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Virtual Screening in Search of Allosteric Modulators of Nav1.1 Channels
242a
Monday, February 13, 2017
disc. INa was modeled using the Hodgkin-Huxley formalism. The intracellular domain of the first cell was subjected to a voltage-clamp activation protocol while the intracellular potential of the second cell was clamped to the resting potential. In the model with one membrane facing a non-conducting obstacle, decreasing cleft width from 1000 to 20 nm resulted in decreasing INa peak intensity at voltage steps R-30 mV. This decrease was caused by the negative Ve in the cleft, which decreased the driving force for INa. However, at voltage steps just above threshold, INa was activated in a ring-shaped peripheral region. This resulted in larger INa in simulations with clefts 10-200 nm wide. When total INa was considered, the negative Ve resulted in a cleft-dependent shift of the steady state activation curve to more negative potentials and a steepening of the curve. Narrowing the cleft thus lowered and accentuated the threshold of INa. These effects were more prominent in larger discs and when Naþ channels were redistributed in the center of the disc. In the two-cell model, the negative Ve caused by INa in the first cell resulted in activation of INa in the second cell after a ~1 ms delay for clefts <70 nm. Therefore, the Ve caused by INa in a narrow restricted extracellular space exerts a major feedback on Naþ channel behavior, modulating the voltage-dependence of activation and the threshold behavior of INa, and thus cellular excitability. These effects are strongly influenced by the spatial distribution of Naþ channels. These findings are relevant for a comprehensive understanding of cardiac excitation. 1190-Pos Board B258 Beta-Pompilidotoxin Adopts a Distinct 3D Structure when Bound to Nav1.2 DIV S3-S4 Paddle Motif Meero Yeu1,2, Boris Arshava1, Jianqin Zhuang1, Se´bastien F. Poget1,2. 1 Department of Chemistry, College of Staten Island, CUNY, Staten Island, NY, USA, 2Program in Biochemistry, The Graduate Center, CUNY, New York, NY, USA. Beta-pompilidotoxin (b-PMTX) is a spider wasp toxin which binds on neurotoxin receptor site 3 of neuronal sodium channels. Even though it lacks disulfide bonds and has no structural homology to the a-scorpion toxins and sea anemone toxins that bind at the same site, b-PMTX not only slows inactivation of voltagegated sodium channels, it also has a high affinity for Nav1.2, but does not affect Nav1.5. Due to its high specificity for Nav1.2 DIV, structural analysis of this 13 amino acid peptide may prove vital in understanding gating mechanism of the channel, because it potentially allows for the channel protein to be ‘‘locked’’ into a specific gating state, thus enabling future studies of the various Nav1.2 channel conformations involved in the gating mechanism. Structural analysis of b-PMTX may also provide valuable insight for the development of future pharmaceutical agents. b-PMTX was synthesized and structures were elucidated using solution state 2D homonuclear NMR. Interestingly, b-PMTX appears to be unfolded in an aqueous environment, but in the presence of a membrane mimetic and a membrane mimetic containing a Nav1.2 fragment, b-PMTX adopted two distinct 3D conformations. Characterization and elucidation of b-PMTX structure were performed in both detergent micelles and lipid bicelles, and interacting residues were identified. 1191-Pos Board B259 The Structural Characterization of the Human Cardiac Sodium Channel Voltage-Sensor Domain IV Paddle Motif Mohammed H. Bhuiyan1,2, Adel K. Hussein1,2, Boris Arshava1, Jianqin Zhuang1, James Aramini3, Fred Naider1,2, Se´bastien F. Poget1,2. 1 Department of Chemistry, College of Staten Island, City University of New York, Staten Island, NY, USA, 2Program in Biochemistry, The Graduate Center, City University of New York, New York, NY, USA, 3CUNY Advanced Science Research Center, City University of New York, New York, NY, USA. Voltage-gated sodium channels (VGSCs) are membrane proteins that serve important functions in the central and peripheral nervous systems (C/PNS) and cardiac and skeletal muscle. Diseases that can be caused by malfunctioning VGSCs (often due to modified gating properties) include epilepsy, chronic pain, and several ailments afflicting the heart. Interestingly, peptide toxins from venomous and poisonous animals have been known to target VGSCs. Site 3 and 4 peptide neurotoxins are known to interact with the voltagesensing domains of VGSCs, modifying their gating properties. It has been proposed that peptide neurotoxins may be used directly or as lead compounds for rational drug design to alleviate symptoms caused by channelopathies. An example of this approach is the drug Prialt, an u-conotoxin, used to alleviate intractable pain in patients. By probing the structural details of the interaction between peptide neurotoxins and VGSCs, we will gain more insight into the function of these toxins and what governs their strong and specific effect. VGSCs are large, highly hydrophobic and heavily post-translationally modified proteins, thus making it unlikely that X-ray crystallography, solution-state NMR or cryo-EM will be successful in producing structural information on intact protein at the atomic level. As an alternative strategy, we are studying
