ORIGINAL RESEARCH article
Front. Pharmacol.
Sec. Drug Metabolism and Transport
In silico and in vitro investigation of the mechanisms of the drug-drug interaction between Fludarabine and Busulfan
Khalil Ben Hassine 1
Shannon Robin 1
Frederic Baleydier 1
Mourad Mseddi 1
Vid Mlakar 1
Jayaraman Muthukumaran 2
Marc Ansari 1
Chakradhara Rao Satyanarayana Uppugunduri 3
1. Cansearch Research Platform for Pediatric Oncology and Hematology, Faculty of Medicine, Department of Pediatrics, Gynecology, and Obstetrics, University of Geneva, Geneva, Switzerland, Geneva, Switzerland
2. Department of Biotechnology, Sharda School of Bio-Science and Technology, Sharda University, Greater Noida 201306, India, Greater Noida, India
3. Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India
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Abstract
Background: Understanding drug interactions in hematopoietic stem cell transplantation (HSCT) is crucial, especially considering the narrow therapeutic index of busulfan (BU), a key conditioning agent. This study investigates the potential impact of fludarabine (Flu), a frequently co-administered agent in HSCT, on Bu pharmacokinetics (PK). Specifically, it examines whether Flu can alter BU's metabolism by affecting the predominantly expressed cytosolic glutathione S-transferases (GSTs), particularly GSTA1, GSTM1, and GSTP1, which are essential for BU detoxification. Methods: We conducted molecular docking and atomistic molecular dynamics (MD) simulations using crystal structures of GSTA1, GSTM1, and GSTP1 to study the binding affinity and structural stability of Flu and its metabolites to these target enzymes. In vitro assays on human recombinant GST enzymes and HepaRG hepatocyte cells were performed, focusing on enzymatic inhibition, GST expression analysis, and glutathione level measurements. Enzymatic assays using 1-Chloro-2,4-dinitrobenzene (CDNB) as a substrate were conducted alongside western blot analyses and cell viability-corrected GSH assays to determine the influence of Flu on GST's enzymatic activity and expression. Results: In silico analysis predicted Flu's binding affinity to GST isoforms, with estimated Ki values of 0.2µM, 5.2µM, and 23.9 µM for GSTA1, GSTM1, and GSTP1, respectively. While these are micromolar (µM) inhibition values, which indicate relatively weaker or moderate inhibition, they are still insightful for understanding potential interactions. However, in vitro assays revealed no significant inhibition of GST enzymes by Flu, even at concentrations up to ten times the clinical peak (Cmax). Furthermore, Flu did not notably alter GSTA1 expression or affect cellular glutathione levels in HepaRG cells. These experimental findings suggest that Flu's influence on the GST-mediated detoxifying pathway may be minimal in the context of cancer treatment. Conclusion: The study underscores the importance of empirically validating in silico observations in pharmacological research, emphasizing the minimal effect of Flu on GST activity and its implications in clinical oncology. Moreover, the findings suggest that Flu is not likely to alter BU pharmacokinetics via the GSH-conjugation pathway.
Summary
Keywords
Drug-Drug Interaction, enzyme inhibition, enzyme kinetics, Estimate binding affinity, GST enzyme, MD simulation, molecular docking
Received
11 November 2025
Accepted
26 January 2026
Copyright
© 2026 Ben Hassine, Robin, Baleydier, Mseddi, Mlakar, Muthukumaran, Ansari and Uppugunduri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chakradhara Rao Satyanarayana Uppugunduri
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