{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T06:33:21Z","timestamp":1780554801484,"version":"3.54.1"},"reference-count":70,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,3,27]],"date-time":"2023-03-27T00:00:00Z","timestamp":1679875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"King Saud University","award":["RSPD2023R724"],"award-info":[{"award-number":["RSPD2023R724"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The interaction of indomethacin with human serum albumin (HSA) has been studied here considering the primary and secondary binding sites. The Stern\u2013Volmer plots were linear in the lower concentration range of indomethacin while a downward curvature was observed in the higher concentration range, suggesting the presence of more than one binding site for indomethacin inside HSA due to which the microenvironment of the fluorophore changed slightly and some of its fraction was not accessible to the quencher. The Stern\u2013Volmer quenching constants (KSV) for the primary and secondary sites were calculated from the two linear portions of the Stern\u2013Volmer plots. There was around a two-fold decrease in the quenching constants for the low-affinity site as compared to the primary binding site. The interaction takes place via a static quenching mechanism and the KSV decreases at both primary and secondary sites upon increasing the temperature. The binding constants were also evaluated, which show strong binding at the primary site and fair binding at the secondary site. The binding was thermodynamically favorable with the liberation of heat and the ordering of the system. In principle, hydrogen bonding and Van der Waals forces were involved in the binding at the primary site while the low-affinity site interacted through hydrophobic forces only. The competitive binding was also evaluated using warfarin, ibuprofen, hemin, and a warfarin + hemin combination as site markers. The binding profile remained unchanged in the presence of ibuprofen, whereas it decreased in the presence of both warfarin and hemin with a straight line in the Stern\u2013Volmer plots. The reduction in the binding was at a maximum when both warfarin and hemin were present simultaneously with the downward curvature in the Stern\u2013Volmer plots at higher concentrations of indomethacin. The secondary structure of HSA also changes slightly in the presence of higher concentrations of indomethacin. Molecular dynamics simulations were performed at the primary and secondary binding sites of HSA which are drug site 1 (located in the subdomain IIA of the protein) and the hemin binding site (located in subdomain IB), respectively. From the results obtained from molecular docking and MD simulation, the indomethacin molecule showed more binding affinity towards drug site 1 followed by the other two sites.<\/jats:p>","DOI":"10.3390\/molecules28072979","type":"journal-article","created":{"date-parts":[[2023,3,27]],"date-time":"2023-03-27T06:46:19Z","timestamp":1679899579000},"page":"2979","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Detailed Experimental and In Silico Investigation of Indomethacin Binding with Human Serum Albumin Considering Primary and Secondary Binding Sites"],"prefix":"10.3390","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0191-9708","authenticated-orcid":false,"given":"Mohd Sajid","family":"Ali","sequence":"first","affiliation":[{"name":"Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jayaraman","family":"Muthukumaran","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida 201310, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Jain","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida 201310, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7795-8194","authenticated-orcid":false,"given":"Mohammad","family":"Tariq","sequence":"additional","affiliation":[{"name":"LAQV-REQUIMTE, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8228-4665","authenticated-orcid":false,"given":"Hamad A.","family":"Al-Lohedan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Science, King Saud University, P.O. 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