the main binding sequence for site 3 and 4 toxins, the S3b-S4 paddle motif, in complex with an interacting gating modifier toxin of the VGSC NaV1.5. VSD IV of NaV1.5 is the known target of many site-3 a-scorpion and sea anemone toxins that inhibit fast inactivation of the channel. We have chemically and biosynthetically synthesized the 37 residue paddle motif peptide and characterized it through circular dichroism spectroscopy, MALDI-TOF mass spectrometry, and solution state NMR, producing the first backbone assignments of a mammalian VGSC paddle motif. 1192-Pos Board B260 Virtual Screening in Search of Allosteric Modulators of Nav1.1 Channels Johnathan Wong1, Syed R. Ali1, Paul Wadsworth1, Aditya K. Singh1, Zhiqing Liu1, Haiying Chen1, Jia Zhou2, Fernanda Laezza2. 1 Department of Pharmacology & Toxicology, UTMB Galveston, Galveston, TX, USA, 2Department of Pharmacology & Toxicology, Center for Addiction Research, UTMB Galveston, Galveston, TX, USA. Voltage-gated Naþ (Nav) channels provide the basis for neuronal excitability in the brain. Of the nine Nav channel isoforms (Nav1.1-Nav1.9), Nav1.1 exhibits cell-specific distribution in fast-spiking parvalbumin (PV) interneurons in the cortical circuit. Reduced function of PV interneurons results in impairment of the cognitive domain, a bio-signature common to a variety of neuropsychiatric disorders. Thus, selective allosteric modulators targeting Nav1.1 might provide new means to rescue PV interneuron function and improve cognition. Previous studies have identified fibroblast growth factor 14 (FGF14) as a functionally relevant regulator of Nav1.1 channels. Binding of FGF14 to the Nav1.1 C-terminal tail results in modulation of Nav1.1-mediated peak transient currents and biophysical properties of the channel activation and steady-state inactivation. The FGF14:Nav1.1 protein:protein interaction (PPI) interface might therefore provide a novel target for the development of Nav1.1 allosteric modulators. In search of small molecules targeting Nav1.1, we conducted a ligand-based high-throughput virtual screening using a FGF14:Nav1.1 homology model based on available crystal structures of homologous proteins. The UCSF chimera software was used to determine the region of interaction between FGF14 and Nav1.1, and Autodock was used to create a grid box surrounding this area to identify critical amino acids at the PPI surface of FGF14.This region of interest was submitted to the Texas Advanced Computing Center (TACC) drug discovery database, which identified 1001 ZINC compounds out of 642,759 possible ligands against the interaction site. Scores ranged from 13 to 0, with lower scores indicating greater likelihood of binding to the FGF14 surface, and thus interfering with Nav 1.1 binding. On-going counter and orthogonal screenings using in heterologous systems as well as Schro¨dinger Advanced Drug Discovery Suite are being used to provide in-cell validation of 14 identified hits and understanding of the target-ligand interactions. These small molecules might represent a new class of PPI-based Nav1.1-specific allosteric modulators with applicability as cognitive enhancers. 1193-Pos Board B261 Tuning the Ion Selectivity of Two-Pore Channels Jiangtao Guo, Weizhong Zeng, Youxing Jiang. UT Southwestern Medical Center, Dallas, TX, USA. Organellar two-pore channels (TPCs) contain two copies of a Shaker-like sixtransmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region yet exhibit drastically different ion selectivity. Plant TPC1 functions as a non-selective, cation channel on the vacuole membrane, while mammalian TPC channels have been shown to be endo/lysosomal Naþ-selective or Ca2þ-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity to that of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is slightly selective for Ca2þ over Naþ, but non-selective among various group I monovalent cations. Our results also confirm that HsTPC2 is a highly Naþ-selective channel activated by PI(3,5) P2. Guided by our recent structure of AtTPC1, we converted the nonselective AtTPC1 to a highly Naþ-selective, HsTPC2-like channel and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Naþ-selective AtTPC1 mutant elucidates the structural basis for Naþ selectivity in mammalian TPCs. 1194-Pos Board B262 Mapping Protein:Protein Interaction of the FGF14:GSK3b Complex Aditya K. Singh, Paul A. Wadsworth, Fernanda Laezza. Pharmacology & Toxicology, UTMB, Galveston, TX, USA. Glycogen synthase kinase 3 (GSK3) is a multifaceted enzyme with ubiquitous expression in the central nervous system (CNS). Increased levels of GSK3 trigger a cascade of serine/threonine (S/T) phosphorylation events that correlates with maladaptive plasticity of neuronal circuitries in neuropsychiatric disorders
