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Discovery of LY314582 and its (+) enantiomer, LY354740: Highly potent and selective group 2 metabotropic glutamate receptor agonists
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DISCOVERY OF LY314582 AND ITS (+) ENANTIOMER, LY354740: HIGHLY POTENT AND SELECTIVE GROUP 2 METABOTROPIC GLUTAMATE RECEPTOR AGONISTS.
PHARMACOLOGICAL STUDIES ON NEW AGONISTS AND ANTAGONISTS OF mGluRs. F. Moroni, G. Lombardi, D.E. Pellegrini-Giampietro, P. Leonardi, S. Attucci, G. Mannaioni, F. Peruginelli, S. Albani, *C. Thomsen and OR. Pellicciari Department of Pharmacology, Univ. of Florence, “Institute of Chemistry, Univ. of Perugia, Italy and *Nova Nordisk, Denmark.
J. A. MOM, M. I. Valli, R. A. Wright, B. G. Johnson, C. R. Salhoff, N. G. Mayne, J. P. Burnett, D. R. Helton, M. J. Kallman, J. P. Tizzano and D. Ill. Schocpp Lilly Research Laboratories, Eli Lilly and Company, Indianapolis IN 46285, USA Metabotropic glutamare receptors (mG1uR.s) are a heterogenous family of G-protein coupled receptors that are linked to multiple second messenger qxtems. We report here the discovery of (k)2-aminobicyclo[3.1.0:@xane-2.6dicarbrboxylate, LY3 14582. This conformationally constrained amino diacid is related to the non selective mGluR agonist L-CCGl. Both LY314582 and L-CCGl are highly potent agonists at group 2 mGluRs in situ (rat hippocampus: EC50 =: 120 j, 7 nM for LY314582 and 1.99 + 0.64 PM for L-CCGl) and in cells espressing recombinant human group 2 mGh&s (mCiluR2: ECso = 32 + 5 nM for LY314582 and 80 + 38 nM for CCG-1). However, while L-CCGl produces agonist responses resulting from activation of group 1 mGluRs (rat hippocampus: EC50 ==42.2 + 14.1 pM; human mGluRla: EC50 = 120 + 11.5 PM), LY314582 (up to 1,000 PM) does not. Furthermore, LY311582 (up to 100 PM) does not displace radioligand binding at NMDA, AMPA or kainate receptors. Resolution of LY3 14582 afforded the (+)-enantiomer, LY354740, which possesses all of the group 2 mGluR agonist-related activity. Systemic administration of either LY314582 or LY354740 in
rodents has led to new insights into the therapeutic utilities for compounds of this vpc.
GROUP I mGlulis COUPLE TO PHOSPHOINOSITIDE MOBILISATION BUT NOT cCMP GENERATION IN THE CEREBELLUM
REVERSAL OF L-CCC-I-MEDIATED LONG TERM DEPRESSION IN THE DENTATE GYRUS BY SUBSEQUENT APPLICATIOS OF MCPG. Vincent Mutel. Jean-Maxime Ducarre &John A. Kemp Pharma Division, Preclinical Research, F. Hoffmann-La AG.. CH-4070 Basel, Switzerland.
New mGluR agents are important tools in understanding the role these receptors play in brain function and the possible existence of new types of mGluRs. We studied commercially available and newly synthesized agonists and antagonists of mGluRs in several biological models such as: I) stable transfected cells (BHK) expressing mGluRla, mGluR2 and mGluR4; 2) brain slices incubated in v&o to evaluate phosphoinositide metabolism, forskolin-induced CAMP accumulation, phospholipase D activity, or transmitter release; 3) cortical wedges exposed to NMDA, AMPA or KA agonists. Concerning characterization of I-aminoindan-1,5dicarboxylic acid (UPF 523) and of several stereoisomers of 2-(2’carboxy-3’-phenylcyclopropyI)glycine (PCCG), we noticed that UPF523, a mGluR1 antagonist, applied for two days to BHK cells transfected with mGluRla, increased PI formation following the application of IS,3R-ACPD, possibly by modifying the expression of these receptors. Nanomolar concentrations of one of the PCCGs (PCCG 16) antagonized IS,3R-ACPD-induced stimulation of phospholipase D, suggesting the existence of new mGluRs. This concept was strengthened by the observation that IS,3R-ACPD potentiated NMDA-induced depolarisation of cortical wedges. The pharmacological profile of the potentiation was different from that of the three mGluR groups so far described in expression systems. Supporred b), E.U. (BMHI-CT93-1033) and the Univ. o/Florence.
Karen E. Neil, FClix Hemandez, David A. Kendall, & Stephen P.H. Molecular & Cellular Pharmacology Group, Alexander, Department of Physiology & Pharmacology, University of Nottingham Medical School, Nottingham, NG7 2UH. UK.
