Mutational and Biochemical Analysis of Isoprenylcysteine Carboxyl Methyltransferase

Ninety percent of pancreatic cancers are attributed to mutations in the Ras protein, making it paramount to concentrate on Ras activity. This study focuses on Ras activity by targeting a post-translational modifying enzyme of Ras called Isoprenylcysteine carboxyl methyltransferase (Icmt). Elucidation of the binding site of Icmt will allow the development of therapeutics that effectively inhibit Icmt causing the mislocalization of Ras, and in turn, aid in the treatment of Ras driven cancers. Currently, the hydrophobic substrate binding site of Icmt is unknown. In order to characterize the substrate binding site of Icmt, site-directed mutagenesis was used to design mutations in the yeast homolog of Icmt, Ste14p, and these mutants were tested on substrate specificity. Residues L33, L34, L40, L176, L190, and L195 were mutated to alanine and residue F80 was mutated to tyrosine. When tested with a methyltransferase assay, all of the mutants lost activity as compared to wild type (WT). Noticeably, L190A had only 44% WT activity. This suggests that L190A is important for either substrate binding or the overall structural integrity of Ste14p. The results from trypsin digestion show that all mutants have cleavage patterns similar to WT. This indicates that the structural integrity of Ste14p remains intact regardless of these mutations. Substrate specificity and photolabeling experiments should be conducted in the future to elucidate if these residues are vital for the substrate binding site of Icmt. These results could be utilized to design more potent and effective drug therapies to minimize Ras signaling in cancer cells

Potential Tools for Eradicating HIV Reservoirs in the Brain: Development of Trojan Horse Prodrugs for the Inhibition of P-Glycoprotein with Anti-HIV-1 Activity

Combination antiretroviral therapy is the mainstay of HIV treatment, lowering plasma viral levels below detection. However, eradication of HIV is a major challenge due to cellular and anatomical viral reservoirs that are often protected from treatment by efflux transporters, such as P-glycoprotein (P-gp) at the blood–brain barrier (BBB). Herein we described a Trojan horse approach to therapeutic evasion of P-gp based on a reversibly linked combination of HIV reverse transcriptase and protease inhibitors. Potent inhibition of P-gp efflux in cells, including human brain endothelial cells, was observed with the linked heterodimeric compounds. In vitro regeneration of active monomeric drugs was observed in a reducing environment with these dimeric prodrugs, with the superior leaving group promoting more facile release from the tether. These release trends were mirrored in the efficacy of the in cyto anti-HIV-1 activity of the Trojan horse heterodimers

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport. © 2005 Blackwell Publishing Ltd

The roles of the human ATP-binding cassette transporters P-glycoprotein and ABCG2 in multidrug resistance in cancer and at endogenous sites: future opportunities for structure-based drug design of inhibitors

The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and ABCG2 are multidrug transporters that confer drug resistance to numerous anti-cancer therapeutics in cell culture. These findings initially created great excitement in the medical oncology community, as inhibitors of these transporters held the promise of overcoming clinical multidrug resistance in cancer patients. However, clinical trials of P-gp and ABCG2 inhibitors in combination with cancer chemotherapeutics have not been successful due, in part, to flawed clinical trial designs resulting from an incomplete molecular understanding of the multifactorial basis of multidrug resistance (MDR) in the cancers examined. The field was also stymied by the lack of high-resolution structural information for P-gp and ABCG2 for use in the rational structure-based drug design of inhibitors. Recent advances in structural biology have led to numerous structures of both ABCG2 and P-gp that elucidated more clearly the mechanism of transport and the polyspecific nature of their substrate and inhibitor binding sites. These data should prove useful helpful for developing even more potent and specific inhibitors of both transporters. As such, although possible pharmacokinetic interactions would need to be evaluated, these inhibitors may show greater effectiveness in overcoming ABC-dependent multidrug resistance in combination with chemotherapeutics in carefully selected subsets of cancers. Another perhaps even more compelling use of these inhibitors may be in reversibly inhibiting endogenously expressed P-gp and ABCG2, which serve a protective role at various blood-tissue barriers. Inhibition of these transporters at sanctuary sites such as the brain and gut could lead to increased penetration by chemotherapeutics used to treat brain cancers or other brain disorders and increased oral bioavailability of these agents, respectively

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 (IC50 ≈ 1.7 μM). 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 (t½ ≈ 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t½ ≈ 21 days). Specific inhibition of [125I]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

Dual Modulation of Human P‑Glycoprotein and ABCG2 with Prodrug Dimers of the Atypical Antipsychotic Agent Paliperidone in a Model of the Blood–Brain Barrier

Many atypical antipsychotic drugs currently prescribed for the treatment of schizophrenia have limited brain penetration due to the efflux activity of ATP-binding cassette (ABC) transporters at the blood–brain barrier (BBB), including P-glycoprotein (P-gp) and ABCG2. Herein, we describe the design and synthesis of the first class of homodimeric prodrug dual inhibitors of P-gp and ABCG2. These inhibitors are based on the structure of the atypical antipsychotic drug paliperidone (Pal), a transport substrate for both transporters. We synthesized and characterized a small library of homodimeric bivalent Pal inhibitors that contain a variety of tethers joining the two monomers via ester linkages. The majority of our compounds were low micromolar to sub-micromolar inhibitors of both P-gp and ABCG2 in cells overexpressing these transporters and in immortalized human hCMEC/D3 cells that are derived from the BBB. Our most potent dual inhibitor also contained an internal disulfide bond in the tether (Pal-8SS) that allowed for rapid reversion to monomer in the presence of reducing agents or plasma esterases. To increase stability against these esterases, we further engineered Pal-8SS to contain two hindering methyl groups alpha to the carbonyl of the ester moiety within the tether. The resulting dimer, Pal-8SSMe, was also a potent dual inhibitor that remained susceptible to reducing conditions but was more resistant to breakdown in human plasma. Importantly, Pal-8SSMe both accumulated and subsequently reverted to the therapeutic Pal monomer in the reducing environment of BBB cells. Thus, these molecules serve two purposes, acting as both inhibitors of P-gp and ABCG2 at the BBB and as prodrugs, effectively delivering therapies to the brain that would otherwise be precluded