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The biophysical interactions between these compounds and human serum albumin (HSA), the primary carrier of exogenous compounds in the human bloodstream, have not yet been thoroughly characterized. Thus, the present study reports the interaction profile between HSA and TFV, TDF, and TAF via UV\u2013Vis, steady-state, and time-resolved fluorescence techniques combined with isothermal titration calorimetry (ITC) and in silico calculations. A spontaneous interaction in the ground state, which does not perturb the microenvironment close to the Trp-214 residue, is classified as weak. In the case of HSA\/TFV and HSA\/TDF, the binding is both enthalpically and entropically driven, while for HSA\/TAF, the binding is only entropically dominated. The binding constant (Ka) and thermodynamic parameters obtained via ITC assays agree with those obtained using steady-state fluorescence quenching measurements, reinforcing the reliability of the data. The small internal cavity known as site I is probably the main binding pocket for TFV due to the low steric volume of the drug. In contrast, most external sites (II and III) can better accommodate TAF due to the high steric volume of this prodrug. The cross-docking approach corroborated experimental drug-displacement assays, indicating that the binding affinity of TFV and TAF might be impacted by the presence of different compounds bound to albumin. Overall, the weak binding capacity of albumin to TFV, TDF, and TAF is one of the main factors for the low residence time of these antiretrovirals in the human bloodstream; however, positive cooperativity for TAF and TDF was detected in the presence of some drugs, which might improve their residence time (pharmacokinetic profile).<\/jats:p>","DOI":"10.3390\/v16040491","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T12:50:49Z","timestamp":1711371049000},"page":"491","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Profiling the Interaction between Human Serum Albumin and Clinically Relevant HIV Reverse Transcriptase Inhibitors"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8555-2370","authenticated-orcid":false,"given":"Andreia","family":"Costa-Tuna","sequence":"first","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6211-7659","authenticated-orcid":false,"given":"Ot\u00e1vio A.","family":"Chaves","sequence":"additional","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"},{"name":"Laboratory of Immunopharmacology, Centro de Pesquisa, Inova\u00e7\u00e3o e Vigil\u00e2ncia em COVID-19 e Emerg\u00eancias Sanit\u00e1rias (CPIV), Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro 21040-361, RJ, Brazil"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4097-2766","authenticated-orcid":false,"given":"Zaida L.","family":"Almeida","sequence":"additional","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"}]},{"given":"Rita S.","family":"Cunha","sequence":"additional","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1848-1167","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Pina","sequence":"additional","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7004-0110","authenticated-orcid":false,"given":"Carlos","family":"Serpa","sequence":"additional","affiliation":[{"name":"CQC-IMS, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"83","DOI":"10.5334\/aogh.4277","article-title":"Advancing the global fight against HIV\/Aids: Strategies, barriers, and the road to eradication","volume":"89","author":"Kumah","year":"2023","journal-title":"Ann. 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