- Email: [email protected]
32. Development of High Throughput Calcium Channel Assays to Accelerate the Discovery of Novel Toxins Targeting Human Cav2.2 Channels
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
The voltage-gated sodium (NaV) channel NaV1.7 has recently been identified as a promising target for treatment of chronic pain. Gain-of-function mutations in the SCN9A gene encoding the pore-forming a-subunit of NaV1.7 cause painful neuropathies whereas, loss-of-function mutations result in a congenital indifference to all forms of pain1. Thus, blockers of Nav1.7 are likely to be useful analgesics for the treatment of persistent pain. However, it will be critical to ensure that such blockers do not inhibit other NaV subtypes with essential physiological functions, such as NaV1.5 that is restricted to the heart and is critical for the rising phase of the cardiac action potential. Many venomous animals have evolved toxins that modulate the activity of NaV channels. Spider venoms in particular are rich in NaV channel modulators, with one third of all known ion channel toxins from spider venoms acting NaV channels.2 Methods: Based on their primary structure and cysteine scaffold, 12 distinct families of spider toxins that modulate the activity of NaV channels (NaSpTxs) were identified3. Several of these families show activity at NaV1.7; one family in particular is interesting from a pharmacologically perspective as some members display subtype selectivity. In order to determine the molecular epitopes that govern the interaction of members of this NaSpTx family with NaV1.7 and other NaV subtypes, we have developed a recombinant expression system for several family members in order to produce isotopically labeled peptides for NMR structural studies and point mutants for functional analyses. Results: We have discovered that one member of this NaSpTx family is the most potent blocker of NaV1.7 discovered to date, and that variations in activity within this family are correlated with subtle changes in the structure and dynamics of the peptide pharmacophore. Discussion: Examining the potency and selectivity of naturally occurring sequence variants within this peptide family, as well as point mutants thereof, will enable us to precisely determine the molecular epitopes that mediate their interaction with NaV1.7 as well as key off-target NaV subtypes. Conclusion: Structure-function relationship studies of this NaSpTx family will allow us to rationally design blockers of NaV1.7 with improved potency, selectivity, and analgesic potential.
111
Background: Voltage-gated calcium channels (Cav) are multidomain membrane proteins that play essential roles in the control of neurotransmitter release and nociceptive transmission. Different Cav auxiliary subunits have been shown to influence the pharmacological and physiological properties of Cav, although the precise role of each subunit is not completely understood. Cav2.2, a validated analgesic target, is potently and selectively inhibited by small peptidic toxins (u-conotoxins) expressed in cone snail venoms. Some of these peptides have become useful pharmacological tools, while others have shown potential as therapeutic leads due to their specificity for Cav2.2. Ziconotide, a synthetic version of u-conotoxin MVIIA, inhibits Cav2.2 with high potency, and it is currently in the market for treatment of severe intractable pain. Unfortunately, the poor selectivity of ziconotide and other available drugs still represent a challenge for the effective treatment of chronic pain, which affects billions of people worldwide. Thus, a better understanding of the mechanisms involved in pain pathways and the identification of novel and more selective drug candidates is urgently needed. Aims: The aims of this study were to provide insights into the contribution of Cav specific subtypes, and auxiliary subunits, to the pharmacology of Cav antagonists, and thus provide a molecular basis for the involvement of Cav2.2 in pain pathways; and to develop high throughput assays to discover novel selective inhibitors for Cav2.2 from cone snail and spider venoms. Key Results and Discussion: We identified the Cav subtypes and auxiliary subunits endogenously expressed in the human neuroblastoma SH SY5Y cell line, and used Cav specific blockers to pharmacologically characterize Cav channels in these cells. These results allowed us to develop high throughput radioligand binding and fluorescent calcium imaging assays to accelerate the discovery and characterization of inhibitors of human Cav2.2. u-Conotoxins, such as CVID, MVIIA and GVIA displaced the highly selective radiolabeled peptide 125I-GVIA from SH SY5Y cell membranes with high affinity (pIC50 w 11). Surprising discrepancies in toxin inhibition were found between the cell membranes and the whole cells in the binding assays, as well as in the calcium imaging assays, suggesting an influence of auxiliary subunits on u-conotoxin affinity.
References Cox, J.J. et al.(2006) Nature 444, 894–898. www.arachnoserver.org. Klint, J.K. et al. (2012) Toxicon. Submitted. Keywords: spider-venom peptides, NaV1.7, therapeutics, analgesics, structure-function relationships, pharmacophore, drug development 10.1016/j.toxicon.2012.04.032
32. Development of High Throughput Calcium Channel Assays to Accelerate the Discovery of Novel Toxins Targeting Human Cav2.2 Channels Silmara R. Sousa, Lotten Ragnarsson, Irina Vetter, Volker Herzig, Glenn F. King, Richard J. Lewis Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, Australia E-mail address: [email protected] (S.R. Sousa).
Keywords: Cav2.2, calcium channels, SH-SY5Y cells, spider and cone snail venom toxins 10.1016/j.toxicon.2012.04.033
33. Mass Spectrometry as a Tool to Search Specific Ligands for G-Protein-Coupled Receptors Camila T. Cologna, Julien Echterbille, Edwin de Pauw, Loïc Quinton Laboratory of Mass Spectrometry, University of Liège, Liège, Belgium E-mail address: [email protected] (C.T. Cologna).
