KINETICS OF DRUG MACROMOLECULE COMPLEX FORMATION

Summary

Principal Investigator: PALMER WILLIAM TAYLOR
Affiliation: University of California
Country: USA
Abstract: DESCRIPTION (provided by applicant): Our continuing investigation of acetylcholinesterase (AChE), the acetylcholine binding protein (AChBP) reflect a long standing and continuous commitment to the study of proteins that affect the intensity and duration of acetylcholine action, a critical neurotransmitter affecting cognition in the CNS and peripheral autonomic and motor function. Our studies, supported by this grant over the past 37 years, have evolved from considerations of structure and function of AChE and the nicotinic acetylcholine receptor (nAChR) and, more recently, from AChE to a related a/b-hydrolase fold protein, neuroligin. In turn, they have spun off separate drug development endeavors directed novel nicotinic receptor ligands for depression, schizophrenia, pain alleviation and nicotine addiction and AChE inhibitor antidotes. Our fundamental studies with the extracellular domain of the three proteins are now based on structure at atomic resolution, that of static crystal structures, but also extend to an analysis of conformational dynamics and assignments of energetic contributions through structural modification and mutant cycle analysis. Conformation and dynamics are examined through fluorescence spectroscopy, decay of fluorescence anisotropy and H/D exchange. Our nicotinic receptor studies focus on an interfacial site between subunits that can be examined by physical methods through the soluble receptor surrogate, AChBP. Crystal structures of the complexes selected from a wide array of ligands with AChBP enable a detailed analysis of the structural determinants of specificity. We propose to expand to presumed non-competitive sites on this molecule, in particular the vestibule leading into the channel constriction and the non-a subunit interfaces that do not bind agonist. With AChE, we propose to continue our analysis of complexes formed by freeze-frame, click chemistry to compare complexes dictated by kinetics of association and by achieving thermodynamic equilibrium. We also will examine the inductive role of the oxyanion hole, the nucleophile rendering catalytic triad and the hydrogen bonding network between the proximal serine hydroxyls at the base of the gorge. Studies with the heterophilic adhesion protein, neuroligin (NL), capitalize on its homologous structure to AChE, both being members of the a/b-hydrolase-fold family, wherein their common globular domains and unique recognition features help direct the study of NL by low angle scattering and high resolution techniques. This approach is helping to uncover the molecular determinants associated with the interaction of NL with its synaptic partner proteins at its structurally unique binding site. Moreover, the common structural fold between AChE and NL allows comparisons in how mutations affect biosynthesis and trafficking of these two molecules. The shared familial structural features enable us to understand how gene mutations, some of which are associated with autism spectrum disorders, affect the biosynthesis and folding in this protein family. PUBLIC HEALTH RELEVANCE: Our proposed research is directed to understanding the structure and function of three proteins, acetylcholinesterase, the nicotinic acetylcholine receptor and neuroligin, that are related to each other in terms of structural homology and coordination of neurotransmitter function. Since these proteins are found at synapses in the nervous system and mediate signaling events and synaptic architecture, they are important potential drug targets and indicators of genetic predisposition to certain disorders in the nervous system. Our structural and functional studies, using X ray crystallography and solution-based spectroscopic and spectrometric techniques, offer new avenues into understanding the roles of these three proteins in physiological function and therapeutic outcomes.
Funding Period: ----------------1975 - ---------------2013-
more information: NIH RePORT

Top Publications

  1. pmc Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion
    Igor P Fabrichny
    Biochimie des Interactions Moléculaires et Cellulaires, CNRS FRE 2738, Institut Federatif de Recherche Jean Roche, Universite de la Mediterranee, Faculte de Medecine Secteur Nord, F 13916 Marseille Cedex 20, France
    Neuron 56:979-91. 2007
  2. pmc Automated docking with protein flexibility in the design of femtomolar "click chemistry" inhibitors of acetylcholinesterase
    Garrett M Morris
    Crysalin, Ltd, Cherwell Innovation Center, 77 Heyford Park, Upper Heyford, Oxfordshire, OX25 5HD, UK
    J Chem Inf Model 53:898-906. 2013
  3. pmc Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging
    Rory Cochran
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093 0650, USA
    J Biol Chem 286:29718-24. 2011
  4. pmc Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases
    Zoran Radic
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, USA
    J Biol Chem 287:11798-809. 2012
  5. pmc Synthesis of selective agonists for the α7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholine-binding protein templates
    John G Yamauchi
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA
    Mol Pharmacol 82:687-99. 2012
  6. pmc Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation
    Giulia Falivelli
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA
    Hum Mol Genet 21:4761-73. 2012
  7. pmc Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates
    Zoran Radic
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093 0650, USA
    Chem Biol Interact 203:67-71. 2013
  8. doi Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins
    Antonella De Jaco
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
    FEBS J 279:4293-305. 2012
  9. pmc Cholinesterase confabs and cousins: approaching forty years
    Palmer Taylor
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, Mail Code 0657, La Jolla, CA 92093, United States
    Chem Biol Interact 203:10-3. 2013
  10. pmc Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system
    Laura P Rodríguez
    Universidad de Santiago de Compostela, Departamento de Farmacologia, Facultad de Veterinaria, 27002 Lugo, Spain
    Anal Chem 85:2340-7. 2013