Monday, February 13, 2017
disc. INa was modeled using the Hodgkin-Huxley formalism. The intracellular domain of the first cell was subjected to a voltage-clamp activation protocol while the intracellular potential of the second cell was clamped to the resting potential. In the model with one membrane facing a non-conducting obstacle, decreasing cleft width from 1000 to 20 nm resulted in decreasing INa peak intensity at voltage steps R-30 mV. This decrease was caused by the negative Ve in the cleft, which decreased the driving force for INa. However, at voltage steps just above threshold, INa was activated in a ring-shaped peripheral region. This resulted in larger INa in simulations with clefts 10-200 nm wide. When total INa was considered, the negative Ve resulted in a cleft-dependent shift of the steady state activation curve to more negative potentials and a steepening of the curve. Narrowing the cleft thus lowered and accentuated the threshold of INa. These effects were more prominent in larger discs and when Naþ channels were redistributed in the center of the disc. In the two-cell model, the negative Ve caused by INa in the first cell resulted in activation of INa in the second cell after a ~1 ms delay for clefts <70 nm. Therefore, the Ve caused by INa in a narrow restricted extracellular space exerts a major feedback on Naþ channel behavior, modulating the voltage-dependence of activation and the threshold behavior of INa, and thus cellular excitability. These effects are strongly influenced by the spatial distribution of Naþ channels. These findings are relevant for a comprehensive understanding of cardiac excitation. 1190-Pos Board B258 Beta-Pompilidotoxin Adopts a Distinct 3D Structure when Bound to Nav1.2 DIV S3-S4 Paddle Motif Meero Yeu1,2, Boris Arshava1, Jianqin Zhuang1, Se´bastien F. Poget1,2. 1 Department of Chemistry, College of Staten Island, CUNY, Staten Island, NY, USA, 2Program in Biochemistry, The Graduate Center, CUNY, New York, NY, USA. Beta-pompilidotoxin (b-PMTX) is a spider wasp toxin which binds on neurotoxin receptor site 3 of neuronal sodium channels. Even though it lacks disulfide bonds and has no structural homology to the a-scorpion toxins and sea anemone toxins that bind at the same site, b-PMTX not only slows inactivation of voltagegated sodium channels, it also has a high affinity for Nav1.2, but does not affect Nav1.5. Due to its high specificity for Nav1.2 DIV, structural analysis of this 13 amino acid peptide may prove vital in understanding gating mechanism of the channel, because it potentially allows for the channel protein to be ‘‘locked’’ into a specific gating state, thus enabling future studies of the various Nav1.2 channel conformations involved in the gating mechanism. Structural analysis of b-PMTX may also provide valuable insight for the development of future pharmaceutical agents. b-PMTX was synthesized and structures were elucidated using solution state 2D homonuclear NMR. Interestingly, b-PMTX appears to be unfolded in an aqueous environment, but in the presence of a membrane mimetic and a membrane mimetic containing a Nav1.2 fragment, b-PMTX adopted two distinct 3D conformations. Characterization and elucidation of b-PMTX structure were performed in both detergent micelles and lipid bicelles, and interacting residues were identified. 1191-Pos Board B259 The Structural Characterization of the Human Cardiac Sodium Channel Voltage-Sensor Domain IV Paddle Motif Mohammed H. Bhuiyan1,2, Adel K. Hussein1,2, Boris Arshava1, Jianqin Zhuang1, James Aramini3, Fred Naider1,2, Se´bastien F. Poget1,2. 1 Department of Chemistry, College of Staten Island, City University of New York, Staten Island, NY, USA, 2Program in Biochemistry, The Graduate Center, City University of New York, New York, NY, USA, 3CUNY Advanced Science Research Center, City University of New York, New York, NY, USA. Voltage-gated sodium channels (VGSCs) are membrane proteins that serve important functions in the central and peripheral nervous systems (C/PNS) and cardiac and skeletal muscle. Diseases that can be caused by malfunctioning VGSCs (often due to modified gating properties) include epilepsy, chronic pain, and several ailments afflicting the heart. Interestingly, peptide toxins from venomous and poisonous animals have been known to target VGSCs. Site 3 and 4 peptide neurotoxins are known to interact with the voltagesensing domains of VGSCs, modifying their gating properties. It has been proposed that peptide neurotoxins may be used directly or as lead compounds for rational drug design to alleviate symptoms caused by channelopathies. An example of this approach is the drug Prialt, an u-conotoxin, used to alleviate intractable pain in patients. By probing the structural details of the interaction between peptide neurotoxins and VGSCs, we will gain more insight into the function of these toxins and what governs their strong and specific effect. VGSCs are large, highly hydrophobic and heavily post-translationally modified proteins, thus making it unlikely that X-ray crystallography, solution-state NMR or cryo-EM will be successful in producing structural information on intact protein at the atomic level. As an alternative strategy, we are studying
