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Ligand Complex Structures as a Guide for Rational Drug Design
Sunday, February 28, 2016 improved single molecule detection. Here, we will review the recent developments of DNA origami nanopores both in lipid [2] and solid-state membranes [3]. These structures have extraordinary versatility and are a new and powerful tool in nanobiotechnology for a wide range of important applications beyond molecular sensing. We discuss the current challenges and possible solutions that would enhance the sensing capabilities of DNA origami nanopores [4]. Finally, we anticipate novel avenues for future research and highlight a range of exciting ideas and applications that could be explored in the near future. These include using these designer nanopores as model systems for protein channels [5] with the aim to build ion-selective and voltage-gated analogues. [1] N. A. W. Bell, C. R. Engst, M. Ablay, G. Divitini, C. Ducati, T. Liedl, and U. F. Keyser. DNA origami nanopores. Nano Letters, 12(1):512-517, 2012 [2] J. R. Burns, K. Goepfrich, J. W. Wood, V. V. Thacker, E. Stulz, U. F. Keyser, and S. Howorka. Lipid-Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor, Angewandte Chemie International Edition, 52(46):12069-12072, 2013. [3] S. Hernandez-Ainsa, N. A. W. Bell, V. V. Thacker, K. Goepfrich, K. Misiunas, M. Fuentes-Perez, F. Moreno-Herrero, and U. F. Keyser. DNA origami nanopores for controlling DNA translocation. ACS nano, 7(7):6024-6030, 2013. [4] N. A. W. Bell and U. F. Keyser. Specific Protein Detection using Designed DNA Carriers and Nanopores. JACS, 137(5):2035-2041, 2015. [5] K. Goepfrich, T. Zettl, A. E. C. Meijering, S. Hernandez-Ainsa, S. Kocabey, T. Liedl, and U. F. Keyser. DNA-Tile Structures Induce Ionic Currents through Lipid Membranes. Nano Letters, 15(5):3134-3138, 2015. 180-Symp Nanoscale Construction and Imaging with DNA Peng Yin. Harvard University, Boston, MA, USA. We have invented a general framework to program synthetic DNA/RNA strands to self-assemble into structures with user-specified geometry or dynamics. By interfacing these nanostructures with other functional molecules, we have introduced digital programmability into diverse application areas, e.g. fabrication of inorganic nanoparticles with arbitrary prescribed shapes, robust DNA/RNA probes with near optimal binding specificity, and RNAbased translational regulators with unprecedented dynamic range and orthogonality. Such digitally precise programmable control on the nanoscale promises to enable diverse applications in biosensing, imaging, diagnostics and therapeutics. In particular, I will discuss our recent work on DNA-based super-resolution imaging. Due to limitations in current optical microscopy, we face three challenges when attempting to image biology on the molecular scale: (1) blurred vision (i.e. difficulty to clearly visualize individual molecules for crowded targets), (2) (partial) color blindness (i.e. difficulty to simultaneously track distinct species with many colors due to spectral overlap), and (3) ambiguous quantification (i.e. difficulty to precisely count the number of targets in a resolution-limited area). Using programmable fluorescent DNA probes, we developed a highly multiplexed (10 demonstrated), precisely quantitative (>90% precision), and ultra-high resolution (sub-5 nm) optical imaging method that promises to potentially simultaneously address these challenges and thus broadly transform biomedical research. See my lab’s research at http://molecular-systems.net.
