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ABSTRACT: Argiotoxin-636 (ArgTX-636), a natural product from the spider Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic glutamate (iGlu) receptors. Here, three series of analogues were designed to exploit selectivity among iGlu receptors, taking advantage of a recently developed solid-phase synthetic methodology for the synthesis of ArgTX-636 and analogues. Initially, the importance of secondary amino groups in the polyamine chain was studied by the synthesis of systematically modified ArgTX-636 analogues, which were evaluated for pharmacological activity at NMDA and AMPA receptors. This led to the identification of two compounds with preference for NMDA and AMPA receptors, respectively. These were further elaborated by systematically changing the aromatic headgroup and linker amino acid leading to compounds with increased potency and selectivity for NMDA and AMPA receptors, respectively. Thus, the first structure-activity relationship study of ArgTX-636 has been carried out and has provided lead compounds for probing the ion channel region of iGlu receptors.
Journal of Medicinal Chemistry 01/2013; · 4.80 Impact Factor
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ABSTRACT: Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further discovery. Here we present a general synthetic strategy for this class of toxins and prepare five structurally varied polyamine toxins. Electrophysiological testing at three ionotropic glutamate receptor subtypes reveals that in particular two of these, Nephila Polyamine Toxins-1 (NPTX-1) and -8 (NPTX-8), comprise intriguing pharmacological activities by having subnanomolar IC50 values at kainate receptors.
Journal of Medicinal Chemistry 10/2012; · 4.80 Impact Factor
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ABSTRACT: The serotonin transporter (SERT) and the norepinephrine transporter (NET) are sodium-dependent neurotransmitter transporters responsible for reuptake of released serotonin and norepinephrine, respectively, into nerve terminals in the brain. A wide range of inhibitors of SERT and NET are used as treatment of depression and anxiety disorders, or as psychostimulant drugs of abuse. Despite their clinical importance, the molecular mechanisms by which various types of antidepressant drugs bind and inhibit SERT and NET are still elusive for the majority of the inhibitors, including the molecular basis for SERT/NET selectivity. Mutational analyses have suggested that a central substrate binding site (denoted the S1 pocket) also harbors an inhibitor binding site. In the present study, we determine the effect of mutating six key S1 residues in human SERT (hSERT) and NET (hNET) on the potency of 15 prototypical SERT/NET inhibitors belonging to different drug classes. Analysis of the resulting drug sensitivity profiles provides novel information on drug binding modes in hSERT and hNET and identifies specific S1 residues as important molecular determinants for inhibitor potency and hSERT/hNET selectivity.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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ABSTRACT: Gaining on pain: Two snake protein toxins combine to form a heteromeric complex, MitTx, which activates acid-sensing ion channels (ASICs) and elicits pain. MitTx is used to explore the roles of specific ASIC subtypes in pain pathways. These insights may provide new pharmacological tools ultimately leading to new treatments for pain.
Angewandte Chemie International Edition 03/2012; 51(17):4009-11. · 13.45 Impact Factor
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Anders Bach,
Bettina H Clausen,
Magda Møller,
Bente Vestergaard,
Celestine N Chi,
Adam Round,
Pernille L Sørensen,
Klaus B Nissen,
Jette S Kastrup,
Michael Gajhede,
Per Jemth, Anders S Kristensen,
Patrik Lundström,
Kate L Lambertsen,
Kristian Strømgaard
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ABSTRACT: Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.
Proceedings of the National Academy of Sciences 02/2012; 109(9):3317-22. · 9.68 Impact Factor
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ABSTRACT: Spiders are important species in ecological systems and as major predators of insects they are endowed with a plethora of low-molecular-weight natural products having intriguing biological activities. The isolation and biological characterization of these entities are well established, however, only very recently have these compounds been used as templates for the design, synthesis, and biological evaluation of synthetic analogues. In contrast, the investigation of compounds responsible for chemical communication between spiders is far less developed, but recently new light has been shed onto the area of pheromones and allomones from spiders. Herein, we recapitulate these recent results, put them into perspective with previous findings, and provide an outlook for future studies of these chemotypes.
Angewandte Chemie International Edition 11/2011; 50(48):11296-311. · 13.45 Impact Factor
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ABSTRACT: The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.
Pharmacological reviews 09/2011; 63(3):585-640. · 17.00 Impact Factor
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Jacob Andersen,
Nicolai Stuhr-Hansen,
Linda Zachariassen,
Søren Toubro,
Stinna M R Hansen,
Jonas N N Eildal,
Andrew D Bond,
Klaus P Bøgesø,
Benny Bang-Andersen, Anders S Kristensen,
Kristian Strømgaard
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ABSTRACT: Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.
Proceedings of the National Academy of Sciences 07/2011; 108(29):12137-42. · 9.68 Impact Factor
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ABSTRACT: The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.