Background: G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins, their importance arises from their role as signal transmitters and regulators of cellular response. They control almost all physiological processes in humans and consequently they
The voltage-gated sodium (NaV) channel NaV1.7 has recently been identified as a promising target for treatment of chronic pain. Gain-of-function mutations in the SCN9A gene encoding the pore-forming a-subunit of NaV1.7 cause painful neuropathies whereas, loss-of-function mutations result in a congenital indifference to all forms of pain1. Thus, blockers of Nav1.7 are likely to be useful analgesics for the treatment of persistent pain. However, it will be critical to ensure that such blockers do not inhibit other NaV subtypes with essential physiological functions, such as NaV1.5 that is restricted to the heart and is critical for the rising phase of the cardiac action potential. Many venomous animals have evolved toxins that modulate the activity of NaV channels. Spider venoms in particular are rich in NaV channel modulators, with one third of all known ion channel toxins from spider venoms acting NaV channels.2 Methods: Based on their primary structure and cysteine scaffold, 12 distinct families of spider toxins that modulate the activity of NaV channels (NaSpTxs) were identified3. Several of these families show activity at NaV1.7; one family in particular is interesting from a pharmacologically perspective as some members display subtype selectivity. In order to determine the molecular epitopes that govern the interaction of members of this NaSpTx family with NaV1.7 and other NaV subtypes, we have developed a recombinant expression system for several family members in order to produce isotopically labeled peptides for NMR structural studies and point mutants for functional analyses. Results: We have discovered that one member of this NaSpTx family is the most potent blocker of NaV1.7 discovered to date, and that variations in activity within this family are correlated with subtle changes in the structure and dynamics of the peptide pharmacophore. Discussion: Examining the potency and selectivity of naturally occurring sequence variants within this peptide family, as well as point mutants thereof, will enable us to precisely determine the molecular epitopes that mediate their interaction with NaV1.7 as well as key off-target NaV subtypes. Conclusion: Structure-function relationship studies of this NaSpTx family will allow us to rationally design blockers of NaV1.7 with improved potency, selectivity, and analgesic potential.
111
Background: Voltage-gated calcium channels (Cav) are multidomain membrane proteins that play essential roles in the control of neurotransmitter release and nociceptive transmission. Different Cav auxiliary subunits have been shown to influence the pharmacological and physiological properties of Cav, although the precise role of each subunit is not completely understood. Cav2.2, a validated analgesic target, is potently and selectively inhibited by small peptidic toxins (u-conotoxins) expressed in cone snail venoms. Some of these peptides have become useful pharmacological tools, while others have shown potential as therapeutic leads due to their specificity for Cav2.2. Ziconotide, a synthetic version of u-conotoxin MVIIA, inhibits Cav2.2 with high potency, and it is currently in the market for treatment of severe intractable pain. Unfortunately, the poor selectivity of ziconotide and other available drugs still represent a challenge for the effective treatment of chronic pain, which affects billions of people worldwide. Thus, a better understanding of the mechanisms involved in pain pathways and the identification of novel and more selective drug candidates is urgently needed. Aims: The aims of this study were to provide insights into the contribution of Cav specific subtypes, and auxiliary subunits, to the pharmacology of Cav antagonists, and thus provide a molecular basis for the involvement of Cav2.2 in pain pathways; and to develop high throughput assays to discover novel selective inhibitors for Cav2.2 from cone snail and spider venoms. Key Results and Discussion: We identified the Cav subtypes and auxiliary subunits endogenously expressed in the human neuroblastoma SH SY5Y cell line, and used Cav specific blockers to pharmacologically characterize Cav channels in these cells. These results allowed us to develop high throughput radioligand binding and fluorescent calcium imaging assays to accelerate the discovery and characterization of inhibitors of human Cav2.2. u-Conotoxins, such as CVID, MVIIA and GVIA displaced the highly selective radiolabeled peptide 125I-GVIA from SH SY5Y cell membranes with high affinity (pIC50 w 11). Surprising discrepancies in toxin inhibition were found between the cell membranes and the whole cells in the binding assays, as well as in the calcium imaging assays, suggesting an influence of auxiliary subunits on u-conotoxin affinity.
References Cox, J.J. et al.(2006) Nature 444, 894–898. www.arachnoserver.org. Klint, J.K. et al. (2012) Toxicon. Submitted. Keywords: spider-venom peptides, NaV1.7, therapeutics, analgesics, structure-function relationships, pharmacophore, drug development 10.1016/j.toxicon.2012.04.032
32. Development of High Throughput Calcium Channel Assays to Accelerate the Discovery of Novel Toxins Targeting Human Cav2.2 Channels Silmara R. Sousa, Lotten Ragnarsson, Irina Vetter, Volker Herzig, Glenn F. King, Richard J. Lewis Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, Australia E-mail address: [email protected] (S.R. Sousa).
Keywords: Cav2.2, calcium channels, SH-SY5Y cells, spider and cone snail venom toxins 10.1016/j.toxicon.2012.04.033
33. Mass Spectrometry as a Tool to Search Specific Ligands for G-Protein-Coupled Receptors Camila T. Cologna, Julien Echterbille, Edwin de Pauw, Loïc Quinton Laboratory of Mass Spectrometry, University of Liège, Liège, Belgium E-mail address: [email protected] (C.T. Cologna).
Background: G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins, their importance arises from their role as signal transmitters and regulators of cellular response. They control almost all physiological processes in humans and consequently they