RESUMEN
Anthracyclines are widely used in patients for anticancer activity. However, one of the limitations for their clinical use is P-gp-mediated drug resistance in cancer therapy. We hypothesize that modified anthracyclines will retain their anticancer activity, avert P-gp binding, and thus overcome P-gp-mediated drug resistance. Twenty-five daunorubicin analogues were synthesized with slight structure modifications in sugar moieties. Molecular docking, cytotoxicity, and P-gp inhibition assays in drug-resistant leukemia cells (K562/Dox) were used to identify several candidates that avert binding to multidrug-resistant protein (MsbA) and overcome drug resistance. Molecular docking showed that daunorubicin bound to the cavity between the intracellular domain (ICD) and nucleoside binding domain (NBD) of MsbA, which might be the "entry site" for the transport of its substrate. The molecular docking accurately predicted the substrates of multidrug-resistant protein. Several aspects are important for daunorubicin analogue binding to MsbA: (1) the substitution pattern and stereochemistry of the tetracyclic ring and sugar moiety; (2) the hydrogen bond donor or acceptor capability of the substituent at C'-3 and C'-4. Molecular docking, cytotoxicity, and P-gp inhibition assays identified ADNR, ADNR-1, and ADNR-3 for averting P-gp binding and overcoming drug resistance. The replacement of C'-3-NH2 with azido group in daunorubicin not only abolishes the hydrogen bond between the sugar moiety and MsbA but also completely changes the overall binding conformation, and thus averts the binding to MsbA. Cytotoxicity assays confirmed that these compounds showed high sensitivity against drug-resistant cancer cells (K562/Dox) with P-gp overexpression. P-gp inhibition assay indeed confirms that these appropriately modified compounds avert P-gp binding and thus overcome P-gp-mediated drug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Metabolismo de los Hidratos de Carbono , Daunorrubicina/metabolismo , Resistencia a Múltiples Medicamentos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ciclosporina/farmacología , Citotoxicidad Inmunológica , Daunorrubicina/análogos & derivados , Daunorrubicina/química , Disacáridos/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Células K562 , Monosacáridos/metabolismo , Estructura Secundaria de ProteínaRESUMEN
To activate prodrugs for cancer treatment, an anti-TAG-72 antibody (HuCC49DeltaCH2) was used for delivery of an activation enzyme (beta-galactosidase) to specifically activate a geldanamycin prodrug (17-AG-C2-Gal) against colon cancer. The geldanamycin prodrug 17-AG-C2-Gal was synthesized by coupling a galactose-amine derivative with geldanamycin at the C-17 position. Molecular docking with two different programs (Affinity and Autodock) showed that the prodrug (17-AG-C2-Gal) was unable to bind to Hsp90; however, the product (17-AG-C2), enzymatically cleaved by beta-galactosidase conjugate, bound to Hsp90 in a similar way as geldanamycin and 17-AG. The computational docking results were further confirmed in experimental testing by the tetrazolium [3-(4,5-dimethythiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and mass spectrometry. HuCC49DeltaCH2 was chemically conjugated to beta-galactosidase. The antibody-enzyme conjugate was able to target tumor antigen TAG-72 with the well-preserved enzymatic activity to activate 17-AG-C2-Gal prodrug. The released active drug 17-AG-C2 was demonstrated to induce up to 70% AKT degradation and enhance anticancer activity by more than 25-fold compared to the prodrug.