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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.
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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.
Assuntos
Azetidinas/farmacologia , Agonismo Inverso de Drogas , Transtornos da Memória/tratamento farmacológico , Oxidiazóis/farmacologia , Receptor CB1 de Canabinoide/agonistas , Animais , Azetidinas/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Oxidiazóis/administração & dosagem , Piperidinas/metabolismo , Ligação Proteica , Pirazóis/metabolismo , EscopolaminaRESUMO
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.
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Receptores de Neuropeptídeos/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/metabolismo , Humanos , Radioisótopos do Iodo/química , Ligação Proteica , Quinolonas/síntese química , Quinolonas/química , Quinolonas/farmacologia , Ratos , Receptores de Neuropeptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Sítio Alostérico/genética , Aminoácidos/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Regulação Alostérica/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Linhagem Celular , Células Cultivadas , Humanos , Masculino , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica/genética , Ratos , Ratos Sprague-DawleyRESUMO
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.
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Acetofenonas/síntese química , Acetofenonas/farmacologia , Regulação Alostérica , Receptores de Glutamato Metabotrópico/agonistas , Animais , Comportamento/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Ácido Glutâmico/farmacologia , Hipercinese/tratamento farmacológico , Norepinefrina/metabolismo , Ratos , Esquizofrenia/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
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.
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Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
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).
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Encéfalo/fisiologia , Butanos/síntese química , Butanos/farmacologia , Propionatos/síntese química , Propionatos/farmacologia , Receptores de Glutamato Metabotrópico/fisiologia , Regulação Alostérica , Animais , Encéfalo/efeitos dos fármacos , Butanos/farmacocinética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Conformação Molecular , Éteres Fenílicos , Propionatos/farmacocinética , Ratos , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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.
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Indanos/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Regulação Alostérica , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Indanos/farmacocinética , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Ratos , Esquizofrenia/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
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).
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Compostos de Bifenilo/síntese química , Indanos/síntese química , Receptores de Glutamato Metabotrópico/agonistas , Regulação Alostérica , Animais , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacocinética , Química Encefálica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indanos/metabolismo , Indanos/farmacocinética , Ratos , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamento farmacológico , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
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.
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Acetofenonas/farmacologia , Modelos Animais de Doenças , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Acetofenonas/química , Acetofenonas/farmacocinética , Regulação Alostérica/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Reagentes de Ligações Cruzadas/química , Humanos , Ketamina/farmacologia , Estrutura Molecular , Ratos , Esquizofrenia/induzido quimicamente , Relação Estrutura-AtividadeRESUMO
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).
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Acetofenonas/síntese química , Receptores de Glutamato Metabotrópico/agonistas , Tetrazóis/síntese química , Acetofenonas/química , Acetofenonas/farmacologia , Regulação Alostérica , Estereoisomerismo , Relação Estrutura-Atividade , Tetrazóis/química , Tetrazóis/farmacologiaRESUMO
Pyrimidine methyl anilines as potent and selective mGlu2 potentiators are described. Findings from the structure-activity-relationship investigations are discussed.
Assuntos
Compostos de Anilina/síntese química , Pirimidinas/síntese química , Receptores de Glutamato Metabotrópico/agonistas , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Ensaio Radioligante , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
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.
Assuntos
Acetofenonas/metabolismo , Piridinas/química , Piridinas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Acetofenonas/química , Regulação Alostérica/fisiologia , Animais , Encéfalo/metabolismo , Linhagem Celular , Humanos , Ligação Proteica/fisiologia , Ratos , Receptores de Glutamato Metabotrópico/agonistas , Relação Estrutura-Atividade , TetrazóisRESUMO
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.