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Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (\n              <jats:italic>Cooperia oncophora<\/jats:italic>\n              ,\n              <jats:italic>Trichostrongylus colubriformis<\/jats:italic>\n              , and\n              <jats:italic>Ostertagia ostertagi<\/jats:italic>\n              ) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In\n              <jats:italic>in vitro<\/jats:italic>\n              phenotypic assays, three kinase inhibitors (digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                Gastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of\n                <jats:italic>Cooperia oncophora<\/jats:italic>\n                ,\n                <jats:italic>Ostertagia ostertagi,<\/jats:italic>\n                and\n                <jats:italic>Trichostrongylus colubriformis<\/jats:italic>\n                for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/mbio.00095-24","type":"journal-article","created":{"date-parts":[[2024,2,15]],"date-time":"2024-02-15T13:49:15Z","timestamp":1708004955000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes"],"prefix":"10.1128","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1083-2882","authenticated-orcid":false,"given":"Hyeim","family":"Jung","sequence":"first","affiliation":[{"name":"Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8845-1724","authenticated-orcid":false,"given":"Dante","family":"Zarlenga","sequence":"additional","affiliation":[{"name":"Animal Parasitic Diseases Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John C.","family":"Martin","sequence":"additional","affiliation":[{"name":"Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"Geldhof","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitology, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kymberlie","family":"Hallsworth-Pepin","sequence":"additional","affiliation":[{"name":"Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9572-3436","authenticated-orcid":true,"given":"Makedonka","family":"Mitreva","sequence":"additional","affiliation":[{"name":"Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA"},{"name":"Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA"},{"name":"McDonnell Genome Institute, Washington University in St. Louis, St. Louis, Missouri, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_7_2_2","first-page":"290","article-title":"Prevalence of internal parasites in beef cows in the United States: results of the national animal health monitoring system\u2019s (NAHMS) beef study, 2007-2008","volume":"79","author":"Stromberg BE","year":"2015","unstructured":"Stromberg BE, Gasbarre LC, Ballweber LR, Dargatz DA, Rodriguez JM, Kopral CA, Zarlenga DS. 2015. Prevalence of internal parasites in beef cows in the United States: results of the national animal health monitoring system\u2019s (NAHMS) beef study, 2007-2008. 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