Design and Characterization of Prodrug-like Inhibitors for Preventing Glutamate Efflux through Reverse Transport.

Glutamate transporters are responsible for active transport of the major excitatory neurotransmitter glutamate across the cell membrane, regulating the extracellular glutamate concentration in the mammalian brain. Extracellular glutamate levels in the brain are usually in the submicromolar range but can increase by exocytosis, inhibition of cellular uptake, or through glutamate release by reverse transport, as well as other mechanisms, which can lead to neurodegeneration and neuronal cell death. Such conditions can be encountered upon energy deprivation during an ischemic stroke. Here, we developed acetoxymethyl (AM) ester prodrug-like derivatives of excitatory amino acid transporter (EAAT) inhibitors that permeate the cell membrane and are activated, most likely through hydrolysis by endogenous cellular esterases, to form the active EAAT inhibitor. Upon increase in external K+ concentration, the inhibitors block glutamate efflux by EAAT reverse transport. Using a novel high-affinity fluorescent prodrug-like inhibitor, dl-threo-9-anthracene-methoxy-aspartate (TAOA) AM ester, we demonstrate that the precursor rapidly accumulates inside cells. Electrophysiological methods and fluorescence assays utilizing the iGluSnFR external glutamate sensor were used to demonstrate the efficacy of AM ester-protected inhibitors in inhibiting K+-mediated glutamate release. Together, our results provide evidence for a novel method to potentially prevent glutamate release by reverse transport under pathophysiological conditions in a model cell system, as well as in human astrocytes, while leaving glutamate uptake under physiological conditions operational. This method could have wide-ranging applications in the prevention of glutamate-induced neuronal cell death.

[Stocked medications in emergency medical service vehicles staffed by physicians-is prehospital treatment according to current guidelines possible?] / Medikamentöse Ausstattung arztbesetzter Rettungsmittel ­ ist eine präklinische Therapie nach aktuellen Leitlinien möglich?

Background: High quality of care in prehospital emergency medicine is characterized by guideline-based therapy. The basic prerequisite for this therapy is the availability of the required drugs in accordance with the current guideline recommendations. It is currently unclear whether this is guaranteed nationwide. There is no uniform standard regarding which drugs must be stocked in emergency medical services (EMS) vehicles staffed by physicians in Germany. The aim of the present study is to identify important diagnoses and the drugs required for their therapy. In a second step, medical directors throughout Germany were interviewed about current drugs available in their physician-staffed EMS vehicles and these were compared with the previously defined diagnosis-dependent drug lists. Materials and methods: After a structured guideline search, tracer diagnoses were defined and relevant drugs were assigned to them. The levels of evidence and recommendations were also considered. In a second step, this was compared with the current drugs available in physician-staffed EMS vehicles. Results: A total of 156 different medications were identified. The median number of medications stocked was 58; the minimum number of medications stocked was 35 at one site, while multiple sites stocked a maximum of 77 medications . Discussion: The present study investigated stocked medications in physician-staffed EMS vehicles. Overall, compared to a 2011 study, drug availability has improved. Most of the recommended medications are available in physician-staffed vehicles in Germany. The data from this study can be used by EMS throughout Germany to evaluate their preparedness.

The Role of Place Attachment in Promoting Refugees' Well-Being and Resettlement: A Literature Review.

Refugees are at great risk of developing mental health problems. Yet, little is known about how to optimally help this vulnerable group as there is a lack of evaluated refugee mental health interventions. The current article presents the results of a literature review which investigates the importance of place attachment for the promotion of refugees' well-being in the resettlement process. This review concentrated on the most recent and current literature regarding the potential role, importance, and relevance of people-place bonds in the dynamic process of refugee resettlement. It examines literature from the field of positive and environmental psychology, highlighting key theoretical concepts and research findings as well as gaps in research. The review revealed that little is known about the dynamics of place bonding, while the debate rages on about the geometry of the psychological constructs of person-place relationships. Yet, knowing more about which needs should be satisfied for easing place bonding could be of crucial importance for facilitating refugee well-being. Ultimately, improving the knowledge and understanding of the phases of this dynamic process could be useful for a more successful implementation of refugee resettlement practices and activities.

WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape.

BACKGROUND: Despite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy. METHODS: Two orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model. RESULTS: Mouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo. CONCLUSIONS: OPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.

