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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

VU0365396 competitively inhibits VU0357121 function but has no effect on CPPHA activity. (A) VU0365396 induces a parallel rightward shift in the VU0357121 concentration−response curve, indicating a competitive interaction between the two compounds. Cells expressing mGlu5 were treated with increasing concentrations of VU0357121 and either vehicle (◼) or VU0365396 (30 μM, △) followed by EC20 glutamate. (B) The calcium mobilization response produced when mGlu5-expressing HEK293 cells were treated with increasing concentrations of CPPHA and EC20 glutamate do not differ in the absence (◼) or presence (△) of VU0365396 (30 μM). Data were obtained from three separate experiments, each performed in duplicate, and are expressed as the mean ± SEM. Data were normalized to the average maximum response obtained from each experiment as determined by 100 μM glutamate. Concentration−response curves were generated by nonlinear curve fitting.
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fig14: VU0365396 competitively inhibits VU0357121 function but has no effect on CPPHA activity. (A) VU0365396 induces a parallel rightward shift in the VU0357121 concentration−response curve, indicating a competitive interaction between the two compounds. Cells expressing mGlu5 were treated with increasing concentrations of VU0357121 and either vehicle (◼) or VU0365396 (30 μM, △) followed by EC20 glutamate. (B) The calcium mobilization response produced when mGlu5-expressing HEK293 cells were treated with increasing concentrations of CPPHA and EC20 glutamate do not differ in the absence (◼) or presence (△) of VU0365396 (30 μM). Data were obtained from three separate experiments, each performed in duplicate, and are expressed as the mean ± SEM. Data were normalized to the average maximum response obtained from each experiment as determined by 100 μM glutamate. Concentration−response curves were generated by nonlinear curve fitting.

Mentions: To date, the only known neutral allosteric ligands or SAMs for mGlu receptors are ligands at the MPEP site on mGlu5. To expand our investigation of putative non-MPEP allosteric sites on mGlu5, it would be helpful to have a neutral ligand that acts at a different site on the receptor and possibly at the same site as VU0357121. We utilized the benzamide analogues that were synthesized earlier to determine whether any neutral ligands were present among these compounds. To screen our compounds for this activity, we determined their ability to block the VU0357121-induced potentiation of mGlu5-mediated responses to glutamate. To accomplish this, we again used a calcium assay, but cells were pretreated with the test compound (10 μM) and VU0357121 (50 nM) followed by stimulation with an EC20 concentration of glutamate. Several compounds significantly inhibited the PAM activity of VU0357121 (Figure 13), but VU0365396 caused the most robust inhibition and was therefore chosen for further characterization. Interestingly, VU0365396 (30 μM) induced a parallel rightward shift in the VU0357121 CRC (Figure 14A), suggesting that this compound is a neutral ligand that acts in a competitive manner at the same allosteric site as VU0357121. In contrast, VU0365396 (30 μM) had no effect on CPPHA-induced potentiation of mGlu5 responses (Figure 14B). The lack of effect on CPPHA PAM activity was somewhat surprising but provides clear evidence that these mGlu5 PAMs do not act at the same site and raises the possibility that these sites may not interact functionally. However, it is possible that limited affinity of the neutral ligand at its own site or limited negative cooperativity led to a lack of effect on CPPHA responses. Unfortunately, limitations with solubility precluded studies with higher concentrations of VU0365396.


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)

VU0365396 competitively inhibits VU0357121 function but has no effect on CPPHA activity. (A) VU0365396 induces a parallel rightward shift in the VU0357121 concentration−response curve, indicating a competitive interaction between the two compounds. Cells expressing mGlu5 were treated with increasing concentrations of VU0357121 and either vehicle (◼) or VU0365396 (30 μM, △) followed by EC20 glutamate. (B) The calcium mobilization response produced when mGlu5-expressing HEK293 cells were treated with increasing concentrations of CPPHA and EC20 glutamate do not differ in the absence (◼) or presence (△) of VU0365396 (30 μM). Data were obtained from three separate experiments, each performed in duplicate, and are expressed as the mean ± SEM. Data were normalized to the average maximum response obtained from each experiment as determined by 100 μM glutamate. Concentration−response curves were generated by nonlinear curve fitting.
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fig14: VU0365396 competitively inhibits VU0357121 function but has no effect on CPPHA activity. (A) VU0365396 induces a parallel rightward shift in the VU0357121 concentration−response curve, indicating a competitive interaction between the two compounds. Cells expressing mGlu5 were treated with increasing concentrations of VU0357121 and either vehicle (◼) or VU0365396 (30 μM, △) followed by EC20 glutamate. (B) The calcium mobilization response produced when mGlu5-expressing HEK293 cells were treated with increasing concentrations of CPPHA and EC20 glutamate do not differ in the absence (◼) or presence (△) of VU0365396 (30 μM). Data were obtained from three separate experiments, each performed in duplicate, and are expressed as the mean ± SEM. Data were normalized to the average maximum response obtained from each experiment as determined by 100 μM glutamate. Concentration−response curves were generated by nonlinear curve fitting.
Mentions: To date, the only known neutral allosteric ligands or SAMs for mGlu receptors are ligands at the MPEP site on mGlu5. To expand our investigation of putative non-MPEP allosteric sites on mGlu5, it would be helpful to have a neutral ligand that acts at a different site on the receptor and possibly at the same site as VU0357121. We utilized the benzamide analogues that were synthesized earlier to determine whether any neutral ligands were present among these compounds. To screen our compounds for this activity, we determined their ability to block the VU0357121-induced potentiation of mGlu5-mediated responses to glutamate. To accomplish this, we again used a calcium assay, but cells were pretreated with the test compound (10 μM) and VU0357121 (50 nM) followed by stimulation with an EC20 concentration of glutamate. Several compounds significantly inhibited the PAM activity of VU0357121 (Figure 13), but VU0365396 caused the most robust inhibition and was therefore chosen for further characterization. Interestingly, VU0365396 (30 μM) induced a parallel rightward shift in the VU0357121 CRC (Figure 14A), suggesting that this compound is a neutral ligand that acts in a competitive manner at the same allosteric site as VU0357121. In contrast, VU0365396 (30 μM) had no effect on CPPHA-induced potentiation of mGlu5 responses (Figure 14B). The lack of effect on CPPHA PAM activity was somewhat surprising but provides clear evidence that these mGlu5 PAMs do not act at the same site and raises the possibility that these sites may not interact functionally. However, it is possible that limited affinity of the neutral ligand at its own site or limited negative cooperativity led to a lack of effect on CPPHA responses. Unfortunately, limitations with solubility precluded studies with higher concentrations of VU0365396.

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