Identification of novel modulators for ionotropic glutamate receptor, iGluA2 by in-silico screening
© Padmanabhan; licensee BioMed Central Ltd. 2013
Received: 1 May 2013
Accepted: 25 June 2013
Published: 15 July 2013
Ionotropic glutamate receptors (iGluAs, IUPHAR nomenclature) are the major excitatory amino acid neurotransmitter receptors in the mammalian central nervous system (CNS). iGluAs are potential therapeutic drug targets for various neurological disorders including ischemia, epilepsy, Parkinson’s and Alzheimer’s diseases. The known iGluA modulators, cyclothiazide (CTZ), IDRA-21, and other benzothiadiazide derivatives (ALTZ, HCTZ, and CLTZ) bind to the ligand-binding domain of flip-form of iGluA2 at the dimer interface, thereby increasing steady-state activation by reducing desensitization.
To discover new modulator compounds, we performed virtual screening for the ligand binding domain (LBD) of iGluA2 against NCI Diversity Set III library containing 1597 compounds, and subsequently performed binding-energy analysis for selected compounds. The crystal structure of rat iGluA2 S1S2J (PDB ID: 3IJO) was used for docking studies.
Results and conclusion
From this study, we obtained four compounds: (1) 10-2(methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, (2) 2-benzo[e]benzotriazol-2-yl-aniline, (3) 9-nitro-6H-indolo-(2,3,-b)quinoxaline, and (4) 1-hydroxy-n-(3-nitrophenyl)-2-napthamide. The binding mode of these four compounds is very similar to that of abovementioned established modulators: two molecules of each compound independently bind to the protein symmetrically at the dimer interface; occupy the subsites B, C, B’ and C’; potentially interact with Ser518 and Ser775. Binding energy analysis shows that all the four hits are comparable to the drug molecule, CTZ, and hence, we propose that the discovered hits may be potential molecules to develop new chemical libraries for modulating the flip form of iGluA2 function.
Ionotropic glutamate receptors (iGluAs) are a family of ligand-gated ion channels that are primarily localized to chemical synapses. They mediate fast excitatory neurotransmission in the mammalian central nervous system (CNS) [1, 2] and references therein. Based on sequence homology and pharmacology, iGluAs are classified into four subfamilies, namely, AMPA, Kainate, NMDA and δ-receptors. iGluAs are critical for normal operations of cellular and synaptic activity and plasticity. Dysregulation of these ion channels is frequently linked to the pathogenesis of a wide range of neurological disorder. For example, dysregulation of AMPA receptors leads to various chronic neuronal diseases such as depression, epilepsy, multiple sclerosis, Parkinson’s and Alzheimer’s diseases . Also, Ischemia (stroke) leads to dysregulation of AMPA receptors. Neuronal death, called excitotoxicity, is induced by excessive stimulation of neuronal glutamate receptors. Ischemia is the most common cause of neuronal activation that induces large increases in glutamate release . It has been clearly shown that inhibiting glutamate receptors using AMPA receptor antagonists can attenuate ischemic injury of neuronal tissue in animal experiments . It has also been demonstrated that the non-competitive AMPA antagonists, in cell cultures, could prevent glutamate-induced neuronal death at any agonist concentration, whereas the protective effect of competitive AMPA antagonists was lost at high agonist concentration . Therefore, inhibition of AMPA receptors using non-competitive method can be more beneficial for treatment than inhibition of AMPA receptors in a competitive manner. Positive allosteric modulators improve short-term memory in humans by slowing-down the deactivation of AMPA receptors , and hence these kinds of modulators may be beneficial for the treatment of depression and other disorders and diseases of CNS [3, 8].