Evaluation of a Positive Psychology Group Intervention in Nature for Young Cancer Survivors to Promote Well-Being and Post-Cancer Identity Development.

Purpose: Adolescent and young adult (AYA) cancer survivors (15-39 years) often struggle with their post-cancer identity. We developed an age-appropriate positive psychology group intervention with fellow AYA cancer survivors in a nature-rich environment. The aim of this study was to examine the needs and expectations of participants of this new, 1-week post-cancer identity intervention, and to evaluate their experiences to identify factors that contribute to the quality and impact of post-cancer mental health care interventions. Methods: This was a mixed-method exploratory pilot study evaluating a new, 1-week post-cancer identity intervention. The participants (n = 13) completed questionnaires at baseline measuring needs and expectations, and on the last day of the intervention and at 1-month follow-up, measuring experiences. Results: Participants' expectations to be supported in finding their post-cancer identity and receiving acknowledgment and recognition of their suffering in fellow AYAs were met. The intervention was perceived as positively contributing to post-cancer identity development (n = 11) and several aspects of well-being. Most appreciated elements of the intervention were peer support and the nature-rich environment on perceived well-being. Conclusion: The group-based positive psychology after care intervention in nature for AYA oncology was well received by participants and provided useful feedback for improvement of future editions. The intervention could offer support to AYA's in the promotion of their well-being and post-cancer identity development, but more research is needed to confirm this.

Designing Policy Solutions to Build a Healthier Rural America.

Disparities exist in the health, livelihood, and opportunities for the 46-60 million people living in America's rural communities. Rural communities across the United States need a new energy and focus concentrated around health and health care that allows for the designing capturing, and spreading of existing and new innovations. This paper aims to provide a framework for policy solutions to build a healthier rural America describing both the current state of rural health policy and the policies and practices in states that could be used as a national model for positive change.

SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth.

Trimethylation of histone 3 lysine 9 (H3K9me3) at gene promoters is a major epigenetic mechanism that silences gene expression. We have developed a small molecule inhibitor for the H3K9me3-specific histone methyltransferase SUV39H1. We report here that FAS expression is significantly down-regulated and SUV39H1 expression is significantly up-regulated in human colorectal carcinoma (CRC) as compared to normal colon. SUV39H1-selective inhibitor F5446 decreased H3K9me3 deposition at the FAS promoter, increased Fas expression, and increased CRC cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, inhibition of SUV39H1 altered the expression of genes with known functions in DNA replication and cell cycle in the metastatic colon carcinoma cells, which is associated with cell cycle arrest at S phase in the metastatic human colon carcinoma cells, resulting in tumor cell apoptosis and growth inhibition in a concentration-dependent manner in vitro. Moreover, F5446 increased 5-FU-resistant human CRC sensitivity to both 5-FU- and FasL-induced apoptosis and inhibited tumor cell growth in vitro. More importantly, F5446 suppressed human colon tumor xenograft growth in vivo. Our data indicate that pharmacological inhibition of SUV39H1 is an effective approach to suppress human CRC.

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion.

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC50 of 0.496 µmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of GZMB, PRF1, FASLG, and IFNG in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.

Pharmacologically targeting the myristoylation of the scaffold protein FRS2α inhibits FGF/FGFR-mediated oncogenic signaling and tumor progression.

Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling facilitates tumor initiation and progression. Although currently approved inhibitors of FGFR kinase have shown therapeutic benefit in clinical trials, overexpression or mutations of FGFRs eventually confer drug resistance and thereby abrogate the desired activity of kinase inhibitors in many cancer types. In this study, we report that loss of myristoylation of fibroblast growth factor receptor substrate 2 (FRS2α), a scaffold protein essential for FGFR signaling, inhibits FGF/FGFR-mediated oncogenic signaling and FGF10-induced tumorigenesis. Moreover, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppressed FRS2α myristoylation and decreased the phosphorylation with mild alteration of FRS2α localization at the cell membrane. B13 inhibited oncogenic signaling induced by WT FGFRs or their drug-resistant mutants (FGFRsDRM). B13 alone or in combination with an FGFR inhibitor suppressed FGF-induced WT FGFR- or FGFRDRM-initiated phosphoinositide 3-kinase (PI3K) activity or MAPK signaling, inducing cell cycle arrest and thereby inhibiting cell proliferation and migration in several cancer cell types. Finally, B13 significantly inhibited the growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo In summary, our study suggests a possible therapeutic approach for inhibiting FGF/FGFR-mediated cancer progression and drug-resistant FGF/FGFR mutants.

Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters.

Glutamate transporters maintain a large glutamate concentration gradient across synaptic membranes and are, thus, critical for functioning of the excitatory synapse. Mammalian glutamate transporters concentrate glutamate inside cells through energetic coupling of glutamate flux to the transmembrane concentration gradient of Na+. Structural models based on an archeal homologue, GltPh, suggest an elevator-like carrier mechanism. However, the energetic determinants of this carrier-based movement are not well understood. Although electrostatics play an important role in governing these energetics, their implication on transport dynamics has not been studied. Here, we combine a pre-steady-state kinetic analysis of the translocation equilibrium with electrostatic computations to gain insight into the energetics of the translocation process. Our results show the biphasic nature of translocation, consistent with the existence of an intermediate on the translocation pathway. In the absence of voltage, the equilibrium is shifted to the outward-facing configuration. Electrostatic computations confirm the intermediate state and show that the elevator-like movement is energetically feasible in the presence of bound Na+ ions, whereas a substrate-hopping model is energetically prohibitive. Our results highlight the critical contribution of charge compensation to transport and add to results from previous molecular dynamics simulations for improved understanding of the glutamate translocation process.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes.

FasL-mediated cytotoxicity is one of the mechanisms that CTLs use to kill tumor cells. However, human colon carcinoma often deregulates the Fas signaling pathway to evade host cancer immune surveillance. We aimed at testing the hypothesis that novel ceramide analogs effectively modulate Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis. We used rational design and synthesized twenty ceramide analogs as Fas function modulators. Five ceramide analogs, IG4, IG7, IG14, IG17, and IG19, exhibit low toxicity and potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. Functional deficiency of Fas limits both FasL and ceramide analogs in the induction of apoptosis. Ceramide enhances FasL-induced activation of the MAPK, NF-κB, and caspase 8 despite induction of potent tumor cell death. Finally, a sublethal dose of several ceramide analogs significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed five novel ceramide analogs that act at a sublethal dose to enhance the efficacy of tumor-specific CTLs, and these ceramide analogs hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.

Multi-kinase inhibitors interact with sildenafil and ERBB1/2/4 inhibitors to kill tumor cells in vitro and in vivo.

We have recently demonstrated that multi-kinase inhibitors such as sorafenib and pazopanib can suppress the detection of chaperones by in situ immuno-fluorescence, which is further enhanced by phosphodiesterase 5 inhibitors. Sorafenib and pazopanib inhibited the HSP90 ATPase activity with IC50 values of ~1.0 µM and ~75 nM, respectively. Pazopanib docked in silico with two possible poses into the HSP90 ATP binding pocket. Pazopanib and sildenafil combined to reduce the total protein levels of HSP1H/p105 and c-MYC and to reduce their co-localization. Sorafenib/pazopanib combined with sildenafil in a [GRP78+HSP27] -dependent fashion to: (i) profoundly activate an eIF2α/Beclin1 pathway; (ii) profoundly inactivate mTOR and increase ATG13 phosphorylation, collectively resulting in the formation of toxic autophagosomes. In a fresh PDX isolate of NSCLC combined knock down of [ERBB1+ERBB3] or use of the ERBB1/2/4 inhibitor afatinib altered cell morphology, enhanced ATG13 phosphorylation, inactivated NFκB, and further enhanced [sorafenib/pazopanib + sildenafil] lethality. Identical data to that with afatinib were obtained knocking down PI3K p110α/ß or using buparlisib, copanlisib or the specific p110α inhibitor BYL719. Afatinib adapted NSCLC clones were resistant to buparlisib or copanlisib but were more sensitive than control clones to [sorafenib + sildenafil] or [pazopanib + sildenafil]. Lapatinib significantly enhanced the anti-tumor effect of [regorafenib + sildenafil] in vivo; afatinib and BYL719 enhanced the anti-tumor effects of [sorafenib + sildenafil] and [pazopanib] in vivo, respectively.

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening.

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models' binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.

Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2.

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na(+) ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na(+)] dependence of ASCT2-associated currents indicates that the Na(+)/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid-free apo form of the transporter. When the substrate and two Na(+) ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na(+) ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na(+) ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na(+) appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.

