Fluorescent probes of the isoxazole–dihydropyridine scaffold: MDR-1 binding and homology model

Abstract

Isoxazole-1,4-Dihydropyridines (IDHPs) were tethered to Fluorescent moieties using double activation via a lanthanide assisted Weibreb amidation. IDHP-fluorophore conjugate 3c exhibits the highest binding to date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP-fluorophore conjugates 3c and 7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis.

Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance transporter

“…Our previous ABCB1 homology model 12,13 was based on the mouse P-gp structure, 37 which has a significantly different topology in its apo conformation than the recent human structure reported by Carpenter’s group ( vide infra ). 38 Our calculation of the NBD distance in our previous human homology model based on the M. musculus P-gp template was 42.5 Å, and the C. elegans distance is 41 Å, 2,38 which means that for our dimeric structures to be effective they would have to intercept the efflux pump in a conformation intermediate at some point between apo and Sav1866 nucleotide bound open-to-outside conformers.…”

“…12,13 B, C, D, calculated distances for n = 2, 3 and 5, tethered dimers, respectively, in energy minimized conformations.…”

“…This suggested additional binding opportunities in proximity to the putative nucleotide binding domain (NBD) of IDHPs ( vide infra ). 12,13 …”

“…Taking into account previous research the tether’s length is critical, however, the MDR undergoes significant conformational reorganization during the ATP driven efflux. In the apo state used in our previous homology model 12,13 the NBDs reside over 40 Å apart using the mouse P-gp data (pdb accession number 3G5U) (Fig. 2, below), while in the ATP bound state exemplified by Sav1866 (pdb accession number 2ONJ), that distance is reported as 4 Å.…”

“…2, below), while in the ATP bound state exemplified by Sav1866 (pdb accession number 2ONJ), that distance is reported as 4 Å. In our previous study of IDHPs tethered to fluorophores, 12 we used hydrocarbon tethers, and we were concerned that such groups could suffer hydrophobic collapse during interaction with the target, given that such folded conformers were evidenced in the NMR. We reasoned that oligo ethyleneoxy linkers (short PEGs), would be more likely to adopt an extended conformation able to intercept the ABC at a stage intermediate to the apo and outward facing ATP bound conformation.…”