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ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.<\/jats:p>","DOI":"10.1038\/s41598-020-64868-7","type":"journal-article","created":{"date-parts":[[2020,5,14]],"date-time":"2020-05-14T06:03:18Z","timestamp":1589436198000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Methodology for the identification of small molecule inhibitors of the Fanconi Anaemia ubiquitin E3 ligase complex"],"prefix":"10.1038","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1019-3729","authenticated-orcid":false,"given":"Michael F.","family":"Sharp","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2791-303X","authenticated-orcid":false,"given":"Vince J.","family":"Murphy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5602-0906","authenticated-orcid":false,"given":"Sylvie Van","family":"Twest","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3229-4157","authenticated-orcid":false,"given":"Winnie","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5875-4820","authenticated-orcid":false,"given":"Jennii","family":"Lui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9136-1781","authenticated-orcid":false,"given":"Kaylene J.","family":"Simpson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5271-4422","authenticated-orcid":false,"given":"Andrew J.","family":"Deans","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0143-8293","authenticated-orcid":false,"given":"Wayne","family":"Crismani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,5,14]]},"reference":[{"key":"64868_CR1","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1038\/nature11368","volume":"489","author":"JI Garaycoechea","year":"2012","unstructured":"Garaycoechea, J. 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