Rhodamine Inhibitors of P-Glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

Abstract

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-His10, 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 KM of 0.087 μM 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 IC50’s of ~2 μM in MDCKII-MDR1 cells.

Targeting T Cell Bioenergetics by Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells To Prevent Graft-versus-Host Disease

“…P-gp (also known as MDR1 or ABCB1) is a member of the ATP-binding cassette (ABC) superfamily and was the first efflux protein identified. Recently, simple substitutions in a series of chalcogenorosamine/rhodamine structures have been shown to create molecules that possess a high affinity for P-gp and are either highly stimulating or inhibiting for ATPase activity (27, 28). Based on this observation, we developed a small library of 24 photosensitive agents that modulate P-gp.…”

Chalcogenopyrylium Dyes as Differential Modulators of Organic Anion Transport by Multidrug Resistance Protein 1 (MRP1), MRP2, and MRP4

“…Modulators have typically been identified by either testing drugs already in clinical practice (or derivatives thereof) or screening libraries of natural product or synthetic chemical entities. As rhodamine derivatives originally developed as photosensitizers and subsequently determined to be modulators of P-glycoprotein and/or MRP1, the CGPs are an example of the latter (Tombline et al, 2006;Sawada et al, 2008;Gannon et al, 2009;Ebert et al, 2012). In the present study, six of the seven molecules comprising a new class of CGPs (class V) tested initially at a single concentration inhibited MRP1-mediated E 2 17bG uptake by .80%, thus identifying class V CGPs with their distinctive methine or trimethine linkage between two disubstituted pyrylium moieties as a particularly effective class of MRP1 modulators (Fig.…”

Identification of the Distance between the Homologous Halves of P-glycoprotein That Triggers the High/Low ATPase Activity Switch

“…Activation of P-gp ATPase activity in the presence of drug substrates appears to be a good measure of P-gp/drug interactions because there is a good correlation with drug transport (50,51). Cross-linking of mutant L175C/N820C was low (Ͻ50% cross-linking) when membranes prepared from transfected cells were treated with oxidant (copper phenanthroline) even at 37°C (data not shown).…”