Scientific Experts

  • PALMER WILLIAM TAYLOR
  • Shelley Camp
  • Davide Comoletti
  • Antonella De Jaco
  • Meghan T Miller
  • Zoran Radic
  • K Barry Sharpless
  • Valery V Fokin
  • John G Yamauchi
  • Akos Nemecz
  • Zrinka Kovarik
  • Edzna Garcia
  • Gabriel Amitai
  • Rakesh K Sit
  • Limin Zhang
  • Kwok Yiu Ho
  • Todd T Talley
  • Neil Grimster
  • Yves Bourne
  • Garrett M Morris
  • Laura P Rodríguez
  • Suzana Berend
  • Neil P Grimster
  • Giulia Falivelli
  • Joseph R Fotsing
  • Anne M Valle
  • Rory Cochran
  • Jaroslaw Kalisiak
  • Alexandre Dobbertin
  • Pascale Marchot
  • Igor P Fabrichny
  • Romulo Araoz
  • Luke G Green
  • Jordi Molgo
  • TODD TALLEY
  • Flavio Grynszpan
  • Luis M Botana
  • Natalia Vilariño
  • M Carmen Louzao
  • Arthur J Olson
  • Timo Weide
  • Carol Green
  • Bozica Radic
  • Hyuck Kim
  • Jennifer Wilson
  • Bernhard Stump
  • Choel Kim
  • Flores Lietta Favaloro
  • Noga Dubi
  • Brett S Abrahams
  • Kimberly Gomez
  • Mikael Dufouil
  • Mark H Ellisman
  • David Kleinfeld
  • Brinda K Rana
  • Jennifer Wessel
  • Fangwen Rao
  • Maja Katalinić
  • Lee F Schroeder
  • Pei an Betty Shih
  • Vafa Mahboubi
  • Arnaud Muller
  • DANIEL T O'CONNOR
  • Valeria Gerardi
  • Tuba Küçükkilinç
  • Quoc Thang Nguyen
  • Jon Lindstrom
  • Li Zhang
  • Korami Dembele
  • Anna Hrabovska
  • Eric Krejci
  • Veronique Bernard
  • Gerlind Sulzenbacher
  • Philippe Leone