Roche
Following reports that agonists of group II mGluRs inhibit the medial perfornnt path (MPP) inpul to the dentate gyrus and th31 IS,3R-ACPD can induce long term depression in this pathway, we investigated further the pharmacology of these effecls. In adult rzu hippocampal slices, IS,3R-ACPD. L-CCG-I and DCG-IV inhibited the MPP-evoked field EPSP with EC=,u’sof
In slices from the guinea-pig cerebellum, NMDA evoked a concentration-dependent stimulalion of cGMP accumulation with a PECKSvalue of 3.87. This response was prevented by dizolcipine, L-NAME and the removal of Ca” from the assay medium, indicating that it is mediated by NMDA receptor-evoked Ca*’ influx activating nitric oxide synthase activity. Group I mGluRs also couple 10 elevation of intracellular Ca” ion concentration through the stimularion of phosphoinositide mobilisation. In the guinea-pig cerebellum, the agonists IS,3RACPD and (RS)-DHPG evoked a concentration-dependent stimulation of phosphoinositide mobilisation with pEC,, values of 4.45 and 4.47, respectively. The phosphoinositide response lo I S,3R-ACPD was antagonised in the presence of (+)-MCPG with a calculated pK, value of 3.55. However, (RS)-DHPG failed IO alter significantly the generation of cGMP in the absence or presence of I mM NMDA. at concentrations ranain!z from IO NM lo I mM. These results’ indicate the presence- of both. ionotropic and metabotropic glutamate receptors coupled to intracellular Ca” elevation mechanisms in the guinea-pig cerebellum. However, it is apparent that the ionotropic, but not the metabotropic receptor is able to mediate the generation of cGMP.
40. 60 and 0.2 FM, respectively. Following washout of DCG-IV the EPSP recovered to control levels over the nexl 30 minutes. After IS,3R-ACPD. the response recovered over approximately IO minutes but followirg high concenlrations (100 & 300 pM) to a plareau level below :hat of the original control. After L-CCG-I. the response stayed at the depressed level 31 each concentration tested and showed ~10 sign of recovery over the following 60 minutes. Applicaticm of MCPG (ImM) following one hour of washout of L-CCG-I (3OpM) returned the response to the original comrol level and following removal of MCPG the EPSP rapidl) returned IO the depressed level. The fact that a competitive antagonist was able :o reverse the long term depressant effects of L-CCG-I suggests the continued presence of the agonist in the receptor vicinity following its removal from the pcrfusate. We speculate thar L-CCG-I, and perhaps also IS,3R-ACPD. may bc taken up during thei: application and subsequently leak hack into the extracellular environment upon washout.
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82
DISCOVERY OF LY314582 AND ITS (+) ENANTIOMER, LY354740: HIGHLY POTENT AND SELECTIVE GROUP 2 METABOTROPIC GLUTAMATE RECEPTOR AGONISTS.
PHARMACOLOGICAL STUDIES ON NEW AGONISTS AND ANTAGONISTS OF mGluRs. F. Moroni, G. Lombardi, D.E. Pellegrini-Giampietro, P. Leonardi, S. Attucci, G. Mannaioni, F. Peruginelli, S. Albani, *C. Thomsen and OR. Pellicciari Department of Pharmacology, Univ. of Florence, “Institute of Chemistry, Univ. of Perugia, Italy and *Nova Nordisk, Denmark.
J. A. MOM, M. I. Valli, R. A. Wright, B. G. Johnson, C. R. Salhoff, N. G. Mayne, J. P. Burnett, D. R. Helton, M. J. Kallman, J. P. Tizzano and D. Ill. Schocpp Lilly Research Laboratories, Eli Lilly and Company, Indianapolis IN 46285, USA Metabotropic glutamare receptors (mG1uR.s) are a heterogenous family of G-protein coupled receptors that are linked to multiple second messenger qxtems. We report here the discovery of (k)2-aminobicyclo[3.1.0:@xane-2.6dicarbrboxylate, LY3 14582. This conformationally constrained amino diacid is related to the non selective mGluR agonist L-CCGl. Both LY314582 and L-CCGl are highly potent agonists at group 2 mGluRs in situ (rat hippocampus: EC50 =: 120 j, 7 nM for LY314582 and 1.99 + 0.64 PM for L-CCGl) and in cells espressing recombinant human group 2 mGh&s (mCiluR2: ECso = 32 + 5 nM for LY314582 and 80 + 38 nM for CCG-1). However, while L-CCGl produces agonist responses resulting from activation of group 1 mGluRs (rat hippocampus: EC50 ==42.2 + 14.1 pM; human mGluRla: EC50 = 120 + 11.5 PM), LY314582 (up to 1,000 PM) does not. Furthermore, LY311582 (up to 100 PM) does not displace radioligand binding at NMDA, AMPA or kainate receptors. Resolution of LY3 14582 afforded the (+)-enantiomer, LY354740, which possesses all of the group 2 mGluR agonist-related activity. Systemic administration of either LY314582 or LY354740 in
rodents has led to new insights into the therapeutic utilities for compounds of this vpc.
