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Discovery of a Novel Chemical Class of mGlu(5) Allosteric Ligands with Distinct Modes of Pharmacology.

Hammond AS, Rodriguez AL, Townsend SD, Niswender CM, Gregory KJ, Lindsley CW, Conn PJ - ACS Chem Neurosci (2010)

Bottom Line: We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu(5)) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu(5), distinct from the classical mGlu(5) negative allosteric modulator (NAM) MPEP allosteric site.An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu(5) PAM activity to afford VU0357121 (EC(50) = 33 nM, 92% Glu(max)), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396).Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu(5), distinct from CPPHA, yet share a functional interaction with the MPEP site.

View Article: PubMed Central - PubMed

ABSTRACT
We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu(5)) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu(5), distinct from the classical mGlu(5) negative allosteric modulator (NAM) MPEP allosteric site. However, a shallow structure-activity relationship (SAR), poor physiochemical properties, and weak PAM activity at rat mGlu(5) limited the utility of CPPHA to explore allosteric activation of mGlu(5) at a non-MPEP site. Thus, we performed a functional high-throughput screen (HTS) and identified a novel mGlu(5) PAM benzamide scaffold, exemplified by VU0001850 (EC(50) = 1.3 μM, 106% Glu(max)) and VU0040237 (EC(50) = 350 nM, 84% Glu Max). An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu(5) PAM activity to afford VU0357121 (EC(50) = 33 nM, 92% Glu(max)), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396). Like CPPHA, PAMs within this class do not appear to bind at the MPEP allosteric site based on radioligand binding studies. Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu(5), distinct from CPPHA, yet share a functional interaction with the MPEP site.

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Unlike MPEP, test compounds do not displace radioligand binding. These novel PAMs have no effect on radioligand binding at concentrations necessary for their function. Membranes prepared from HEK cells expressing mGlu5 were incubated with the radiolabeled MPEP analogue [3H] methoxyPEPy (2 nM final concentration in 50 mM Tris and 0.9% NaCl, pH 7.4) for 60 min at room temperature in the presence of varying concentrations of test compound. Equilibrium binding was terminated by rapid filtration. Data are plotted as a percentage of total [3H]methoxyPEPy binding. Data were obtained from three separate experiments, each performed in triplicate, and are expressed as the mean ± SEM.
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fig9: Unlike MPEP, test compounds do not displace radioligand binding. These novel PAMs have no effect on radioligand binding at concentrations necessary for their function. Membranes prepared from HEK cells expressing mGlu5 were incubated with the radiolabeled MPEP analogue [3H] methoxyPEPy (2 nM final concentration in 50 mM Tris and 0.9% NaCl, pH 7.4) for 60 min at room temperature in the presence of varying concentrations of test compound. Equilibrium binding was terminated by rapid filtration. Data are plotted as a percentage of total [3H]methoxyPEPy binding. Data were obtained from three separate experiments, each performed in triplicate, and are expressed as the mean ± SEM.

Mentions: Because of the structural similarity between these novel mGlu5 PAMs and CPPHA and the shallow SAR, we hypothesized that they might interact with a site on the receptor that is distinct from the MPEP binding site. In order to test this hypothesis, we assessed the ability of these test compounds to compete for binding of the radiolabeled MPEP analogue [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine) ([3H]methoxy-PEPy) (22) to evaluate their interaction with the allosteric MPEP site on mGlu5. The radioligand was added to membranes prepared from HEK293 cells stably expressing mGlu5 with or without the test compound, and reactions were allowed to incubate for 1 h. It was found that, unlike MPEP (Ki = 35 ± 10.4 nM), test compounds did not inhibit steady state [3H]methoxyPEPy binding at concentrations of up to 100 μM, 3 orders of magnitude higher than those required for their PAM activity, suggesting that the ability of these agents to enhance glutamate sensitivity of mGlu5 is likely due to the interaction at a site on the receptor distinct from the MPEP binding site (Figure 9). Slight inhibition of radioligand binding at higher concentrations may indicate some interaction between the MPEP site and the putative site of action of these PAMs.


