 2, we will use reverse genetics and chemical approaches to assess the role of ES-localized GPCRs in the regulation of B. malayi secretory function. In Aim 3, we will use whole-organism model nematode and mammalian single-cell heterologous expression platforms to de?ne the pharmacology of ES-localized GPCRs and to establish functional assays for GPCR screening. Completion of this project will produce fundamental new knowledge about the ?larial nematode ES system and deliver new lead targets and validated screens for novel anti-?larial drug discovery.