the main binding sequence for site 3 and 4 toxins, the S3b-S4 paddle motif, in complex with an interacting gating modifier toxin of the VGSC NaV1.5. VSD IV of NaV1.5 is the known target of many site-3 a-scorpion and sea anemone toxins that inhibit fast inactivation of the channel. We have chemically and biosynthetically synthesized the 37 residue paddle motif peptide and characterized it through circular dichroism spectroscopy, MALDI-TOF mass spectrometry, and solution state NMR, producing the first backbone assignments of a mammalian VGSC paddle motif. 1192-Pos Board B260 Virtual Screening in Search of Allosteric Modulators of Nav1.1 Channels Johnathan Wong1, Syed R. Ali1, Paul Wadsworth1, Aditya K. Singh1, Zhiqing Liu1, Haiying Chen1, Jia Zhou2, Fernanda Laezza2. 1 Department of Pharmacology & Toxicology, UTMB Galveston, Galveston, TX, USA, 2Department of Pharmacology & Toxicology, Center for Addiction Research, UTMB Galveston, Galveston, TX, USA. Voltage-gated Naþ (Nav) channels provide the basis for neuronal excitability in the brain. Of the nine Nav channel isoforms (Nav1.1-Nav1.9), Nav1.1 exhibits cell-specific distribution in fast-spiking parvalbumin (PV) interneurons in the cortical circuit. Reduced function of PV interneurons results in impairment of the cognitive domain, a bio-signature common to a variety of neuropsychiatric disorders. Thus, selective allosteric modulators targeting Nav1.1 might provide new means to rescue PV interneuron function and improve cognition. Previous studies have identified fibroblast growth factor 14 (FGF14) as a functionally relevant regulator of Nav1.1 channels. Binding of FGF14 to the Nav1.1 C-terminal tail results in modulation of Nav1.1-mediated peak transient currents and biophysical properties of the channel activation and steady-state inactivation. The FGF14:Nav1.1 protein:protein interaction (PPI) interface might therefore provide a novel target for the development of Nav1.1 allosteric modulators. In search of small molecules targeting Nav1.1, we conducted a ligand-based high-throughput virtual screening using a FGF14:Nav1.1 homology model based on available crystal structures of homologous proteins. The UCSF chimera software was used to determine the region of interaction between FGF14 and Nav1.1, and Autodock was used to create a grid box surrounding this area to identify critical amino acids at the PPI surface of FGF14.This region of interest was submitted to the Texas Advanced Computing Center (TACC) drug discovery database, which identified 1001 ZINC compounds out of 642,759 possible ligands against the interaction site. Scores ranged from 13 to 0, with lower scores indicating greater likelihood of binding to the FGF14 surface, and thus interfering with Nav 1.1 binding. On-going counter and orthogonal screenings using in heterologous systems as well as Schro¨dinger Advanced Drug Discovery Suite are being used to provide in-cell validation of 14 identified hits and understanding of the target-ligand interactions. These small molecules might represent a new class of PPI-based Nav1.1-specific allosteric modulators with applicability as cognitive enhancers. 1193-Pos Board B261 Tuning the Ion Selectivity of Two-Pore Channels Jiangtao Guo, Weizhong Zeng, Youxing Jiang. UT Southwestern Medical Center, Dallas, TX, USA. Organellar two-pore channels (TPCs) contain two copies of a Shaker-like sixtransmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region yet exhibit drastically different ion selectivity. Plant TPC1 functions as a non-selective, cation channel on the vacuole membrane, while mammalian TPC channels have been shown to be endo/lysosomal Naþ-selective or Ca2þ-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity to that of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is slightly selective for Ca2þ over Naþ, but non-selective among various group I monovalent cations. Our results also confirm that HsTPC2 is a highly Naþ-selective channel activated by PI(3,5) P2. Guided by our recent structure of AtTPC1, we converted the nonselective AtTPC1 to a highly Naþ-selective, HsTPC2-like channel and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Naþ-selective AtTPC1 mutant elucidates the structural basis for Naþ selectivity in mammalian TPCs. 1194-Pos Board B262 Mapping Protein:Protein Interaction of the FGF14:GSK3b Complex Aditya K. Singh, Paul A. Wadsworth, Fernanda Laezza. Pharmacology & Toxicology, UTMB, Galveston, TX, USA. Glycogen synthase kinase 3 (GSK3) is a multifaceted enzyme with ubiquitous expression in the central nervous system (CNS). Increased levels of GSK3 trigger a cascade of serine/threonine (S/T) phosphorylation events that correlates with maladaptive plasticity of neuronal circuitries in neuropsychiatric disorders