GROUP I mGlulis COUPLE TO PHOSPHOINOSITIDE MOBILISATION BUT NOT cCMP GENERATION IN THE CEREBELLUM
REVERSAL OF L-CCC-I-MEDIATED LONG TERM DEPRESSION IN THE DENTATE GYRUS BY SUBSEQUENT APPLICATIOS OF MCPG. Vincent Mutel. Jean-Maxime Ducarre &John A. Kemp Pharma Division, Preclinical Research, F. Hoffmann-La AG.. CH-4070 Basel, Switzerland.
New mGluR agents are important tools in understanding the role these receptors play in brain function and the possible existence of new types of mGluRs. We studied commercially available and newly synthesized agonists and antagonists of mGluRs in several biological models such as: I) stable transfected cells (BHK) expressing mGluRla, mGluR2 and mGluR4; 2) brain slices incubated in v&o to evaluate phosphoinositide metabolism, forskolin-induced CAMP accumulation, phospholipase D activity, or transmitter release; 3) cortical wedges exposed to NMDA, AMPA or KA agonists. Concerning characterization of I-aminoindan-1,5dicarboxylic acid (UPF 523) and of several stereoisomers of 2-(2’carboxy-3’-phenylcyclopropyI)glycine (PCCG), we noticed that UPF523, a mGluR1 antagonist, applied for two days to BHK cells transfected with mGluRla, increased PI formation following the application of IS,3R-ACPD, possibly by modifying the expression of these receptors. Nanomolar concentrations of one of the PCCGs (PCCG 16) antagonized IS,3R-ACPD-induced stimulation of phospholipase D, suggesting the existence of new mGluRs. This concept was strengthened by the observation that IS,3R-ACPD potentiated NMDA-induced depolarisation of cortical wedges. The pharmacological profile of the potentiation was different from that of the three mGluR groups so far described in expression systems. Supporred b), E.U. (BMHI-CT93-1033) and the Univ. o/Florence.
Karen E. Neil, FClix Hemandez, David A. Kendall, & Stephen P.H. Molecular & Cellular Pharmacology Group, Alexander, Department of Physiology & Pharmacology, University of Nottingham Medical School, Nottingham, NG7 2UH. UK.
Roche
Following reports that agonists of group II mGluRs inhibit the medial perfornnt path (MPP) inpul to the dentate gyrus and th31 IS,3R-ACPD can induce long term depression in this pathway, we investigated further the pharmacology of these effecls. In adult rzu hippocampal slices, IS,3R-ACPD. L-CCG-I and DCG-IV inhibited the MPP-evoked field EPSP with EC=,u’sof
In slices from the guinea-pig cerebellum, NMDA evoked a concentration-dependent stimulalion of cGMP accumulation with a PECKSvalue of 3.87. This response was prevented by dizolcipine, L-NAME and the removal of Ca” from the assay medium, indicating that it is mediated by NMDA receptor-evoked Ca*’ influx activating nitric oxide synthase activity. Group I mGluRs also couple 10 elevation of intracellular Ca” ion concentration through the stimularion of phosphoinositide mobilisation. In the guinea-pig cerebellum, the agonists IS,3RACPD and (RS)-DHPG evoked a concentration-dependent stimulation of phosphoinositide mobilisation with pEC,, values of 4.45 and 4.47, respectively. The phosphoinositide response lo I S,3R-ACPD was antagonised in the presence of (+)-MCPG with a calculated pK, value of 3.55. However, (RS)-DHPG failed IO alter significantly the generation of cGMP in the absence or presence of I mM NMDA. at concentrations ranain!z from IO NM lo I mM. These results’ indicate the presence- of both. ionotropic and metabotropic glutamate receptors coupled to intracellular Ca” elevation mechanisms in the guinea-pig cerebellum. However, it is apparent that the ionotropic, but not the metabotropic receptor is able to mediate the generation of cGMP.
40. 60 and 0.2 FM, respectively. Following washout of DCG-IV the EPSP recovered to control levels over the nexl 30 minutes. After IS,3R-ACPD. the response recovered over approximately IO minutes but followirg high concenlrations (100 & 300 pM) to a plareau level below :hat of the original control. After L-CCG-I. the response stayed at the depressed level 31 each concentration tested and showed ~10 sign of recovery over the following 60 minutes. Applicaticm of MCPG (ImM) following one hour of washout of L-CCG-I (3OpM) returned the response to the original comrol level and following removal of MCPG the EPSP rapidl) returned IO the depressed level. The fact that a competitive antagonist was able :o reverse the long term depressant effects of L-CCG-I suggests the continued presence of the agonist in the receptor vicinity following its removal from the pcrfusate. We speculate thar L-CCG-I, and perhaps also IS,3R-ACPD. may bc taken up during thei: application and subsequently leak hack into the extracellular environment upon washout.
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