title: Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1.
abstract: Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors--[(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR), dilazep, and dipyridamole--and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [(3)H]NBMPR association was relatively large (20 kcal mol(-1)) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (ΔH°) and entropy (ΔS°) contributions to the reaction energetics were determined by van't Hoff analysis to be roughly similar (25-75% ΔG°). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter.
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
authors.forename: Shahid
authors.initials: S
authors.lastname: Rehan
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
authors.forename: Yashwanth
authors.initials: Y
authors.lastname: Ashok
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
authors.forename: Rahul
authors.initials: R
authors.lastname: Nanekar
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland. Electronic address: veli-pekka.jaakola@oulu.fi.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
mesh_terms: D000818:Animals; D004109:Dilazep / Q000493:pharmacokinetics; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D011485:Protein Binding / Q000502:physiology; D013816:Thermodynamics
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; C438649:SLC29A1 protein, human; D004109:Dilazep
keywords: Activation energy; Adenosine (PubChem CID: 60961); Anti-cancer; Conformational change; Dilazep (PubChem CID: 29976); Dipyridamole (PubChem CID: 3108); NBMPR (PubChem CID: 5129); Nucleoside transporter; Thermodynamic