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Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive\/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 \u00b5M. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.<\/jats:p>","DOI":"10.3390\/ijms25074084","type":"journal-article","created":{"date-parts":[[2024,4,8]],"date-time":"2024-04-08T10:11:55Z","timestamp":1712571115000},"page":"4084","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8852-6071","authenticated-orcid":false,"given":"Johan","family":"Gising","sequence":"first","affiliation":[{"name":"The Beijer Laboratory, Science for Life Laboratory, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4558-501X","authenticated-orcid":false,"given":"Saman","family":"Honarnejad","sequence":"additional","affiliation":[{"name":"Pivot Park Screening Centre, Kloosterstraat 9, 5349 AB Oss, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maaike","family":"Bras","sequence":"additional","affiliation":[{"name":"Pivot Park Screening Centre, Kloosterstraat 9, 5349 AB Oss, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gemma L.","family":"Baillie","sequence":"additional","affiliation":[{"name":"BioAscent Discovery Ltd., Bo\u2018Ness Road, Newhouse, Motherwell ML1 5UH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5182-2616","authenticated-orcid":false,"given":"Stuart P.","family":"McElroy","sequence":"additional","affiliation":[{"name":"BioAscent Discovery Ltd., Bo\u2018Ness Road, Newhouse, Motherwell ML1 5UH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip S.","family":"Jones","sequence":"additional","affiliation":[{"name":"BioAscent Discovery Ltd., Bo\u2018Ness Road, Newhouse, Motherwell ML1 5UH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angus","family":"Morrison","sequence":"additional","affiliation":[{"name":"BioAscent Discovery Ltd., Bo\u2018Ness Road, Newhouse, Motherwell ML1 5UH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9425-8832","authenticated-orcid":false,"given":"Julia","family":"Beveridge","sequence":"additional","affiliation":[{"name":"The Beijer Laboratory, Science for Life Laboratory, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mathias","family":"Hallberg","sequence":"additional","affiliation":[{"name":"The Beijer Laboratory, Department of Pharmaceutical Biosciences, Neuropharmacology and Addiction Research, Biomedical Centre, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6258-0635","authenticated-orcid":false,"given":"Mats","family":"Larhed","sequence":"additional","affiliation":[{"name":"The Beijer Laboratory, Science for Life Laboratory, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1016\/0306-4522(88)90182-0","article-title":"Angiotensin Ii-(3\u20138)-Hexapeptide Affects Motor Activity, Performance of Passive Avoidance and a Conditioned Avoidance Response in Rats","volume":"27","author":"Braszko","year":"1988","journal-title":"Neuroscience"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/0361-9230(93)90297-O","article-title":"Angiotensin II(3\u20138) (ANG IV) Hippocampal Binding: Potential Role in the Facilitation of Memory","volume":"32","author":"Wright","year":"1993","journal-title":"Brain Res. 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