Novel EGFR-MAPK kinase and ABC transporter inhibitors for HepG2 resistant to Erlotinib.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer death in the world, with outlook for most patients having a 5-year survivability of less than 5%. In a previous study from our laboratory, novel estrone inspired analogs act as epidermal growth factor receptor (EGFR) inhibitors in HepG2 cells. This study focuses on the effect of these analogs on an HCC cell line resistance to Erlotinib. Lead compounds MMA132 and MMA102 showed 13 and 20 µM IC50 values, respectively against HepG2-R resistant to Erlotinib. These compounds showed cell cycle arrest of the G2 phase up to 54%, and inhibited cell migration of HepG2-R cells up to 48 h. Western blot analysis revealed that MMA132 reduced total EGFR content after 48 h, while MMA102 inhibited MEK kinase by 84% after 48 h. Western blot analysis also revealed that multidrug resistance protein 2 (MRP2) is overexpressed in HepG2-R, suggesting that ABC transporters play a likely cause in drug resistance. MMA102 showed significant inhibition of both P-glycoprotein (83%) and ABCG2 (53%), two additional ABC transporters. Additionally, MMA102 and MMA132 were used in a combination therapy with MK571(MRP1/2 inhibitor) and produced IC50 values of 18 and 10 µM, respectively, better than either MMA102/132 or MK571 alone. To validate our findings, we conducted molecular dynamic simulations with MMA102 and MMA132 in MEK, P-glycoprotein, MRP1, and MRP2. Results coincided with biological findings in which MMA102 orientation is favored in both MEK and P-glycoprotein pockets, whereas MMA132 likely binds with MRP2, as likely suggested by the combinatorial study.

A critical review on modulators of Multidrug Resistance Protein 1 in cancer cells.

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux transporter, and responsible for the transport of a broad spectrum of xenobiotics, toxins, and physiological substrates across the plasma membrane. As an efflux pump, it plays a significant role in the absorption and disposition of drugs including anticancer drugs, antivirals, antimalarials, and antibiotics and their metabolites across physiological barriers in cells. MRP1 is also known to aid in the regulation of several physiological processes such as redox homeostasis, steroid metabolism, and tissue defense. However, its overexpression has been reported to be a key clinical marker associated with multidrug resistance (MDR) of several types of cancers including lung cancer, childhood neuroblastoma, breast and prostate carcinomas, often resulting in a higher risk of treatment failure and shortened survival rates in cancer patients. Aside MDR, overexpression of MRP1 is also implicated in the development of neurodegenerative and cardiovascular diseases. Due to the cellular importance of MRP1, the identification and biochemical/molecular characterization of modulators of MRP1 activity and expression levels are of key interest to cancer research and beyond. This review primarily aims at highlighting the physiological and pharmacological importance of MRP1, known MRP1 modulators, current challenges encountered, and the potential benefits of conducting further research on the MRP1 transporter.

Triazole-estradiol analogs: A potential cancer therapeutic targeting ovarian and colorectal cancer.

1,2,3-triazoles have continuously shown effectiveness as biologically active systems towards various cancers, and when used in combination with steroid skeletons as a carrier, which can act as a drug delivery system, allows for a creation of a novel set of analogs that may be useful as a pharmacophore leading to a potential treatment option for cancer. A common molecular target for cancer inhibition is that of the Epidermal Growth Factor Receptor/Mitogen Activated Protein Kinase pathways, as inhibition of these proteins is associated with a decrease in cell viability. Estradiol-Triazole analogs were thus designed using a molecular modeling approach. Thirteen of the high scoring analogs were then synthesized and tested in-vitro on an ovarian cancer cell line (A2780) and colorectal cancer cell line (HT-29). The most active compound, Fz25, shows low micromolar activity in both the ovarian (15.29 ± 2.19 µM) and colorectal lines (15.98 ± 0.39 µM). Mechanism of action studies proved that Fz25 moderately arrests cells in the G1 phase of the cell cycle, specifically inhibiting STAT3 in both cell lines. Additionally, Fz57 shows activity in the colorectal line (24.19 ± 1.37 µM). Inhibition studies in both cell lines show inhibition against various proteins in the EGFR pathway, namely EGFR, STAT3, ERK, and mTOR. To further study their effects as therapeutics, Fz25 and Fz57 were studied against drug efflux proteins, which are associated with drug resistance, and were found to inhibit the ABC transporter P-glycoprotein. We can conclude that these estradiol-triazole analogs provide a key for future studies targeting protein inhibition and drug resistance in cancer.

