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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.

No MeSH data available.


Related in: MedlinePlus

Benzamide compounds are able to potentiate the calcium mobilization response of mGlu5 to glutamate. Raw traces show the effect of a fixed concentration (10 μM) of test compound or vehicle when added to calcium-sensitive dye-loaded cells and allowed to incubate for 5 min. A suboptimal (EC20) concentration of glutamate was added and the calcium response measured by the FDSS plate reader. Responses are expressed as a fluorescence ratio.
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fig2: Benzamide compounds are able to potentiate the calcium mobilization response of mGlu5 to glutamate. Raw traces show the effect of a fixed concentration (10 μM) of test compound or vehicle when added to calcium-sensitive dye-loaded cells and allowed to incubate for 5 min. A suboptimal (EC20) concentration of glutamate was added and the calcium response measured by the FDSS plate reader. Responses are expressed as a fluorescence ratio.

Mentions: Our laboratory performed a functional high-throughput screen (HTS) (18−20) that led to the identification of about 20 structural classes of mGlu5 PAMs. The present study focuses on a novel benzamide chemical scaffold that was identified in that screen. Sixty-five compounds were identified that belong to this structural class, and 10 were found to have high potencies with EC50 values ≤1 μM from HTS stock solutions. This class is of particular interest because these compounds share a benzamide backbone that is similar to that of CPPHA yet are distinct. These compounds display little intrinsic mGlu5 agonist activity in the calcium mobilization assay and induce a robust enhancement of the response to a suboptimal concentration (EC20) of glutamate (Figure 2). To test their PAM activity, HEK293 cells expressing rat mGlu5 were preloaded with calcium-sensitive fluorescent dye and received vehicle or compound (10 μM), followed by an EC20 concentration of glutamate 5 min later. Receptor-induced calcium mobilization responses were monitored using Functional Drug Screening System (FDSS, Hamamatsu, Japan).


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)

Benzamide compounds are able to potentiate the calcium mobilization response of mGlu5 to glutamate. Raw traces show the effect of a fixed concentration (10 μM) of test compound or vehicle when added to calcium-sensitive dye-loaded cells and allowed to incubate for 5 min. A suboptimal (EC20) concentration of glutamate was added and the calcium response measured by the FDSS plate reader. Responses are expressed as a fluorescence ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Benzamide compounds are able to potentiate the calcium mobilization response of mGlu5 to glutamate. Raw traces show the effect of a fixed concentration (10 μM) of test compound or vehicle when added to calcium-sensitive dye-loaded cells and allowed to incubate for 5 min. A suboptimal (EC20) concentration of glutamate was added and the calcium response measured by the FDSS plate reader. Responses are expressed as a fluorescence ratio.
Mentions: Our laboratory performed a functional high-throughput screen (HTS) (18−20) that led to the identification of about 20 structural classes of mGlu5 PAMs. The present study focuses on a novel benzamide chemical scaffold that was identified in that screen. Sixty-five compounds were identified that belong to this structural class, and 10 were found to have high potencies with EC50 values ≤1 μM from HTS stock solutions. This class is of particular interest because these compounds share a benzamide backbone that is similar to that of CPPHA yet are distinct. These compounds display little intrinsic mGlu5 agonist activity in the calcium mobilization assay and induce a robust enhancement of the response to a suboptimal concentration (EC20) of glutamate (Figure 2). To test their PAM activity, HEK293 cells expressing rat mGlu5 were preloaded with calcium-sensitive fluorescent dye and received vehicle or compound (10 μM), followed by an EC20 concentration of glutamate 5 min later. Receptor-induced calcium mobilization responses were monitored using Functional Drug Screening System (FDSS, Hamamatsu, Japan).

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