Reversible dimers of the atypical antipsychotic quetiapine inhibit p-glycoprotein-mediated efflux in vitro with increased binding affinity and in situ at the blood-brain barrier.

The multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed in the capillary endothelial cells of the blood-brain barrier (BBB) where it functions to limit the brain penetration of many drugs, including antipsychotic agents used to treat schizophrenia. Therefore, in an effort to inhibit the transporter, we designed dimers of the antipsychotic drug and P-gp substrate quetiapine (QT), linked by variable length tethers. In P-gp overexpressing cells and in human brain capillary endothelial hCMEC/D3 cells, the dimer with the shortest tether length (QT2C2) (1) was the most potent inhibitor showing >80-fold better inhibition of P-gp-mediated transport than monomeric QT. The dimers, which are linked via ester moieties, are designed to revert to the therapeutic monomer once inside the target cells. We demonstrated that the addition of two sterically blocking methyl groups to the linker (QT2C2Me2, 8) increased the half-life of the molecule in plasma 10-fold as compared to the dimer lacking methyl groups (QT2C2, 1), while retaining inhibitory potency for P-gp transport and sensitivity to cellular esterases. Experiments with purified P-gp demonstrated that QT2C2 (1) and QT2C2Me2 (8) interacted with both the H- and R-binding sites of the transporter with binding affinities 20- to 30-fold higher than that of monomeric QT. Using isolated rat brain capillaries, QT2C2Me2 (8) was a more potent inhibitor of P-gp transport than QT. Lastly, we showed that QT2C2Me2 (8) increased the accumulation of the P-gp substrate verapamil in rat brain in situ three times more than QT. Together, these results indicate that the QT dimer QT2C2Me2 (8) strongly inhibited P-gp transport activity in human brain capillary endothelial cells, in rat brain capillaries, and at the BBB in an animal model.

Homodimers of the Antiviral Abacavir as Modulators of P-glycoprotein Transport in Cell Culture: Probing Tether Length.

A major hurdle in permanently eliminating HIV from the body is the persistence of viral reservoirs, including those of the brain. One potential strategy towards eradicating HIV reservoirs of the brain is to block efflux transporters, such as P-glycoprotein (P-gp), that contribute to the limited penetration of antiviral agents across the blood-brain barrier (BBB). Herein, we described a series of dimeric inhibitors of P-gp based on the nucleoside reverse transcriptase inhibitor and P-gp substrate, abacavir. Varying tether lengths were used to generate abacavir dimers to probe tether requirements for inhibitory potency. These dimeric agents were evaluated in two cell lines that express P-gp at varying levels: a P-gp over-expressing CD4+ T-lymphocyte cell line (12D7-MDR) and a human brain capillary endothelial cell line as an in vitro model of the BBB (hCMEC/D3) that expresses endogenous levels of P-gp. All dimeric abacavir analogs were inhibitors of P-gp efflux in the two cell lines with potencies that varied with tether length; the most potent agents displayed low micromolar inhibition. P-gp inhibition in a highly P-gp over-expressing cell line (MCF-7/DX1) was also observed with a range of therapeutic substrates. Competition studies with the photoaffinity substrate [125I]iodoarylazidoprazosin demonstrated that abacavir dimers act by competing for the substrate binding sites of P-gp. These data demonstrate that the tether length of dimeric abacavir derivatives has a significant effect on inhibition of P-gp drug efflux, with up to a 35-fold increase in potency observed with longer tether linkages.

Toward eradicating HIV reservoirs in the brain: inhibiting P-glycoprotein at the blood-brain barrier with prodrug abacavir dimers.

Eradication of HIV reservoirs in the brain necessitates penetration of antiviral agents across the blood-brain barrier (BBB), a process limited by drug efflux proteins such as P-glycoprotein (P-gp) at the membrane of brain capillary endothelial cells. We present an innovative chemical strategy toward the goal of therapeutic brain penetration of the P-gp substrate and antiviral agent abacavir, in conjunction with a traceless tether. Dimeric prodrugs of abacavir were designed to have two functions: inhibit P-gp efflux at the BBB and revert to monomeric therapeutic within cellular reducing environments. The prodrug dimers are potent P-gp inhibitors in cell culture and in a brain capillary model of the BBB. Significantly, these agents demonstrate anti-HIV activity in two T-cell-based HIV assays, a result that is linked to cellular reversion of the prodrug to abacavir. This strategy represents a platform technology that may be applied to other therapies with limited brain penetration due to P-glycoprotein.

Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study.

Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-ß-D-glucopyranoside and n-dodecyl-ß-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.

Inhibition of human P-glycoprotein transport and substrate binding using a galantamine dimer.

The human multidrug resistance transporter P-glycoprotein (P-gp) prevents the entry of compounds into the brain by an active efflux mechanism at the blood-brain barrier (BBB). Treatment of neurodegenerative diseases, therefore, has become a challenge and the development of new reversible inhibitors of P-gp is pertinent to overcome this problem. We report the design and synthesis of a crosslinked agent based on the Alzheimer's disease treatment galantamine (Gal-2) that inhibits P-gp-mediated efflux from cultured cells. Gal-2 was found to inhibit the efflux of the fluorescent P-gp substrate rhodamine 123 in cancer cells that over-express P-gp with an IC(50) value of approximately 0.6 microM. In addition, Gal-2 was found to inhibit the efflux of therapeutic substrates of P-gp, such as doxorubicin, daunomycin and verapamil with IC(50) values ranging from 0.3 to 1.6 microM. Through competition experiments, it was determined that Gal-2 modulates P-gp mediated efflux by competing for the substrate binding sites. These findings support a potential role of agents, such as Gal-2, as inhibitors of P-gp at the BBB to augment treatment of neurodegenerative diseases.

Inhibition of P-glycoprotein-mediated paclitaxel resistance by reversibly linked quinine homodimers.

P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers as a result of its overexpression. In this work, rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC(50) approximately 1.7 microM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and BODIPY-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t(1/2) approximately 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t(1/2) approximately 21 days). Specific inhibition of [(125)I]iodoarylazidoprazosin binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. In addition, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled paclitaxel (Taxol) in MCF-7/DX1 cells, and it completely reversed the P-gp-mediated paclitaxel resistance phenotype.

Engineering unnatural nucleotide specificity to probe G protein signaling.

G proteins comprise approximately 0.5% of proteins encoded by mammalian genomes. To date, there exists a lack of small-molecule modulators that could contribute to their functional study. In this report, we present the use of H-Ras to develop a system that answers this need. Small molecules that allow for the highly specific inhibition or activation of the engineered G protein were developed. The rational design preserved binding of the natural substrates to the G protein, and the mutations were functionally innocuous in a cellular context. This tool can be used for isolating specific G protein effectors, as we demonstrate with the identification of Nol1 as a putative effector of H-Ras. Finally, the generalization of this system was confirmed by applying it to Rap1B, suggesting that this method will be applicable to other G proteins.