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The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by<jats:italic>in vitro<\/jats:italic>testing of the best virtual hits in recombinant human Caspase-6 activity assays identified novel allosteric Caspase-6 inhibitors with IC<jats:sub>50<\/jats:sub>and<jats:italic>K<\/jats:italic><jats:sub>i<\/jats:sub>values ranging from ~2 to 13\u2009\u00b5M. This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.<\/jats:p>","DOI":"10.1038\/s41598-019-41930-7","type":"journal-article","created":{"date-parts":[[2019,4,2]],"date-time":"2019-04-02T10:03:17Z","timestamp":1554199397000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Identification of Allosteric Inhibitors against Active Caspase-6"],"prefix":"10.1038","volume":"9","author":[{"given":"Agne","family":"Tubeleviciute-Aydin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandre","family":"Beautrait","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9211-2733","authenticated-orcid":false,"given":"Jeffrey","family":"Lynham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gyanesh","family":"Sharma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6236-4633","authenticated-orcid":false,"given":"Alexei","family":"Gorelik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ludovic J.","family":"Deny","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0086-4389","authenticated-orcid":false,"given":"Naoto","family":"Soya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gergely L.","family":"Lukacs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bhushan","family":"Nagar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0116-8345","authenticated-orcid":false,"given":"Anne","family":"Marinier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea C.","family":"LeBlanc","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,4,2]]},"reference":[{"key":"41930_CR1","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1038\/cdd.2014.216","volume":"22","author":"S Shalini","year":"2015","unstructured":"Shalini, S., Dorstyn, L., Dawar, S. & Kumar, S. 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