Preliminary studies on drug delivery of polymeric primaquine microparticles using the liver high uptake effect based on size of particles to improve malaria treatment.

Malaria is the most common parasitic disease around the world, especially in tropical and sub-tropical regions. This parasitic disease can have a rapid and severe evolution. It is transmitted by female anopheline mosquitoes. There is no reliable vaccine or diagnostic test against malaria; instead, Artesunate is used for the treatment of severe malaria and Artemisinin is used for uncomplicated falciparum malaria. However, these treatments are not efficient against severe malaria and improvements are needed. Primaquine (PQ) is one of the most widely used antimalarial drugs. It is the only available drug to date for combating the relapsing form of malaria. Nevertheless, it has severe side effects. Particle drug-delivery systems present the ability to enhance the therapeutic properties of drugs and decrease their side effects. Here, we report the development of Polymeric Primaquine Microparticles (PPM) labeled with 99mTc for therapeutic strategy against malaria infection. The amount of primaquine encapsulated into the PPM was 79.54%. PPM presented a mean size of 929.47 ± 37.72 nm, with a PDI of 0.228 ± 0.05 showing a homogeneous size for the microparticles and a monodispersive behavior. Furthermore, the biodistribution test showed that primaquine microparticles have a high liver accumulation. In vivo experiments using mice show that the PPM treatments resulted in partial efficacy and protection against the development of the parasite compared to free Primaquine. These results suggest that microparticles drug delivery systems of primaquine could be a possible approach for malaria prevention and treatment.

Rheumatoid arthritis treatment using hydroxychloroquine and methotrexate co-loaded nanomicelles: In vivo results.

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. It is a long-lasting autoimmune disease, which mainly affects the joints causing inflammation and swelling of the synovial joint. RA has a significant impact on the ability to perform daily activities including simple work and household chores. Nonetheless, due to the long periods of pain and the continuous use of anti-inflammatory drugs, RA can debilitate the quality of life and increases mortality. Current therapeutic approaches to treat RA aim to achieve prolonged activity and early and persistent remission of the disease, with the gradual adoption of different drugs available. In this study, we developed a novel hydroxychloroquine and methotrexate co-loaded Pluronic® F-127 nanomicelle and evaluated its therapeutic effects against RA. Our results showed that drug-loaded nanomicelles were capable of modulating the inflammatory process of RA and reducing osteoclastogenesis, edema, and cell migration to the joint. Overall, compared to the free drugs, the drug-loaded nanomicelles showed a 2-fold higher therapeutic effect.

Radioactive gold nanocluster (198-AuNCs) showed inhibitory effects on cancer cells lines.

Cancer is a global epidemic disease responsible for over ten millions death worldwide. The early diagnosis and the precise treatment with reduced adverse reactions are the main goal worldwide. In this study, we produced, characterized and evaluated (in vitro) in three different cancer cell lines (protaste, breast and melanoma) a radioactive gold nanocluster (R-AuNC) (198Au25(Capt)18). The pharmacokinetics as the influence in the ABC transporter (MRP1 Efflux Transporter Protein) was also evaluated. The results showed that R-AuNC (198Au25(Capt)18) are capable to kill the cancer cells lines of protaste, breast and melanoma. The pharmacokinetics showed a fast clearance and great volume of distribution, confirming the use of R-AuNC as nanomedicine for cancer treatment. Finally, the ABC transporter assay corroborated that the R-AuNC (198Au25(Capt)18) has no risk of being pumped out of cells by this efflux transporter. The results validate the use of gold nanoparticles as therapeutic nanomedicine for cancer treatment.

