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de Econom\u00eda y Competitividad of Spain","award":["PID2019-109395GB-I00"],"award-info":[{"award-number":["PID2019-109395GB-I00"]}]},{"name":"Ministerio de Econom\u00eda y Competitividad of Spain","award":["PY20_01176"],"award-info":[{"award-number":["PY20_01176"]}]},{"name":"Ministerio de Econom\u00eda y Competitividad of Spain","award":["BB\/P010660\/1"],"award-info":[{"award-number":["BB\/P010660\/1"]}]},{"name":"Ministerio de Econom\u00eda y Competitividad of Spain","award":["CTQ2012-31247"],"award-info":[{"award-number":["CTQ2012-31247"]}]},{"name":"Ministerio de Econom\u00eda y Competitividad of Spain","award":["CTQ2016-77270-R"],"award-info":[{"award-number":["CTQ2016-77270-R"]}]},{"name":"Ministerio de Econom\u00eda y Competitividad of Spain","award":["FQM-345"],"award-info":[{"award-number":["FQM-345"]}]},{"name":"Junta de Andaluc\u00eda","award":["PID2019-109395GB-I00"],"award-info":[{"award-number":["PID2019-109395GB-I00"]}]},{"name":"Junta de 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Novel STD NMR approaches relying on \u201cmulti-frequency\u201d irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries.<\/jats:p>","DOI":"10.3390\/ph15081030","type":"journal-article","created":{"date-parts":[[2022,8,22]],"date-time":"2022-08-22T01:56:40Z","timestamp":1661133400000},"page":"1030","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Inter-Ligand STD NMR: An Efficient 1D NMR Approach to Probe Relative Orientation of Ligands in a Multi-Subsite Protein Binding Pocket"],"prefix":"10.3390","volume":"15","author":[{"given":"Serena","family":"Monaco","sequence":"first","affiliation":[{"name":"School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Ram\u00edrez-C\u00e1rdenas","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Qu\u00edmicas (CSIC\u2014Universidad de Sevilla), 41092 Seville, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1058-7127","authenticated-orcid":false,"given":"Ana Teresa","family":"Carmona","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica Org\u00e1nica, Facultad de Qu\u00edmica, Universidad de Sevilla, 41012 Sevilla, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1447-8032","authenticated-orcid":false,"given":"Inmaculada","family":"Robina","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica Org\u00e1nica, Facultad de Qu\u00edmica, Universidad de Sevilla, 41012 Sevilla, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7250-5639","authenticated-orcid":false,"given":"Jesus","family":"Angulo","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK"},{"name":"Instituto de Investigaciones Qu\u00edmicas (CSIC\u2014Universidad de Sevilla), 41092 Seville, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,21]]},"reference":[{"key":"ref_1","first-page":"9","article-title":"Javier Ca\u00f1ada and Jes\u00fas Jim\u00e9nez-Barbero, NMR and molecular recognition. 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Reson."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/15\/8\/1030\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:13:20Z","timestamp":1760141600000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/15\/8\/1030"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,8,21]]},"references-count":19,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2022,8]]}},"alternative-id":["ph15081030"],"URL":"https:\/\/doi.org\/10.3390\/ph15081030","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,8,21]]}}}