Three structural classes of positive AMPA receptor modulators are available as of now: 1) pyrrolidinone and related piperidine compounds (e.g., aniracetam, CX614, CX516, CX516), 2) benzothiadiazide derivatives (e.g., IDRA-21 and S18986), and 3) biarylpropylsulfonamide compounds (e.s., PEPA, LY404187). The LBD-GluA2– cyclothiazide (CTZ) complex was the first crystal structure which described how an allosteric modulator inhibits desensitization through interacting with LBD of iGluA2 . The CTZ compound is moderately selective for the flip-form of AMPA receptors . Subsequently, structures of several other allosteric modulators complexed with LBD of GluA2 have been determined [13–15]. Moreover, crystal structures of the iGluA2-LBD complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) were recently determined for the flip-form of AMPA receptors .
To find the structurally diverse potential compounds, we performed virtual screening for LBD of iGluA2 against NCI Diversity Set III containing 1597 compounds. Here, we discuss four compounds which could be potential allosteric modulators for the flip-form of iGluA2: 10-(2-methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, 2-benzo[e]benzotriazol−2-yl-aniline, 1-hydroxy-N-(3-nitrophyenyl)-2-napthamide, and 9-nitro-6H-indolo-(2,3,-b) quinoxaline.
Virtual screening calculations
The AutoDock Vina 1.1.2  was used for virtual screening. The crystal structure of rat iGluA2 S1S2J (ligand-binding domain: N392-K506 and P632 – S775; PDB ID: 3IJO)  was used as a model for the docking procedure. The docking protocol was set to rigid condition and the size of the dock grid 21 Å × 17 Å × 25 Å, which encompassed the dimer interface of iGluA2-LBD. Exhaustiveness was set to 20 with all other parameters set on default values. About 1597 structurally diversified compounds of the NCI Diversity Set III were used for virtual screening. All calculations were done on the Intel core i5 processor and 4 GB of RAM running on the Ubuntu 12.04 operating system. The top-ranked compounds, sorted by binding energy values, were visually inspected for good chemical geometry and docking. For visualization, docking poses generated by AutoDock Vina were directly loaded into PyMol (http://www.pymol.org) through PyMOL Autodock/Vina Plugin . Pictures of the modeled protein-ligands complex were produced by PyMol.
Docking energy analysis
To further confirm the docking results, another robust program, DSX (DrugScore eXtented)  was used to estimate the binding energy of the ligands bound to the LBD of iGluA2. DSX uses a knowledge-based scoring function based on the DrugScore formalism . The ligand with the larger negative score has a theoretical higher affinity.
Results and discussion
The initial step in activation of glutamate receptor is the binding of the agonist (glycine, D-serine, aspartate, and glutamate analogues) to the ligand-binding domain (LBD) of the receptor. The LBD comprises a dimer of dimers relative to the more symmetrical assembly of the tetramer channel-forming domain. The dimer interface of the iGluA2-LBD forms an inverted U-shaped crevice with two-fold symmetry (Figure 1). At the dimer interface, as defined by Ptak et al. , the binding site region can be subdivided into five overlapping subsites (Figure 1A). The central subsite (subsite A) and two symmetrical copies of two subsites (subsites B, C, B’ and C’) are bordered by residues from each of the two LBD chains.
Compound #1: 10-(2-methoxyethyl)-3-phenyl benzo [g] pteridine-2,4-dione
Compound #2: 2-benzo [e] benzotriazol-2-yl-aniline
Two molecules of #2 independently bind to iGluA2 as observed in compound #1 (Figures 3C,D). The three-membered ring is sandwiched between the loop connecting the β-strand, β4 in chain A and β-strand, β9’ in chain B. The polar benzotriazole group of three-membered cyclic ring makes hydrogen bonds with Ser518 and Ser775 (Figure 3D). Moreover, Ser775 contributes another hydrogen bond to a nitrogen atom of aniline moiety which sits in the subsite B. The aniline group also possesses hydrophobic interactions with Leu780. In this complex, the subsite C is partially occupied by a non-polar naphthalene group. Incorporating a small hydrophobic group by connecting naphthalene moiety may contribute additional binding affinity with the protein. We observed that the subsite A is free from ligand contacts.