Platform: Voltage-gated Channels (Na and Ca) 181-Plat Rational Design and Synthesis of a Novel Membrane Binding NaV1.8 Selective Inhibitor with in vivo Activity in Pain Models Christina I. Schroeder1, Jennifer Deuis1, Sonia Troeria Henriques1, Zoltan Dekan1, Marco Inserra1, Mehdi Mobli2, Irina Vetter1. 1 The University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia, 2The University of Queensland, Centre for Advanced Imaging, Brisbane, Australia. Voltage-gated sodium channels (NaV), including subtype NaV1.8, are emerging as promising therapeutic targets to treat chronic pain. However, as these channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors are suitable as drug leads. We have recently isolated the mO-conotoxin, MfVIA, which inhibits NaV1.8 with high potency through interaction with the voltage sensor domain of the NaV1.8 channel. mO-conotoxin peptides are extremely hydrophobic and difficult to synthesize. In light of this we have pioneered a novel sophisticated approach to obtain synthetic MfVIA, allowing us to produce analogues
33a
of mO-conotoxin to conduct pharmacological characterization of mO-conotoxin peptides. Peptide-interaction with the voltage sensor domains is likely driven by the peptide initially inserting into the cell membrane surrounding this domain. With this in mind, analogs with improved membrane-binding properties compared to MfVIA were designed. One of these analogs showed a striking improvement in selectivity towards NaV1.8 over all the other NaV subtypes, including the skeletal muscle subtype NaV1.4. Therefore we believe to have found the first peptide drug lead with potential to selectively target NaV1.8. Synthesis, structure-activity in vitro results, membrane-binding interactions and in vivo results from animal pain studies will be discussed, highlighting that MfVIA binding sites on NaV1.8 and NaV1.4 are distinct and that that selectivity for NaV1.8 over NaV1.4 can be achieved resulting in peptides with proven efficacy in validated animal pain models. 182-Plat Sodium Channel/Ligand Complex Structures as a Guide for Rational Drug Design Altin Sula1, Paul DeCaen2, Claire Naylor1, Geancarlo Zanatta1, Claire Bagneris1, David E. Clapham2, David Pryde3, B.A. Wallace1. 1 Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, United Kingdom, 2Neurobiology, Harvard Medical School, Boston, MA, USA, 3Pfizer Neusentis, Cambridge, United Kingdom. The initiation of the action potential in excitable cells results from the opening of voltage-gated sodium channels. These channels represent key targets for development of pharmaceutical drugs as mutations in human sodium channels produce a wide range of neurological and cardiovascular diseases. Channel blockers such as lamotrigine and lidocaine have been shown to have efficacy, respectively, as anti-epileptic and local anaesthetic drugs, and are widely used clinically. We have shown that these and other drugs and ligands which block human sodium channels also bind to and block the NavMs channel (a prokaryotic orthologue of human sodium channels) with similar affinities and kinetics. We have determined the crystal structures of several open pore forms of NavMs in complex with human drugs and other channel blockers, and have further examined their interactions with these channels using circular dichroism spectroscopy and molecular modelling methods. The binding sites we have identified have been validated by both structure and function studies on designed mutants. This information should be valuable for the design of new specific and selective drugs. 183-Plat Structural Basis of Nav1.7 Inhibition by an Isoform-Selective Small Molecule Antagonist David H. Hackos1, Shivani Ahuja2, Susmith Mukund2, Lunbin Deng1, Kuldip Khakh3, Elaine Chang3, Clint Young3, Sophia Lin3, J.P. Johnson, Jr.3, Daniel F. Ortwine4, Brian S. Safina4, Daniel P. Sutherlin4, Charles J. Cohen3, Christopher M. Koth2, Jian Payandeh2. 1 Neuroscience, Genentech, South San Francisco, CA, USA, 2Structural Biology, Genentech, South San Francisco, CA, USA, 3Biology, Xenon Pharmaceuticals, Vancouver, BC, Canada, 4Discovery Chemistry, Genentech, South San Francisco, CA, USA. Voltage-gated sodium (Nav) channels are therapeutic targets for many cardiovascular and neurological disorders. Unfortunately, selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Recently, a series of highly selective small-molecule Nav channel antagonists (the aryl-sulfonamide series) has been identified that is able to achieve isoform selectivity by binding to the relatively poorly-conserved surface of voltage-sensor domain IV (VSD4). Understanding how these antagonists work and the details of their binding site has been a recent focus of the pharmaceutical industry which has hopes of identifying selective inhibitors of Nav1.7, an important target for novel pain drugs. Here, we describe the structural basis of Nav1.7 inhibition by this novel class of small molecule antagonist using X-ray crystallography, and thus present important experimental structures of a gating modifier in complex with a voltage-gated ion channel. To enable these unique mammalian Nav channel crystal structures, we exploited the established portability of VSDs and the presumed structural relatedness between human and bacterial Nav channels to develop a robust protein production and crystallization strategy. GX-936 and related aryl-sulfonamide inhibitors bind to the activated state of VSD4, where their anionic aryl sulfonamide warhead directly engages the R4 gating charge on the S4 helix. By opposing VSD4 deactivation, which stabilizes inactivated states of the channel, these
