In silico exploration of phenytoin binding site in two catalytic states of human P-glycoprotein models.

P-glycoprotein (P-gp), an ATP-dependant efflux pump transports a wide range of substrates across cellular membranes. Earlier studies have identified drug efflux due to the over-expression of P-gp as one of the causes for the resistance of phenytoin, an anti-epileptic drug (AED). While no clear evidence exists on the specific characteristics of phenytoin association with the human P-gp, this study employed structure-based computational approaches to identify its binding site and the underlying interactions. The identified site was validated with that of rhodamine, a widely accepted reference and an experimental probe. Further, an in silico proof-of-concept for phenytoin interactions and its decreased binding affinity with the closed-state of human P-gp model was provided in comparison with other AEDs. This is the first report to provide insights into the phenytoin binding site and possibly better explain its efflux by P-gp.

Cinética del enlazamiento de H33342 a la p-glicoproteína: detección y análisis de curvas de progreso / Kinetic of the binding of H33342 to the p-glycoprotein: detection and analysis of progress curves

El transportador multidrogas P-glicoproteína (Pgp) lleva a cabo el eflujo celular, ATP-dependiente, de muchas drogas hidrofóbicas, productos naturales y péptidos. Se propone que la Pgp contiene dos sitios de transporte, conocidos como los sitios -H y -R por sus preferencias por Hoechst 33342 (H33342) y rodamina 123, respectivamente. Cuando H33342 interactúa con la Pgp purificada, su rendimiento cuántico incrementa debido al ambiente hidrofóbico del bolsillo de enlazamiento –H. En este trabajo, estudiamos el enlazamiento de H33342 a la Pgp empleando experimentos cinéticos en la modalidad de stoppedflow. El curso temporal de la reacción fue seguido por el incremento de la fluorescencia del colorante y analizado con la herramienta computacional DYNAFIT, usando un modelo donde una reacción bimolecular rápida, es seguida por tres isomerizaciones secuenciales. Adicionalmente, bajo condiciones de seudo-primer-orden (exceso del ligando), la reacción presentó cuatro relajaciones caracterizadas por cuatro constantes de tiempo (á`s) y cuatro amplitudes (A¡`s) Estos parámetros fueron analizados utilizando la técnica de la matriz de los operadores de proyección. Esta aproximación aportó, por primera vez, información acerca de las constantes de velocidad y propiedades fluorescentes de los diversos intermediarios formados durante el enlazamiento de H33342 a la Pgp.

The P-glycoprotein multidrug transporter (Pgp) carries out ATP-driven cellular efflux of many different hydrophobic drugs, natural products, and peptides. Pgp is proposed to contain two drug transport sites, known as the H site and the R site for their preference for Hoechst 33342 (H33342) and rhodamine 123, respectively. When H33342 interacts with purified Pgp, its quantum yield is increased due to transfer to a hydrophobic environment within the H binding pocket, as shown by the steady-state fluorescence emission. In this work, we studied the binding of H33342 to Pgp using stopped-flow kinetic experiments. The time course of the reaction was followed by enhancement of dye fluorescence and analyzed by the computational tool DYNAFIT, using the model of a fast bimolecular reaction, followed by a three-step sequential isomerization. Additionally, under pseudo-first-order conditions (excess ligand), the reaction presented five normal modes, characterized by four relaxation times (á`s) and four amplitudes (A¡`s) These parameters were analyzed using the matrix projection operator technique, considering a four-step sequential reaction. This approach provides, for the first time, information about the rate constants and fluorescent properties of the diverse intermediates formed during the binding of H33342 to Pgp.