Compound #3: 9-nitro-6H-indolo (2,3,-b) quinoxaline
In this complex, the rigid cyclic ring structure of indolo-quinoxaline group is sandwiched at the dimer interface, and occupying subsites B and C (Figures 3E,F). The quinoxaline group is positioned in the subsite C. The hydroxyl group of Ser775 is hydrogen bonded to a nitrogen atom of quinoxaline. The Ser518 residue contributes hydrogen bond interactions with the nitro group and another nitrogen atom of quinoxaline through its main-chain and side-chain, respectively (Figure 3F). The nitro group also makes a potential salt bridge with Lys784 which is positioned in the subsite B. The subsite A is less occupied from ligand binding. The second molecule also binds in a similar mode to the symmetry subsites, B’ and C’ of the LBD domain.
Compound #4: 1-hydroxy-N-(3-nitrophenyl)-2-napthamide
In this complex, two molecules are independently sandwiched between the two monomers of the β-strand, β9’ and a loop connecting the β-strand, β4 (Figures 3G,H). The naphthalene moiety binds to the subsite C. The hydroxyl group of naphthalene moiety shares a hydrogen bond with Ser775. The subsite B is occupied by the nitrophenyl group. The phenyl ring makes hydrophobic interactions with Leu780. Ser518 contributes two hydrogen bonds with carbonyl and nitro-groups (Figure 3H). The nitro-group contributes additional salt bridge with Lys784. The subsite A is free from ligand interactions.
Comparison between compounds #1–4
Comparison of compounds #1–4 with the other AMPA modulators
The known positive AMPA receptor modulators fall into three structural classes, namely: 1) pyrrolidinone and related piperidine compounds (e.g., aniracetam, CX614, CX516, CX516), 2) benzothiadiazide derivatives (e.g., IDRA-21 and S18986), and 3) biarylpropylsulfonamide compounds (e.g., PEPA, LY404187). The first crystal structure of the iGluA2-LBD complex was solved using the compound, cyclothiazide (CTZ) . The CTZ compound is moderately selective for the flip-form of AMPA receptors . Subsequently, structures of several other allosteric modulators complexed with LBD of iGluA2 have been determined [13–15]. Moreover, crystal structures of the iGluA2-LBD complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) were recently determined for the flip-form of AMPA receptors .
Docking energy analysis by DSX procedure
Ionotropic glutamate receptors are the major excitatory amino acid neurotransmitter receptors in the vertebrate central nervous system (CNS). AMPA receptors, a subfamily of iGluAs, mediate the majority of the fast excitatory amino acid synaptic transmission in the CNS. Discovering modulators to regulate the function of AMPA receptors may provide numerous therapeutic avenues in the field of CNS drug discovery. In this aspect, we have discovered four compounds from virtual screening and docking studies using NCI Diversity Set III library containing 1597 compounds. All of these compounds, 1–4 bind to the dimer interface of iGluA2-LBD, and the binding mode of these compounds are essentially similar to the known established compounds such as cyclothiazide (CTZ), IDRA-21 and benzothiadiazide derivatives of ALTZ, HCTZ, CLTZ and HCTZ. Moreover, the binding energy analysis using DSX method revealed that all the predicted four compounds are unexpectedly comparable to the potential drug molecule, CTZ and much better than IDRA-21 and benzothiadiazide derivatives. Hence, we speculate that predicted molecules (compounds 1–4) are potential hits to develop new chemical libraries as modulators for AMPA receptors.
We thank Prof. Satishchandra, Director/Vice Chancellor to provide the finance support for computational facility.