Platform: Voltage-gated Channels (Na and Ca) 181-Plat Rational Design and Synthesis of a Novel Membrane Binding NaV1.8 Selective Inhibitor with in vivo Activity in Pain Models Christina I. Schroeder1, Jennifer Deuis1, Sonia Troeria Henriques1, Zoltan Dekan1, Marco Inserra1, Mehdi Mobli2, Irina Vetter1. 1 The University of Queensland, Institute for Molecular Bioscience, Brisbane, Australia, 2The University of Queensland, Centre for Advanced Imaging, Brisbane, Australia. Voltage-gated sodium channels (NaV), including subtype NaV1.8, are emerging as promising therapeutic targets to treat chronic pain. However, as these channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors are suitable as drug leads. We have recently isolated the mO-conotoxin, MfVIA, which inhibits NaV1.8 with high potency through interaction with the voltage sensor domain of the NaV1.8 channel. mO-conotoxin peptides are extremely hydrophobic and difficult to synthesize. In light of this we have pioneered a novel sophisticated approach to obtain synthetic MfVIA, allowing us to produce analogues
33a
of mO-conotoxin to conduct pharmacological characterization of mO-conotoxin peptides. Peptide-interaction with the voltage sensor domains is likely driven by the peptide initially inserting into the cell membrane surrounding this domain. With this in mind, analogs with improved membrane-binding properties compared to MfVIA were designed. One of these analogs showed a striking improvement in selectivity towards NaV1.8 over all the other NaV subtypes, including the skeletal muscle subtype NaV1.4. Therefore we believe to have found the first peptide drug lead with potential to selectively target NaV1.8. Synthesis, structure-activity in vitro results, membrane-binding interactions and in vivo results from animal pain studies will be discussed, highlighting that MfVIA binding sites on NaV1.8 and NaV1.4 are distinct and that that selectivity for NaV1.8 over NaV1.4 can be achieved resulting in peptides with proven efficacy in validated animal pain models. 182-Plat Sodium Channel/Ligand Complex Structures as a Guide for Rational Drug Design Altin Sula1, Paul DeCaen2, Claire Naylor1, Geancarlo Zanatta1, Claire Bagneris1, David E. Clapham2, David Pryde3, B.A. Wallace1. 1 Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, United Kingdom, 2Neurobiology, Harvard Medical School, Boston, MA, USA, 3Pfizer Neusentis, Cambridge, United Kingdom. The initiation of the action potential in excitable cells results from the opening of voltage-gated sodium channels. These channels represent key targets for development of pharmaceutical drugs as mutations in human sodium channels produce a wide range of neurological and cardiovascular diseases. Channel blockers such as lamotrigine and lidocaine have been shown to have efficacy, respectively, as anti-epileptic and local anaesthetic drugs, and are widely used clinically. We have shown that these and other drugs and ligands which block human sodium channels also bind to and block the NavMs channel (a prokaryotic orthologue of human sodium channels) with similar affinities and kinetics. We have determined the crystal structures of several open pore forms of NavMs in complex with human drugs and other channel blockers, and have further examined their interactions with these channels using circular dichroism spectroscopy and molecular modelling methods. The binding sites we have identified have been validated by both structure and function studies on designed mutants. This information should be valuable for the design of new specific and selective drugs. 183-Plat Structural Basis of Nav1.7 Inhibition by an Isoform-Selective Small Molecule Antagonist David H. Hackos1, Shivani Ahuja2, Susmith Mukund2, Lunbin Deng1, Kuldip Khakh3, Elaine Chang3, Clint Young3, Sophia Lin3, J.P. Johnson, Jr.3, Daniel F. Ortwine4, Brian S. Safina4, Daniel P. Sutherlin4, Charles J. Cohen3, Christopher M. Koth2, Jian Payandeh2. 1 Neuroscience, Genentech, South San Francisco, CA, USA, 2Structural Biology, Genentech, South San Francisco, CA, USA, 3Biology, Xenon Pharmaceuticals, Vancouver, BC, Canada, 4Discovery Chemistry, Genentech, South San Francisco, CA, USA. Voltage-gated sodium (Nav) channels are therapeutic targets for many cardiovascular and neurological disorders. Unfortunately, selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Recently, a series of highly selective small-molecule Nav channel antagonists (the aryl-sulfonamide series) has been identified that is able to achieve isoform selectivity by binding to the relatively poorly-conserved surface of voltage-sensor domain IV (VSD4). Understanding how these antagonists work and the details of their binding site has been a recent focus of the pharmaceutical industry which has hopes of identifying selective inhibitors of Nav1.7, an important target for novel pain drugs. Here, we describe the structural basis of Nav1.7 inhibition by this novel class of small molecule antagonist using X-ray crystallography, and thus present important experimental structures of a gating modifier in complex with a voltage-gated ion channel. To enable these unique mammalian Nav channel crystal structures, we exploited the established portability of VSDs and the presumed structural relatedness between human and bacterial Nav channels to develop a robust protein production and crystallization strategy. GX-936 and related aryl-sulfonamide inhibitors bind to the activated state of VSD4, where their anionic aryl sulfonamide warhead directly engages the R4 gating charge on the S4 helix. By opposing VSD4 deactivation, which stabilizes inactivated states of the channel, these