ABSTRACT
Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.
Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.
ABSTRACT
Cannabinoid receptors (CBRs) play an important role in a variety of physiological functions and have been considered drug targets for obesity and psychiatric disorders. In particular, the CB1R is highly expressed in brain regions crucial to learning and memory processes, and several lines of evidence indicate that pharmacological blockade of this receptor could have therapeutic applications in the treatment of cognitive disorders. In this study, we investigated whether MK-7128 (0.1, 0.3, and 1 mg/kg, orally), a novel and selective CB1R inverse agonist, could improve learning and memory deficits induced by scopolamine (1 mg/kg, subcutaneously) in mice. The investigators also assessed CB1R occupancy in the brain to ensure target engagement of MK-7128, and showed that MK-7128 significantly improved both Y-maze spontaneous alternation and object habituation performance in scopolamine-treated mice and inhibits the binding of radioiodinated AM251 in murine cortex and hippocampus. These data indicate that MK-7128 improves cognitive performance in a model of cholinergic hypofunction and suggest that efficacy is achieved at relatively low levels of CB1R occupancy in the brain. Our results extend earlier findings suggesting a role of CB1Rs in the modulation of memory processes and a potential therapeutic application for CB1R inverse agonists in cognitive disorders.
Subject(s)
Azetidines/pharmacology , Drug Inverse Agonism , Memory Disorders/drug therapy , Oxadiazoles/pharmacology , Receptor, Cannabinoid, CB1/agonists , Animals , Azetidines/administration & dosage , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cognition Disorders/drug therapy , Cognition Disorders/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Hippocampus/drug effects , Hippocampus/metabolism , Male , Maze Learning/drug effects , Memory Disorders/physiopathology , Mice , Mice, Inbred C57BL , Oxadiazoles/administration & dosage , Piperidines/metabolism , Protein Binding , Pyrazoles/metabolism , ScopolamineABSTRACT
Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited (125)I-NPS binding in the CNS when administered to rats.
Subject(s)
Receptors, Neuropeptide/antagonists & inhibitors , Amides/chemical synthesis , Amides/chemistry , Amides/pharmacology , Animals , Blood-Brain Barrier/metabolism , Central Nervous System/metabolism , Humans , Iodine Radioisotopes/chemistry , Protein Binding , Quinolones/chemical synthesis , Quinolones/chemistry , Quinolones/pharmacology , Rats , Receptors, Neuropeptide/metabolism , Structure-Activity RelationshipABSTRACT
Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.
Subject(s)
Allosteric Site/genetics , Amino Acids/metabolism , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/genetics , Allosteric Regulation/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Amino Acids/chemistry , Amino Acids/genetics , Animals , Cell Line , Cells, Cultured , Humans , Male , Molecular Sequence Data , Point Mutation , Protein Binding/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Herein we disclose the discovery of a new class of positive allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2), phenyl-tetrazolyl acetophenones, e.g. 1-(2-hydroxy-3-propyl-4-[4-[4-(2H-tetrazol-5-yl)phenoxy]butoxy]phenyl) ethanone (4). These potentiators were shown to have no effect in the absence of glutamate as well as no effect at mGlu3 or the other mGlu receptors. The compounds were also evaluated in rodent models with potential relevance for schizophrenia, and 4 was shown to have activity in the inhibition of ketamine-induced norepinephrine release and ketamine-induced hyperactivity. This represents the first example of the efficacy of mGlu2 receptor potentiators in these models.
Subject(s)
Acetophenones/chemical synthesis , Acetophenones/pharmacology , Allosteric Regulation , Receptors, Metabotropic Glutamate/agonists , Animals , Behavior/drug effects , Brain/metabolism , Dose-Response Relationship, Drug , Glutamic Acid/pharmacology , Hyperkinesis/drug therapy , Norepinephrine/metabolism , Rats , Schizophrenia/drug therapy , Structure-Activity RelationshipABSTRACT
INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.
Subject(s)
Enzyme Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Animals , Benzamides/pharmacology , Brain/drug effects , Brain/enzymology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Female , Male , Models, Animal , Rats , Rats, Sprague-DawleyABSTRACT
We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC50=1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk i.p., brain level of 5700 nM).
Subject(s)
Brain/physiology , Butanes/chemical synthesis , Butanes/pharmacology , Propionates/chemical synthesis , Propionates/pharmacology , Receptors, Metabotropic Glutamate/physiology , Allosteric Regulation , Animals , Brain/drug effects , Butanes/pharmacokinetics , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Kinetics , Molecular Conformation , Phenyl Ethers , Propionates/pharmacokinetics , Rats , Receptors, Metabotropic Glutamate/drug effects , Structure-Activity RelationshipABSTRACT
We have identified and synthesized a series of biphenyl-carboxylic acid indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency and the brain to plasma ratio of the initial lead led to the discovery of 5 and 23 (EC50=111 and 5 nM, respectively).