Discovery of a Novel Chemical Class of mGlu(5) Allosteric Ligands with Distinct Modes of Pharmacology.

Hammond AS, Rodriguez AL, Townsend SD, Niswender CM, Gregory KJ, Lindsley CW, Conn PJ - ACS Chem Neurosci (2010)

Unlike MPEP, test compounds do not displace radioligand binding. These novel PAMs have no effect on radioligand binding at concentrations necessary for their function. Membranes prepared from HEK cells expressing mGlu5 were incubated with the radiolabeled MPEP analogue [3H] methoxyPEPy (2 nM final concentration in 50 mM Tris and 0.9% NaCl, pH 7.4) for 60 min at room temperature in the presence of varying concentrations of test compound. Equilibrium binding was terminated by rapid filtration. Data are plotted as a percentage of total [3H]methoxyPEPy binding. Data were obtained from three separate experiments, each performed in triplicate, and are expressed as the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2957851&req=5

fig9: Unlike MPEP, test compounds do not displace radioligand binding. These novel PAMs have no effect on radioligand binding at concentrations necessary for their function. Membranes prepared from HEK cells expressing mGlu5 were incubated with the radiolabeled MPEP analogue [3H] methoxyPEPy (2 nM final concentration in 50 mM Tris and 0.9% NaCl, pH 7.4) for 60 min at room temperature in the presence of varying concentrations of test compound. Equilibrium binding was terminated by rapid filtration. Data are plotted as a percentage of total [3H]methoxyPEPy binding. Data were obtained from three separate experiments, each performed in triplicate, and are expressed as the mean ± SEM.
Mentions: Because of the structural similarity between these novel mGlu5 PAMs and CPPHA and the shallow SAR, we hypothesized that they might interact with a site on the receptor that is distinct from the MPEP binding site. In order to test this hypothesis, we assessed the ability of these test compounds to compete for binding of the radiolabeled MPEP analogue [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine) ([3H]methoxy-PEPy) (22) to evaluate their interaction with the allosteric MPEP site on mGlu5. The radioligand was added to membranes prepared from HEK293 cells stably expressing mGlu5 with or without the test compound, and reactions were allowed to incubate for 1 h. It was found that, unlike MPEP (Ki = 35 ± 10.4 nM), test compounds did not inhibit steady state [3H]methoxyPEPy binding at concentrations of up to 100 μM, 3 orders of magnitude higher than those required for their PAM activity, suggesting that the ability of these agents to enhance glutamate sensitivity of mGlu5 is likely due to the interaction at a site on the receptor distinct from the MPEP binding site (Figure 9). Slight inhibition of radioligand binding at higher concentrations may indicate some interaction between the MPEP site and the putative site of action of these PAMs.

Bottom Line: We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu(5)) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu(5), distinct from the classical mGlu(5) negative allosteric modulator (NAM) MPEP allosteric site.An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu(5) PAM activity to afford VU0357121 (EC(50) = 33 nM, 92% Glu(max)), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396).Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu(5), distinct from CPPHA, yet share a functional interaction with the MPEP site.

View Article: PubMed Central - PubMed

ABSTRACT
We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu(5)) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu(5), distinct from the classical mGlu(5) negative allosteric modulator (NAM) MPEP allosteric site. However, a shallow structure-activity relationship (SAR), poor physiochemical properties, and weak PAM activity at rat mGlu(5) limited the utility of CPPHA to explore allosteric activation of mGlu(5) at a non-MPEP site. Thus, we performed a functional high-throughput screen (HTS) and identified a novel mGlu(5) PAM benzamide scaffold, exemplified by VU0001850 (EC(50) = 1.3 μM, 106% Glu(max)) and VU0040237 (EC(50) = 350 nM, 84% Glu Max). An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu(5) PAM activity to afford VU0357121 (EC(50) = 33 nM, 92% Glu(max)), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396). Like CPPHA, PAMs within this class do not appear to bind at the MPEP allosteric site based on radioligand binding studies. Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu(5), distinct from CPPHA, yet share a functional interaction with the MPEP site.

No MeSH data available.


Related in: MedlinePlus