The Effect of Nanosystems on ATP-Binding Cassette Transporters: Understanding the Influence of Nanosystems on Multidrug Resistance Protein-1 and P-glycoprotein.

The cancer multidrug resistance is involved in the failure of several treatments during cancer treatment. It is a phenomenon that has been receiving great attention in the last years due to the sheer amount of mechanisms discovered and involved in the process of resistance which hinders the effectiveness of many anti-cancer drugs. Among the mechanisms involved in the multidrug resistance, the participation of ATP-binding cassette (ABC) transporters is the main one. The ABC transporters are a group of plasma membrane and intracellular organelle proteins involved in the process of externalization of substrates from cells, which are expressed in cancer. They are involved in the clearance of intracellular metabolites as ions, hormones, lipids and other small molecules from the cell, affecting directly and indirectly drug absorption, distribution, metabolism and excretion. Other mechanisms responsible for resistance are the signaling pathways and the anti- and pro-apoptotic proteins involved in cell death by apoptosis. In this study we evaluated the influence of three nanosystem (Graphene Quantum Dots (GQDs), mesoporous silica (MSN) and poly-lactic nanoparticles (PLA)) in the main mechanism related to the cancer multidrug resistance such as the Multidrug Resistance Protein-1 and P-glycoprotein. We also evaluated this influence in a group of proteins involved in the apoptosis-related resistance including cIAP-1, XIAP, Bcl-2, BAK and Survivin proteins. Last, colonogenic and MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assays have also been performed. The results showed, regardless of the concentration used, GQDs, MSN and PLA were not cytotoxic to MDA-MB-231 cells and showed no impairment in the colony formation capacity. In addition, it has been observed that P-gp membrane expression was not significantly altered by any of the three nanomaterials. The results suggest that GQDs nanoparticles would be suitable for the delivery of other multidrug resistance protein 1 (MRP1) substrate drugs that bind to the transporter at the same binding pocket, while MSN can strongly inhibit doxorubicin efflux by MRP1. On the other hand, PLA showed moderate inhibition of doxorubicin efflux by MRP1 suggesting that this nanomaterial can also be useful to treat MDR (Multidrug resistance) due to MRP1 overexpression.

Lycopene used as Anti-inflammatory Nanodrug for the Treatment of Rheumathoid Arthritis: Animal assay, Pharmacokinetics, ABC Transporter and Tissue Deposition.

Lycopene is a carotenoid drug that has demonstrated several properties, including antioxidant and anti-inflammatory activity. The absorption in human body is very low (10-30 % only). In order to increase the bioavailability, lycopene nanoemulsion was formulated and characterized (atomic force microscopy, thermogravimetric analysis dynamic light scattering and differential scanning calorimetry). Also in vitro assay to evaluate the at-binding with MPR1 was performed. Finally, in vivo assay in animals inducted with rheumathoid arthritis were performed. The results showed that the formulated nanolycopene had superior efficacy when compared with the conventional lycopene (not nano-formulated) in inducted animals (rheumatoid arthritis). The results support the use of nanolycopene as an anti-inflammatory agent for rheumatoid arthritis therapy.

Doxorubicin as a fluorescent reporter identifies novel MRP1 (ABCC1) inhibitors missed by calcein-based high content screening of anticancer agents.

