ABSTRACT
Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.
Subject(s)
Alternative Splicing/physiology , Ligands , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Amino Acid Motifs , Animals , Binding Sites , Calcium/metabolism , Cell Adhesion Molecules, Neuronal , Crystallography, X-Ray , Membrane Proteins/metabolism , Models, Biological , Models, Molecular , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Rats , Substrate SpecificityABSTRACT
Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhibitors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent pharmacokinetic properties.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Imides/chemistry , Serine/chemistry , Threonine/chemistry , ortho-Aminobenzoates/chemistry , Animals , Aryl Hydrocarbon Hydroxylases/chemistry , Crystallography, X-Ray , Cytochrome P-450 CYP2C9 , Drug Design , Glycine/chemistry , Glycogen Phosphorylase/metabolism , Humans , Inhibitory Concentration 50 , Liver/enzymology , Models, Chemical , RatsABSTRACT
Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Carboxylic Acids/chemistry , Chemistry, Pharmaceutical/methods , Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Imides/pharmacology , ortho-Aminobenzoates/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/chemistry , Crystallography, X-Ray , Cytochrome P-450 CYP2C9 , Dogs , Drug Design , Glycine/chemistry , Glycogen Phosphorylase/metabolism , Humans , Imides/chemistry , Inhibitory Concentration 50 , Liver/enzymology , Molecular Conformation , Rats , ortho-Aminobenzoates/chemistryABSTRACT
Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Hypoglycemic Agents/chemical synthesis , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/pharmacology , Animals , Blood Glucose/analysis , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Disease Models, Animal , Hypoglycemic Agents/blood , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Mice , Molecular Conformation , Molecular Structure , Structure-Activity Relationship , Urea/pharmacology , ortho-Aminobenzoates/blood , ortho-Aminobenzoates/chemistryABSTRACT
Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1alpha_LNS#2 (the second LNS/LG domain of bovine neurexin 1alpha) reveals large structural differences compared with n1alpha_LNS#6 (or n1beta_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1alpha_LNS#2 has low affinity (Kd approximately 400 microm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.