ABSTRACT
A discriminating pharmacophore model for noncompetitive metabotropic glutamate receptor antagonists of subtype 1 (mGluR1) was developed that facilitated the discovery of moderately active mGluR1 antagonists. One scaffold was selected for the design of several focused libraries where different substitution patterns were introduced. This approach facilitated the discovery of potent mGluR1 antagonists, as well as positive and negative mGluR5 modulators, because both receptor subtypes share similar binding pockets. For mGluR1 antagonists, a homology model of the mGlu1 receptor was established, and a putative binding mode within the receptor's transmembrane domain was visualized.
Subject(s)
Excitatory Amino Acid Agents/chemical synthesis , Nitriles/chemical synthesis , Quinolines/chemical synthesis , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/physiology , Acetylene/chemical synthesis , Acetylene/chemistry , Acetylene/pharmacology , Allosteric Regulation , Animals , Azepines/chemical synthesis , Azepines/chemistry , Azepines/pharmacology , Binding Sites , Calcium/metabolism , Cells, Cultured , Cerebellum/cytology , Cricetinae , Cricetulus , Cyclopentanes/chemical synthesis , Cyclopentanes/chemistry , Cyclopentanes/pharmacology , Excitatory Amino Acid Agents/chemistry , Excitatory Amino Acid Agents/pharmacology , Inositol Phosphates/biosynthesis , Models, Molecular , Nitriles/chemistry , Nitriles/pharmacology , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Radioligand Assay , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/agonists , Structure-Activity RelationshipABSTRACT
Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their H(CC) (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.
Subject(s)
Botulinum Toxins/metabolism , Synaptotagmin II/metabolism , Synaptotagmin I/metabolism , Animals , Binding Sites , Botulinum Toxins/chemistry , Botulinum Toxins/toxicity , Botulinum Toxins, Type A , Circular Dichroism , Gangliosides/metabolism , Mice , Mice, Inbred C57BL , N-Acetylneuraminic Acid/metabolismABSTRACT
Mildronate (3-(2,2,2-trimethylhydrazinium)propionate; MET-88; meldonium, quaterine) is an antiischemic drug developed at the Latvian Institute of Organic Synthesis. Mildronate was designed to inhibit carnitine biosynthesis in order to prevent accumulation of cytotoxic intermediate products of fatty acid beta-oxidation in ischemic tissues and to block this highly oxygen-consuming process. Mildronate is efficient in the treatment of heart ischemia and its consequences. Extensive evaluation of pharmacological activities of mildronate revealed its beneficial effect on cerebral circulation disorders and central nervous system (CNS) functions. The drug is used in neurological clinics for the treatment of brain circulation disorders. It appears to improve patients' mood; they become more active, their motor dysfunction decreases, and asthenia, dizziness and nausea become less pronounced. Since the brain does not utilize fatty acids as fuel other mechanisms of action of mildronate in CNS should be considered. Several reports indicate the possible existence of an alternative, non-carnitine dependent mechanism of action of mildronate. Our recent findings suggest that CNS effects of mildronate could be mediated by stimulation of the nitric oxide production in the vascular endothelium by modification of the gamma-butyrobetaine and its esters pools. It is hypothesized that mildronate may increase the formation of the gamma-butyrobetaine esters. The latter are potent cholinomimetics and may activate eNOS via acetylcholine receptors or specific gamma-butyrobetaine ester receptors. This article summarizes known pharmacological effects of mildronate, its pharmacokinetics, toxicology, as well as the proposed mechanisms of action.