Nature Neuroscience 06/2011; 14(6):727-35. · 15.53 Impact Factor
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ABSTRACT: The protein--protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treating ischemic brain diseases, neuropathic pain, and Alzheimer's disease. We have previously demonstrated that N-alkylated tetrapeptides are potent inhibitors of this interaction, and here, this template is exploited for the development of blood plasma-stable and cell-permeable inhibitors. Initially, we explored both the amino acid sequence of the tetrapeptide and the nature of the N-alkyl groups, which consolidated N-cyclohexylethyl-ETAV (1) as the most potent and selective compound. Next, the amide moieties of N-methylated ETAV were systematically replaced with thioamides, demonstrating that one of three amide bonds could be replaced without compromising the affinity. Subsequent optimization of the N-alkyl groups and evaluation of cell permeability led to identification of N-cyclohexylethyl-ETA(S)V (54) as the most potent, plasma-stable and cell-permeable inhibitor, which is a promising tool in unraveling the therapeutic potential of the PSD-95/NMDA receptor interaction.
Journal of Medicinal Chemistry 02/2011; 54(5):1333-46. · 4.80 Impact Factor
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ABSTRACT: Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology. Biological evaluation revealed that JSTX-4 inhibits iGlu receptors only in high μM concentrations, thereby being substantially less potent than structurally related polyamine toxins.
Journal of Natural Products 12/2010; 74(3):483-6. · 3.13 Impact Factor
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ABSTRACT: An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.
Journal of Medicinal Chemistry 10/2010; 53(20):7441-51. · 4.80 Impact Factor
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ABSTRACT: Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.
Journal of Biological Chemistry 09/2010; 285(36):28252-60. · 4.77 Impact Factor
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ABSTRACT: The serotonin transporter (SERT) regulates extracellular levels of the neurotransmitter serotonin (5-hydroxytryptamine) in
the brain by facilitating uptake of released 5-hydroxytryptamine into neuronal cells. SERT is the target for widely used antidepressant
drugs, including imipramine, fluoxetine, and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant
binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the
(S)-citalopram binding pocket in human SERT (hSERT) using mutational and computational approaches. Comparative modeling and
ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes
is functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational
mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)-citalopram binding to hSERT and demonstrates that the antidepressant binding site overlaps with the substrate binding site.
Journal of Biological Chemistry 01/2010; 285(3):2051-2063. · 4.77 Impact Factor
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Angewandte Chemie International Edition 11/2009; 48(51):9685-9. · 13.45 Impact Factor
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ABSTRACT: The serotonin transporter (SERT) regulates extracellular levels of the neurotransmitter serotonin (5-hydroxytryptamine) in the brain by facilitating uptake of released 5-hydroxytryptamine into neuronal cells. SERT is the target for widely used antidepressant drugs, including imipramine, fluoxetine, and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the (S)-citalopram binding pocket in human SERT (hSERT) using mutational and computational approaches. Comparative modeling and ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes is functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)-citalopram binding to hSERT and demonstrates that the antidepressant binding site overlaps with the substrate binding site.
Journal of Biological Chemistry 11/2009; 285(3):2051-63. · 4.77 Impact Factor
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Rasmus P. Clausen,
Peter Naur, Anders S. Kristensen,
Jeremy R. Greenwood,
Mette Strange,
Hans Bräuner-Osborne,
Anders A. Jensen,
Anne Sophie T. Nielsen,
Ulla Geneser,
Lone M. Ringgaard,
Birgitte Nielsen,
Darryl S. Pickering,
Lotte Brehm,
Michael Gajhede,
Povl Krogsgaard-Larsen,
Jette S. Kastrup
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ABSTRACT: The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.
08/2009;
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ABSTRACT: The biogenic monoamine transporters are integral membrane proteins that perform active transport of extracellular dopamine, serotonin and norepinephrine into cells. These transporters are targets for therapeutic agents such as antidepressants, as well as addictive substances such as cocaine and amphetamine. Seminal advances in the understanding of the structure and function of this transporter family have recently been accomplished by structural studies of a bacterial transporter, as well as medicinal chemistry and pharmacological studies of mammalian transporters. This feature article focuses on antidepressant drugs that act on the serotonin and/or the norepinephrine transporters. Specifically, we focus on structure-activity relationships of these drugs with emphasis on relationships between their molecular properties and the current knowledge of transporter structure.
Chemical Communications 08/2009; · 6.17 Impact Factor
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Rasmus P Clausen,
Peter Naur, Anders S Kristensen,
Jeremy R Greenwood,
Mette Strange,
Hans Bräuner-Osborne,
Anders A Jensen,
Anne Sophie T Nielsen,
Ulla Geneser,
Lone M Ringgaard,
Birgitte Nielsen,
Darryl S Pickering,
Lotte Brehm,
Michael Gajhede,
Povl Krogsgaard-Larsen,
Jette S Kastrup
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ABSTRACT: The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.
Journal of Medicinal Chemistry 08/2009; 52(15):4911-22. · 4.80 Impact Factor
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ABSTRACT: The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting
5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional
disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter
(LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human
transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However,
the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still
elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant
for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic
antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively
increased the Ki values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket
by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these
antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrate-
and inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion
of the 5HT-binding site.
Journal of Biological Chemistry 04/2009; 284(15):10276-10284. · 4.77 Impact Factor