Subject(s)
Biphenyl Compounds/chemical synthesis , Indans/chemical synthesis , Receptors, Metabotropic Glutamate/agonists , Allosteric Regulation , Animals , Biphenyl Compounds/metabolism , Biphenyl Compounds/pharmacokinetics , Brain Chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Indans/metabolism , Indans/pharmacokinetics , Rats , Receptors, Metabotropic Glutamate/metabolism , Schizophrenia/drug therapy , Structure-Activity Relationship , Tissue DistributionABSTRACT
We have identified and synthesized a series of phenyl-tetrazolyl and 4-thiopyridyl indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation, as well as PK properties, led to the discovery of 28 (EC50=186 nM), which displayed activity in a rodent model for schizophrenia.
Subject(s)
Indans/pharmacology , Receptors, Metabotropic Glutamate/agonists , Allosteric Regulation , Animals , Brain/metabolism , Disease Models, Animal , Indans/pharmacokinetics , Models, Chemical , Molecular Structure , Protein Binding , Rats , Schizophrenia/drug therapy , Structure-Activity RelationshipABSTRACT
Metabotropic glutamate receptor 2 (mGluR2) has been implicated in a variety of CNS disorders, including schizophrenia. Disclosed herein is the development of a new series of allosteric potentiators of mGluR2. Structure-activity relationship studies in conjunction with pharmacokinetic data led to the discovery of indole 5, which is active in an animal model for schizophrenia.
Subject(s)
Acetophenones/pharmacology , Disease Models, Animal , Receptors, Metabotropic Glutamate/metabolism , Schizophrenia/drug therapy , Schizophrenia/metabolism , Acetophenones/chemistry , Acetophenones/pharmacokinetics , Allosteric Regulation/drug effects , Animals , Brain/drug effects , Brain/metabolism , Cross-Linking Reagents/chemistry , Humans , Ketamine/pharmacology , Molecular Structure , Rats , Schizophrenia/chemically induced , Structure-Activity RelationshipABSTRACT
We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation led to the discovery of 22 (EC(50)=93nM, 128% potentiation).
Subject(s)
Acetophenones/chemical synthesis , Receptors, Metabotropic Glutamate/agonists , Tetrazoles/chemical synthesis , Acetophenones/chemistry , Acetophenones/pharmacology , Allosteric Regulation , Stereoisomerism , Structure-Activity Relationship , Tetrazoles/chemistry , Tetrazoles/pharmacologyABSTRACT
In the present study, we describe the characterization of a positive allosteric modulator at metabotropic glutamate subtype 2 receptors (mGluR2). N-(4-(2-Methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379) is a selective positive allosteric modulator at human mGluR2 and is without activity at human mGluR3. Furthermore, LY487379 has no intrinsic agonist or antagonist activity at hmGluR2, as determined by functional guanosine 5'(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding, single-cell Ca2+ imaging, and electrophysiological studies. However, LY487379 markedly potentiated glutamate-stimulated [35S]GTPgammaS binding in a concentration-dependent manner at hmGluR2, shifting the glutamate dose-response curve leftward by 3-fold and increasing the maximum levels of [35S]GTPgammaS stimulation. This effect of LY487479 was also observed to a greater extent on the concentration-response curves to selective hmGluR2/3 agonists. In radioligand binding studies to rat cortical membranes, LY487379 increased the affinity of the radiolabeled agonist, [3H]DCG-IV, without affecting the binding affinity of the radiolabeled antagonist, [3H]LY341495. In rat hippocampal slices, coapplication of LY487379 potentiated synaptically evoked mGluR2 responses. Finally, to elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR2 and hmGluR3. Substitution of Ser688 and/or Gly689 in transmembrane IV along with Asn735 located in transmembrane segment V, with the homologous amino acids of hmGluR3, completely eliminated LY487379 allosteric modulation of hmGluR2. We propose that this allosteric binding site defines a pocket that is different from the orthosteric site located in the amino terminal domain.
Subject(s)
Glycine/analogs & derivatives , Pyridines/pharmacology , Receptors, Metabotropic Glutamate/metabolism , Sulfonamides/pharmacology , Allosteric Regulation , Amino Acid Sequence , Amino Acids/pharmacology , Animals , Binding Sites , CHO Cells , Calcium/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cerebral Cortex/cytology , Cricetinae , Cyclopropanes/pharmacology , Dentate Gyrus/drug effects , Dentate Gyrus/metabolism , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Glycine/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Molecular Sequence Data , Perforant Pathway/drug effects , Perforant Pathway/metabolism , Protein Structure, Tertiary , Rats , Sequence Homology, Amino Acid , Tritium , Xanthenes/pharmacologyABSTRACT
We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC(50)=340 nM), which showed improved brain penetration over the initial lead.
Subject(s)
Acetophenones/metabolism , Pyridines/chemistry , Pyridines/metabolism , Receptors, Metabotropic Glutamate/metabolism , Acetophenones/chemistry , Allosteric Regulation/physiology , Animals , Brain/metabolism , Cell Line , Humans , Protein Binding/physiology , Rats , Receptors, Metabotropic Glutamate/agonists , Structure-Activity Relationship , TetrazolesABSTRACT
Pyrimidine methyl anilines as potent and selective mGlu2 potentiators are described. Findings from the structure-activity-relationship investigations are discussed.