Protonation state of a conserved acidic amino acid involved in Na(+) binding to the glutamate transporter EAAC1.

Substrate transport by glutamate transporters is coupled to the co-transport of 3 Na(+) ions and counter-transport of 1 K(+) ion. The highly conserved Asp454, which may be negatively charged, is of interest as its side chain may coordinate cations and/or contribute to charge compensation. Mutation to the nonionizable Asn resulted in a transporter that no longer catalyzed forward transport. However, Na(+)/glutamate exchange was still functional, as demonstrated by the presence of transient currents following rapid substrate application and voltage jumps. While the kinetics of Na(+)/glutamate exchange were slowed, the apparent valence (z) of the charge moved in EAAC1 D454N (0.71) was similar to that of EAAC1 WT (0.64). Valences calculated using the Poisson-Boltzmann equation were close to the experimental values for EAAC1 D454N (0.55), and with D454 protonated (0.45). In addition, pK(a) calculations performed for the bacterial homologue GltPh revealed a highly perturbed pK(a) (7.6 to >14) for D405 residue (analogous to D454), consistent with this site being protonated at physiological pH. In contrast to the D454N mutation, substitution to alanine resulted in a transporter that still bound glutamate, but could not translocate it. The results are consistent with molecular dynamics simulations, showing that the alanine but not the asparagine mutation resulted in defective Na(+) coordination. Our results raise the possibility that the protonated state of D454 supports transporter function.

Charge compensation mechanism of a Na+-coupled, secondary active glutamate transporter.

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.

Defining substrate and blocker activity of alanine-serine-cysteine transporter 2 (ASCT2) Ligands with Novel Serine Analogs.

The neutral amino acid transporter alanine-serine-cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of solute transporters and transports small, neutral amino acids across the membrane, including the physiologically important and ubiquitous amino acid glutamine. Our understanding of the involvement of ASCT2 in the physiological processes involving glutamine is hampered by a lack of understanding of its pharmacology and the absence of high-affinity inhibitors. In this study, we combined an in silico docking approach with experimental investigation of binding parameters to develop new ASCT2 inhibitors and substrates, a series of serine esters, and to determine structural parameters that govern their functional effects. The series of compounds was synthesized using standard methods and exhibited a range of properties, from inhibitors to partial substrates and full substrates. Our results suggest that amino acid derivatives with small side-chain volume and low side-chain hydrophobicity interact strongly with the closed-loop form of the binding site, in which re-entrant loop 2, the presumed extracellular gate for the substrate binding site, is closed off. However, these derivatives bind weakly to the open-loop form (external gate open to the extracellular side), acting as transported substrates. In contrast, inhibitors bind preferentially to the open-loop form. An aromatic residue in the side chain is required for high-affinity interaction. One of the compounds, the l-serine ester serine biphenyl-4-carboxylate reversibly inhibits ASCT2 function with an apparent affinity of 30 µM.

Iron(II) template synthesis of benzannulated triphospha- and triarsamacrocycles.

Nine-membered 1,4,7-triphospha- and triarsamacrocycles with unsaturated benzo-backbones have been prepared using the [Cp(R)Fe](+) unit as a template. The cyclisation involves the attack of a coordinated phosphide (or arsenide) nucleophile at an activated, electrophilic ortho-fluorophenyl substituent on a neighbouring pnictide donor. The macrocycle assembly is of the 2 + 1 type where two new chelate rings are formed from appropriately derivatised bidentate and monodentate phosphines/arsines. Both [(η(5)-C(5)H(5))Fe](+) and [(η(5)-C(5)Me(5))Fe](+) may be employed for the cyclisation with higher yields generally being observed with the unsubstituted Cp. All new compounds have been characterised by spectroscopic and analytical methods including the single-crystal X-ray structure determination of [(η(5)-C(5)H(5))Fe(tribenzo-9aneP(3)-Ph,Ph(F)(2))](+), 3a, and [(η(5)-C(5)H(5))Fe(tribenzo-9aneAs(3)-Ph,Ph(F)(2))](+), 5, as the tetraphenylborate salts. The crystal structures are isomorphous and show the unique conformation of these new macrocycles with a 'cup shaped' cavity formed by the rigid benzo-backbones. The 9aneAs(3) derivative is the first example of a nine-membered triarsamacrocycle.