RESUMO
Multidrug resistance proteins (MRPs) mediate the ATP-dependent efflux of structurally diverse compounds, including anticancer drugs and physiologic organic anions. Five classes of chalcogenopyrylium dyes (CGPs) were examined for their ability to modulate transport of [(3)H]estradiol glucuronide (E(2)17ßG; a prototypical MRP substrate) into MRP-enriched inside-out membrane vesicles. Additionally, some CGPs were tested in intact transfected cells using a calcein efflux assay. Sixteen of 34 CGPs inhibited MRP1-mediated E(2)17ßG uptake by >50% (IC50 values: 0.7-7.6 µM). Of 9 CGPs with IC50 values ≤2 µM, two belonged to class I, two to class III, and five to class V. When tested in the intact cells, only 4 of 16 CGPs (at 10 µM) inhibited MRP1-mediated calcein efflux by >50% (III-1, V-3, V-4, V-6), whereas a fifth (I-5) inhibited efflux by just 23%. These five CGPs also inhibited [(3)H]E(2)17ßG uptake by MRP4. In contrast, their effects on MRP2 varied, with two (V-4, V-6) inhibiting E(2)17ßG transport (IC(50) values: 2.0 and 9.2 µM) and two (V-3, III-1) stimulating transport (>2-fold), whereas CGP I-5 had no effect. Strikingly, although V-3 and V-4 had opposite effects on MRP2 activity, they are structurally identical except for their chalcogen atom (Se versus Te). This study is the first to identify class V CGPs, with their distinctive methine or trimethine linkage between two disubstituted pyrylium moieties, as a particularly potent class of MRP modulators, and to show that, within this core structure, differences in the electronegativity associated with a chalcogen atom can be the sole determinant of whether a compound will stimulate or inhibit MRP2.
Assuntos
Corantes/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Linhagem Celular Transformada , Calcogênios/química , Calcogênios/farmacologia , Corantes/química , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His(10), for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave K(M) of 0.087 microM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC(50)'s of approximately 2 microM in MDCKII-MDR1 cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Amidas/farmacologia , Rodaminas/farmacologia , Tioamidas/farmacologia , Adenosina Trifosfatases/efeitos dos fármacos , Amidas/química , Animais , Transporte Biológico , Linhagem Celular , Cães , Resistência a Múltiplos Medicamentos , Fluoresceínas/farmacocinética , Compostos Heterocíclicos com 3 Anéis , Humanos , Cinética , Ligação Proteica , Rodaminas/química , Relação Estrutura-Atividade , Tioamidas/química , Vimblastina/farmacocinéticaRESUMO
The multidrug efflux pump P-glycoprotein (Pgp) couples drug transport to ATP hydrolysis. Previously, using a synthetic library of tetramethylrosamine ( TMR) analogues, we observed significant variation in ATPase stimulation ( V m (D)). Concentrations required for half-maximal ATPase stimulation ( K m (D)) correlated with ATP hydrolysis transition-state stabilization and ATP occlusion (EC 50 (D)) at a single site. Herein, we characterize several TMR analogues that elicit modest turnover ( k cat Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo
, Trifosfato de Adenosina/metabolismo
, Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química
, Adenosina Trifosfatases/metabolismo
, Trifosfato de Adenosina/química
, Ativação Enzimática/efeitos dos fármacos
, Compostos Heterocíclicos com 3 Anéis/química
, Compostos Heterocíclicos com 3 Anéis/farmacologia
, Hidrólise
, Estrutura Molecular
, Rodaminas
, Verapamil/química
, Verapamil/farmacologia