Genomes and Genes
KINETICS OF DRUG MACROMOLECULE COMPLEX FORMATION
Principal Investigator: PALMER WILLIAM TAYLOR
Affiliation: University of California
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
- Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesionIgor 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...
- Automated docking with protein flexibility in the design of femtomolar "click chemistry" inhibitors of acetylcholinesteraseGarrett 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...
- Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist agingRory 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....
- Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterasesZoran 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...
- Synthesis of selective agonists for the α7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholine-binding protein templatesJohn 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....
- Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activationGiulia 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...
- Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugatesZoran 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....
- Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteinsAntonella 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...
- Cholinesterase confabs and cousins: approaching forty yearsPalmer 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....
- Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry systemLaura 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...
- New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterasesRakesh 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....
- Acetylcholinesterase expression in muscle is specifically controlled by a promoter-selective enhancesome in the first intronShelley 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....
- Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline stateYves 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...
- 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 subunitAlexandre 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...
- Synthesis and reactivity of rhodium(II) N-triflyl azavinyl carbenesNeil 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...
- Neuroligin trafficking deficiencies arising from mutations in the alpha/beta-hydrolase fold protein familyAntonella 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...
- Interaction kinetics of oximes with native, phosphylated and aged human acetylcholinesteraseZoran 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...
- Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensorsJohn 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...
- Contributions of selective knockout studies to understanding cholinesterase disposition and functionShelley 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...
- From Split to Sibenik: the tortuous pathway in the cholinesterase fieldPalmer 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...
- Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein templateNeil 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...
- Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analysesAkos 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...
- Processing of cholinesterase-like α/β-hydrolase fold proteins: alterations associated with congenital disordersAntonella De Jaco
Dipartimento di Biologia e Biotecnologie Charles Darwin, Universita di Roma La Sapienza, Italy
Protein Pept Lett 19:173-9. 2012....
- The macromolecular architecture of extracellular domain of alphaNRXN1: domain organization, flexibility, and insights into trans-synaptic dispositionDavide 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...
- Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traitsAnne 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...
- The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and functionMeghan 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...