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum multidrug resistance and can decrease the bioavailability of many important drugs. Substrates of MRP1 include anti-cancer agents, antibiotics, antivirals, antidepressants and anti-inflammatory drugs. Using calcein as a fluorescent reporter in a high content uptake assay, we recently reported the identification of 12 MRP1 inhibitors after screening an anti-cancer library of 386 compounds. Here, we describe the development of a new high content imaging-based uptake assay using doxorubicin as a fluorescent reporter. Screening the same anti-cancer library of 386 compounds, the new assay identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. Inhibition of MRP1 activity was confirmed using flow cytometry and confocal microscopy-based transport assays. Six drugs (afatinib, celecoxib, doramapimod, mifepristone, MK-2206 and rosiglitazone) were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against vincristine, doxorubicin and etoposide. Mifepristone and doramapimod were most effective in reversal of resistance against vincristine while mifepristone and rosiglitazone were most successful in resensitizing H69AR cells against doxorubicin. Furthermore, resistance towards etoposide was completely reversed in the presence of celecoxib or doramapimod. Selected drugs were also evaluated for resistance reversal in HEK cells that overexpress P-glycoprotein or breast cancer resistance protein. Our results indicate mifepristone and doramapimod as pan inhibitors of these three drug transporters while celecoxib exhibited selective MRP1 inhibition. Together, our findings signify the importance of MRP1 in drug discovery and demonstrate the effectiveness and value of doxorubicin-based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies. On the other hand, anti-cancer drugs that did not interact with MRP1 carry a low risk for developing MRP1-mediated resistance.

Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators.

Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies.

Calcitriol and Calcipotriol Modulate Transport Activity of ABC Transporters and Exhibit Selective Cytotoxicity in MRP1-overexpressing Cells.

Efflux transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) can affect the efficacy and toxicity of a wide variety of drugs and are implicated in multidrug resistance (MDR). Eight test compounds, recently identified from an intramolecular FRET-based high throughput screening, were characterized for their interaction with MRP1. We report that the active metabolite of vitamin D3, calcitriol, and its analog calcipotriol are selectively cytotoxic to MRP1-overexpressing cells, besides inhibiting transport function of P-gp, MRP1, and BCRP. Calcitriol and calcipotriol consistently displayed a potent inhibitory activity on MRP1-mediated doxorubicin and calcein efflux in MRP1-overexpressing H69AR and HEK293/MRP1 cells. Vesicular transport studies confirmed a strong inhibitory effect of calcitriol and calcipotriol on MRP1-mediated uptake of tritium-labeled estradiol glucuronide and leukotriene C4 In cytotoxicity assays, MRP1-overexpressing cells exhibited hypersensitivity toward calcitriol and calcipotriol. Such collateral sensitivity, however, was not observed in HEK293/P-gp and HEK293/BCRP cells, although the vitamin D3 analogs inhibited calcein efflux in P-gp-overexpressing cells, and mitoxantrone efflux in BCRP-overexpressing cells. The selective cytotoxicity of calcitriol and calpotriol toward MRP1 over-expressing cells can be eliminated with MRP1 inhibitor MK571. Our data indicate a potential role of calcitriol and its analogs in targeting malignancies in which MRP1 expression is prominent and contributes to MDR.

Biophysical Approaches Facilitate Computational Drug Discovery for ATP-Binding Cassette Proteins.

Although membrane proteins represent most therapeutically relevant drug targets, the availability of atomic resolution structures for this class of proteins has been limited. Structural characterization has been hampered by the biophysical nature of these polytopic transporters, receptors, and channels, and recent innovations to in vitro techniques aim to mitigate these challenges. One such class of membrane proteins, the ATP-binding cassette (ABC) superfamily, are broadly expressed throughout the human body, required for normal physiology and disease-causing when mutated, yet lacks sufficient structural representation in the Protein Data Bank. However, recent improvements to biophysical techniques (e.g., cryo-electron microscopy) have allowed for previously "hard-to-study" ABC proteins to be characterized at high resolution, providing insight into molecular mechanisms-of-action as well as revealing novel druggable sites for therapy design. These new advances provide ample opportunity for computational methods (e.g., virtual screening, molecular dynamics simulations, and structure-based drug design) to catalyze the discovery of novel small molecule therapeutics that can be easily translated from computer to bench and subsequently to the patient's bedside. In this review, we explore the utility of recent advances in biophysical methods coupled with well-established in silico techniques towards drug development for diseases caused by dysfunctional ABC proteins.

