Julien Dumond, Jean-Marcel Julien Tronchet, Serge Kirkiacharian, Michel Seman and Michèle Reboud-Ravaux* Pages 818 - 825 ( 8 )
Background: Reverse Transcriptase (RT) of immunodeficiency virus type-1 (HIV-1) remains an essential target for new antiretroviral therapies. Non-nucleoside reverse transcriptase inhibitors (or NNRTIs) constitute a major class of RT inhibitors whose characterization is essential.
Introduction: Several biochemical, biological, and biophysical methods have been previously used to analyze the biological effects of NNRTIs. We explored here the use of surface plasmonic resonance to characterize the affinity of RT towards selected NNRTIs and compared the results with those obtained with in vitro and in cellulo assays.
Methods: The solubility and stability in buffers of the tested NNRTIs were assessed by spectrophotometry and fluorescence. Surface plasmonic resonance experiments to study direct NNRTIs binding to immobilized RT and intramolecular quenching of RT tryptophan fluorescence were used to determine the KA association constants (= 1/KD) between RT and the inhibitors. The in vitro inhibition constants of RT were determined using kinetics and the effects on three other potential targets (proteasome, HIV-1 integrase, and HIV-1 protease) were analyzed.
Results: The results obtained with two typical molecules belonging to our previous N-hydroxyureido acylnucleoside derivatives series using the above biophysical assays matched those obtained in in vitro and previous in cellulo assays.
Conclusion: Surface plasmonic resonance provides reliable thermodynamic information on the interaction of RT with NNRTIs and appears as a useful method for understanding their inhibitory mechanism.
HIV-1 reverse transcriptase, non-nucleoside reverse transcriptase inhibitors (NNRTIs), surface plasmonic resonance, flourescence, N-hydroxyureido acylnucleoside derivatives, double-stranded, DNA (dsDNA).
Sorbonne Universite, CNRS, INSERM, Institut de Biologie Paris Seine, IBPS, Adaptation biologique et Vieillissement, 7 quai Saint Bernard 75252 Paris Cedex 05, Institut de Chimie Pharmaceutique de l’Universite, Sciences II, CH-1211, Geneve 4, Suisse, Faculte de Pharmacie Paris-Sud. Laboratoire de Chimie Therapeutique. 5 rue Jean- Baptiste Clement 92290 Chatenay-Malabry, Institut National de la Sante et de la Recherche Medicale U905, Pathophysiology Biotherapy of Inflammatory and Autoimmune Diseases, Faculte de Médecine et de Pharmacie de Rouen, 22 Boulevard Gambetta 76183 Rouen Cedex, Sorbonne Universite, CNRS, INSERM, Institut de Biologie Paris Seine, IBPS, Adaptation biologique et Vieillissement, 7 quai Saint Bernard 75252 Paris Cedex 05