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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

Mutations that inhibit the function of MPEP/VU-29 also inhibit VU0357121 function, but mutations that inhbit CPPHA activity do not. The ability of VU0357121 (1 μM) to function when specific mGlu5 mutants were transiently expressed in HEK cells was assessed. The calcium mobilization assay was used to determine whether the mGlu5 PAM was able to enhance the glutamate concentration−response relationship at either the A809V/rmGlu5 mutant or the F585I/rmGlu5 mutant. The A809V mutation led to an inhibition of VU0357121 activity (A), whereas the F585I mutation had no effect on its function (B). Concentration−response relationships were generated by adding either vehicle or a fixed concentration (1 μM) of VU0357121 to transiently transfected HEK cells, followed by increasing concentrations of glutamate. 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.
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fig15: Mutations that inhibit the function of MPEP/VU-29 also inhibit VU0357121 function, but mutations that inhbit CPPHA activity do not. The ability of VU0357121 (1 μM) to function when specific mGlu5 mutants were transiently expressed in HEK cells was assessed. The calcium mobilization assay was used to determine whether the mGlu5 PAM was able to enhance the glutamate concentration−response relationship at either the A809V/rmGlu5 mutant or the F585I/rmGlu5 mutant. The A809V mutation led to an inhibition of VU0357121 activity (A), whereas the F585I mutation had no effect on its function (B). Concentration−response relationships were generated by adding either vehicle or a fixed concentration (1 μM) of VU0357121 to transiently transfected HEK cells, followed by increasing concentrations of glutamate. 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.

Mentions: We previously reported the effects of single point mutations in mGlu5 which can inhibit the responses of mGlu5 to specific PAMs and NAMs (8,14,20). These mutants were tested for their ability to alter responses to VU0357121 (1 μM) in transiently transfected HEK293 cells using the calcium mobilization assay. Mutation of the alanine at the 809 position of rat mGlu5 to a valine (A809V/rmGlu5) reduces the binding and function of MPEP and inhibits functional responses to PAMs that act at the MPEP site, such as VU-29 (8,14,20) but has no effect on responses to CPPHA (14). In contrast, the F585I/rmGlu5 mutation inhibits the PAM activity of CPPHA but has no effect on responses to VU-29 activity (14). We replicated these previous findings and found that the response to VU-29 but not CPPHA is lost in the A809V/rmGlu5 mutant, whereas the response to CPPHA but not VU29 is lost in the F585I/rmGlu5 mutant (data not shown). Interestingly, we found that the A809V/rmGlu5 mutation inhibited the ability of VU0357121 to shift the glutamate concentration−response curve (Figure 15A), whereas the response to VU0357121 was not altered by the F585I/rmGlu5 mutation (Figure 15B). The finding that the F585I mutation was without effect is consistent with studies above suggesting that CPPHA and VU0357121 do not act at identical sites. The finding that the response was inhibited by the A809V mutation is somewhat surprising in light of the results from the molecular pharmacology studies and further illustrates the complexity of the relationships between putative allosteric sites on mGlu5. In addition, these data highlight the fact that mutagenesis studies must be interpreted with caution. While previous studies and the current data provide strong evidence that multiple mGlu5 modulators do not bind to identical binding sites, it is possible that they interact with overlapping sites that share critical residues. Also, in the absence of direct structural information about the class C GPCRs, models of class C GPCR structure and functional effects of specific mutations do not provide definitive information about the nature of the binding of these ligands. However, the results of these mutagenesis studies combined with the current and previous molecular pharmacology studies provide further evidence that there is some interaction between the putative binding sites of CPPHA, VU0357121, and the MPEP binding site that are important for the action of these structurally and functionally diverse allosteric modulators.


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)

Mutations that inhibit the function of MPEP/VU-29 also inhibit VU0357121 function, but mutations that inhbit CPPHA activity do not. The ability of VU0357121 (1 μM) to function when specific mGlu5 mutants were transiently expressed in HEK cells was assessed. The calcium mobilization assay was used to determine whether the mGlu5 PAM was able to enhance the glutamate concentration−response relationship at either the A809V/rmGlu5 mutant or the F585I/rmGlu5 mutant. The A809V mutation led to an inhibition of VU0357121 activity (A), whereas the F585I mutation had no effect on its function (B). Concentration−response relationships were generated by adding either vehicle or a fixed concentration (1 μM) of VU0357121 to transiently transfected HEK cells, followed by increasing concentrations of glutamate. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig15: Mutations that inhibit the function of MPEP/VU-29 also inhibit VU0357121 function, but mutations that inhbit CPPHA activity do not. The ability of VU0357121 (1 μM) to function when specific mGlu5 mutants were transiently expressed in HEK cells was assessed. The calcium mobilization assay was used to determine whether the mGlu5 PAM was able to enhance the glutamate concentration−response relationship at either the A809V/rmGlu5 mutant or the F585I/rmGlu5 mutant. The A809V mutation led to an inhibition of VU0357121 activity (A), whereas the F585I mutation had no effect on its function (B). Concentration−response relationships were generated by adding either vehicle or a fixed concentration (1 μM) of VU0357121 to transiently transfected HEK cells, followed by increasing concentrations of glutamate. 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.
Mentions: We previously reported the effects of single point mutations in mGlu5 which can inhibit the responses of mGlu5 to specific PAMs and NAMs (8,14,20). These mutants were tested for their ability to alter responses to VU0357121 (1 μM) in transiently transfected HEK293 cells using the calcium mobilization assay. Mutation of the alanine at the 809 position of rat mGlu5 to a valine (A809V/rmGlu5) reduces the binding and function of MPEP and inhibits functional responses to PAMs that act at the MPEP site, such as VU-29 (8,14,20) but has no effect on responses to CPPHA (14). In contrast, the F585I/rmGlu5 mutation inhibits the PAM activity of CPPHA but has no effect on responses to VU-29 activity (14). We replicated these previous findings and found that the response to VU-29 but not CPPHA is lost in the A809V/rmGlu5 mutant, whereas the response to CPPHA but not VU29 is lost in the F585I/rmGlu5 mutant (data not shown). Interestingly, we found that the A809V/rmGlu5 mutation inhibited the ability of VU0357121 to shift the glutamate concentration−response curve (Figure 15A), whereas the response to VU0357121 was not altered by the F585I/rmGlu5 mutation (Figure 15B). The finding that the F585I mutation was without effect is consistent with studies above suggesting that CPPHA and VU0357121 do not act at identical sites. The finding that the response was inhibited by the A809V mutation is somewhat surprising in light of the results from the molecular pharmacology studies and further illustrates the complexity of the relationships between putative allosteric sites on mGlu5. In addition, these data highlight the fact that mutagenesis studies must be interpreted with caution. While previous studies and the current data provide strong evidence that multiple mGlu5 modulators do not bind to identical binding sites, it is possible that they interact with overlapping sites that share critical residues. Also, in the absence of direct structural information about the class C GPCRs, models of class C GPCR structure and functional effects of specific mutations do not provide definitive information about the nature of the binding of these ligands. However, the results of these mutagenesis studies combined with the current and previous molecular pharmacology studies provide further evidence that there is some interaction between the putative binding sites of CPPHA, VU0357121, and the MPEP binding site that are important for the action of these structurally and functionally diverse allosteric modulators.

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