Detail Information

Publications26

  1. pmc Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion
    Igor P Fabrichny
    Biochimie des Interactions Moléculaires et Cellulaires, CNRS FRE 2738, Institut Federatif de Recherche Jean Roche, Universite de la Mediterranee, Faculte de Medecine Secteur Nord, F 13916 Marseille Cedex 20, France
    Neuron 56:979-91. 2007
    ..These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism...
  2. pmc Automated docking with protein flexibility in the design of femtomolar "click chemistry" inhibitors of acetylcholinesterase
    Garrett M Morris
    Crysalin, Ltd, Cherwell Innovation Center, 77 Heyford Park, Upper Heyford, Oxfordshire, OX25 5HD, UK
    J Chem Inf Model 53:898-906. 2013
    ..Here, we also used a version of AutoDock which permits additional conformational flexibility in selected amino acid side chains of the target protein...
  3. pmc Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging
    Rory Cochran
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093 0650, USA
    J Biol Chem 286:29718-24. 2011
    ....
  4. pmc Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases
    Zoran Radic
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, USA
    J Biol Chem 287:11798-809. 2012
    ..Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult...
  5. pmc Synthesis of selective agonists for the α7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholine-binding protein templates
    John G Yamauchi
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA
    Mol Pharmacol 82:687-99. 2012
    ....
  6. pmc Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation
    Giulia Falivelli
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA
    Hum Mol Genet 21:4761-73. 2012
    ..Our data support a complex genetic architecture in which multiple distinct risk factors interact with others to shape autism risk and presentation...
  7. pmc Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates
    Zoran Radic
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093 0650, USA
    Chem Biol Interact 203:67-71. 2013
    ....
  8. doi Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins
    Antonella De Jaco
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
    FEBS J 279:4293-305. 2012
    ..More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination...
  9. pmc Cholinesterase confabs and cousins: approaching forty years
    Palmer Taylor
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, Mail Code 0657, La Jolla, CA 92093, United States
    Chem Biol Interact 203:10-3. 2013
    ....
  10. pmc Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system
    Laura P Rodríguez
    Universidad de Santiago de Compostela, Departamento de Farmacologia, Facultad de Veterinaria, 27002 Lugo, Spain
    Anal Chem 85:2340-7. 2013
    ..This microsphere-based assay provides a rapid, sensitive, and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins...
  11. pmc New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterases
    Rakesh K Sit
    Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
    J Biol Chem 286:19422-30. 2011
    ....
  12. pmc Acetylcholinesterase expression in muscle is specifically controlled by a promoter-selective enhancesome in the first intron
    Shelley Camp
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093 0650, USA
    J Neurosci 28:2459-70. 2008
    ....
  13. pmc Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state
    Yves Bourne
    Architecture et Fonction des Macromolécules Biologiques AFMB, CNRS UMR 6098, Universités d Aix Marseille, Campus Luminy Case 932, F 13288 Marseille Cedex 09, France
    J Am Chem Soc 132:18292-300. 2010
    ..Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals...
  14. pmc Targeting of acetylcholinesterase in neurons in vivo: a dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit
    Alexandre Dobbertin
    Universite Paris Descartes, Paris, France
    J Neurosci 29:4519-30. 2009
    ..These unexpected findings open new avenues to modulating AChE activity and its distribution in CNS disorders...
  15. pmc Synthesis and reactivity of rhodium(II) N-triflyl azavinyl carbenes
    Neil Grimster
    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
    J Am Chem Soc 132:2510-1. 2010
    ..They rapidly and selectively react with olefins, providing cyclopropane carboxaldehydes and 2,3-dihydropyrroles in generally excellent yields and high enantio- and diastereoselectivity...
  16. pmc Neuroligin trafficking deficiencies arising from mutations in the alpha/beta-hydrolase fold protein family
    Antonella De Jaco
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, USA
    J Biol Chem 285:28674-82. 2010
    ..Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems...
  17. pmc Interaction kinetics of oximes with native, phosphylated and aged human acetylcholinesterase
    Zoran Radic
    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093 0650, United States
    Chem Biol Interact 187:163-6. 2010
    ..Dealkylation of phosphonylated enzyme, however opens space in the gorge allowing oximes to bind tighter...
  18. pmc Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors
    John G Yamauchi
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 6:e16519. 2011
    ..The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters...
  19. pmc Contributions of selective knockout studies to understanding cholinesterase disposition and function
    Shelley Camp
    Department Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences 0657, University of California San Diego, La Jolla, CA 92093 0657, USA
    Chem Biol Interact 187:72-7. 2010
    ..The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro...
  20. pmc From Split to Sibenik: the tortuous pathway in the cholinesterase field
    Palmer Taylor
    Department of Pharmacology 0636, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093 0650, USA
    Chem Biol Interact 187:3-9. 2010
    ..Those engaged in cholinesterase research should take great pride in our accomplishments punctuated by the series of ten meetings. The momentum established and initial studies with related proteins all hold great promise for the future...
  21. pmc Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template
    Neil P Grimster
    Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
    J Am Chem Soc 134:6732-40. 2012
    ..Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels...
  22. pmc Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses
    Akos Nemecz
    Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0650, USA
    J Biol Chem 286:42555-65. 2011
    ..We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site...
  23. ncbi Processing of cholinesterase-like α/β-hydrolase fold proteins: alterations associated with congenital disorders
    Antonella De Jaco
    Dipartimento di Biologia e Biotecnologie Charles Darwin, Universita di Roma La Sapienza, Italy
    Protein Pept Lett 19:173-9. 2012
    ....
  24. pmc The macromolecular architecture of extracellular domain of alphaNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition
    Davide Comoletti
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA
    Structure 18:1044-53. 2010
    ..We thus provide the first structural insights into the architecture of the extracellular region of neurexin-1alpha, show how the protein may fit in the synaptic cleft, and how partnering proteins could bind simultaneously...
  25. pmc Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traits
    Anne M Valle
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093 0657, USA
    J Pharmacol Exp Ther 338:125-33. 2011
    ..A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood...
  26. pmc The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function
    Meghan T Miller
    Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA
    Structure 19:767-78. 2011
    ..These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1...