High-content screening of clinically tested anticancer drugs identifies novel inhibitors of human MRP1 (ABCC1).

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2-6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.

ATP-Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening.

Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a "two-color MRP1" construct by fusing green fluorescent protein (GFP) and TagRFP to MRP1 nucleotide-binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. Two-color MRP1 transport activity was normal, as shown by vesicular transport of [(3)H]17ß-estradiol-17-ß-(D-glucuronide) and doxorubicin efflux in AAV-293 cells. We quantified fluorescence resonance energy transfer (FRET) from GFP to TagRFP as an index of NBD conformational changes. Our results show that ATP binding induces a large-amplitude conformational change that brings the NBDs into closer proximity. FRET was further increased by substrate in the presence of ATP but not by substrate alone. The data suggest that substrate binding is required to achieve a fully closed and compact structure. ATP analogs bind MRP1 with reduced apparent affinity, inducing a partially closed conformation. The results demonstrate the utility of the two-color MRP1 construct for investigating ATP-binding cassette transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1.

Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.

Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1.

Mutation of Glu521 or Glu535 in cytoplasmic loop 5 causes differential misfolding in multiple domains of multidrug and organic anion transporter MRP1 (ABCC1).

The polytopic 5-domain multidrug resistance protein 1 (MRP1/ABCC1) extrudes a variety of drugs and organic anions across the plasma membrane. Four charged residues in the fifth cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 are critical for stable expression of MRP1 at the plasma membrane. Thus Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A. However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A. On the other hand, 4-PBA treatment allowed both E521A and E535A to exit the endoplasmic reticulum and be stably expressed at the plasma membrane. However, the 4-PBA-rescued E535A mutant exhibited decreased transport activity associated with reduced substrate affinity and conformational changes in both halves of the transporter. By contrast, E521A exhibited reduced transport activity associated with alterations in the mutant interactions with ATP as well as a distinct conformational change in the COOH-proximal half of MRP1. These findings illustrate the critical and complex role of CL5 for stable expression of MRP1 at the plasma membrane and more specifically show the differential importance of Glu(521) and Glu(535) in interdomain interactions required for proper folding and assembly of MRP1 into a fully transport competent native structure.

Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase.

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

Structure of the Bateman2 domain of yeast Snf4: dimeric association and relevance for AMP binding.

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. AMP is believed to control the activity of AMPK by binding to the gamma subunit of this heterotrimeric enzyme. This subunit contains two Bateman domains, each of which is composed of a tandem pair of cystathionine beta-synthase (CBS) motifs. No structural information is currently available on this subunit, and the molecular basis for its interactions with AMP is not well understood. We report here the crystal structure at 1.9 Angstrom resolution of the Bateman2 domain of Snf4, the gamma subunit of the yeast ortholog of AMPK. The structure revealed a dimer of the Bateman2 domain, and this dimerization is supported by our light-scattering, mutagenesis, and biochemical studies. There is a prominent pocket at the center of this dimer, and most of the disease-causing mutations are located in or near this pocket.

The beta-oxidation systems of Escherichia coli and Salmonella enterica are not functionally equivalent.

Based on its genome sequence, the pathway of beta-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to grow, whereas S. enterica grew well. Exchange of the wild-type fadR genes between the two organisms showed this to be a property of S. enterica rather than of the FadR proteins per se. This difference in growth was attributed to S. enterica having higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid. S. enterica cleanly converted all of the acid to acetyl-CoA, whereas E. coli accumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that the S. enterica FadE and FadBA enzymes were responsible for the greater efficiency of beta-oxidation relative to that of E. coli.