- Dingledine R, Borges K, Bowie D, Traynelis SF: The glutamate receptor ion channels. Pharmacol Rev. 1999, 51: 7-61.PubMedGoogle Scholar
- Traynelis SF, Wollmuth LP, McBain CJ: Glutamate receptor ion channels: structure, regulation and function. Pharmacol Rev. 2010, 62: 405-496. 10.1124/pr.109.002451.PubMed CentralView ArticlePubMedGoogle Scholar
- Black MD: Therapeutic potential of positive AMPA modulators and their relationship to AMPA receptor subunits. A review of preclinical data. Psychopharmacology. 2005, 179: 154-163. 10.1007/s00213-004-2065-6.View ArticlePubMedGoogle Scholar
- Lipton P: Ischemic cell death in brain neurons. Physiol Rev. 1999, 79: 143-1568.Google Scholar
- Weiser T: AMPA receptor antagonists for the treatment of stroke. Curr Drug Targets CNS Neurol Disord. 2005, 4: 153-159. 10.2174/1568007053544129.View ArticlePubMedGoogle Scholar
- Kovács AD, Szabó G: Comparison of neuroprotective efficacy of competitive and non-competitive AMPA antagonists in vitro. Environ Toxicol Pharmacol. 1997, 3: 69-72. 10.1016/S1382-6689(96)00134-2.View ArticlePubMedGoogle Scholar
- Ingvar M, Ambros-Ingerson J, Davis M, Granger R, Kessler M, Rogers GA, Schehr RS, Lynch G: Enhancement by an ampakine of memory encoding in humans. Exp Neurol. 1997, 146: 553-555. 10.1006/exnr.1997.6581.View ArticlePubMedGoogle Scholar
- O’Neill MJ, Bleakman D, Zimmerman DM, Nisenbaum ES: AMPA receptor potentiators for the treatment of CNS disorders. Curr Drug Targets. 2004, 3: 181-194.Google Scholar
- Sobolevsky AI, Roscon MP, Gouaux E: X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature. 2009, 462: 745-756. 10.1038/nature08624.PubMed CentralView ArticlePubMedGoogle Scholar
- Ptak CP, Ahmed AH, Oswald RE: Probing the allosteric modulator binding site of GluR2 with thiazide derivatives. Biochemistry. 2009, 48: 8594-8602. 10.1021/bi901127s.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun Y, Olson R, Horning M, Armstrong N, Mayer M, Gouaux E: Mechanism of glutamate receptor desensitization. Nature. 2002, 417: 245-253. 10.1038/417245a.View ArticlePubMedGoogle Scholar
- Partin KM, Fleck MW, Mayer ML: AMPA receptor flip/flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocynate. J Neurosci. 1996, 16: 6634-6647.PubMedGoogle Scholar
- Jin R, Clark S, Weeks AM, Dudman JT, Gouaux E, Partin KM: Mechanism of positive allosteric modulators acting on AMPA receptors. J Neurosci. 2005, 25: 9027-9036. 10.1523/JNEUROSCI.2567-05.2005.View ArticlePubMedGoogle Scholar
- Kaae BH, Harpsoe K, Kastrup JS, Sanz AC: Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites. Chem Biol. 2007, 14: 1294-1303. 10.1016/j.chembiol.2007.10.012.View ArticlePubMedGoogle Scholar
- Hald H, Ahring PK, Timmermann DB, Liljefors T, Gajhede M, Kastrup JS: Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iGluA2. J Mol Biol. 2009, 391: 906-917. 10.1016/j.jmb.2009.07.002.View ArticlePubMedGoogle Scholar
- Trott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010, 31: 455-461.PubMed CentralPubMedGoogle Scholar
- Seelier D, de Bert GL: Ligand docking and binding site analysis with PyMol and Autodock/Vina. J Comput Aided Mol Des. 2010, 24: 417-422. 10.1007/s10822-010-9352-6.View ArticleGoogle Scholar
- Neudert G, Klebe G: DSX: a knowledge-based scoring function for the assessment of protein-ligand complexes. J Chem Inf Model. 2011, 51: 2731-2745. 10.1021/ci200274q.View ArticlePubMedGoogle Scholar
- Velec HF, Gohlke H, Klebe G: DrugScore (CSO) knowledge-based scoring function derived from small molecule crystal data with superior recognition rate of near-native ligand poses and better affinity prediction. J Med Chem. 2005, 48: 6296-6303. 10.1021/jm050436v.View ArticlePubMedGoogle Scholar
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