Probing the Metabotropic Glutamate Receptor 5 (mGlu(5)) Positive Allosteric Modulator (PAM) Binding Pocket: Discovery of Point Mutations That Engender a "Molecular Switch" in PAM Pharmacology

1 Vanderbilt University
Molecular pharmacology (Impact Factor: 4.13). 02/2013; 83(5). DOI: 10.1124/mol.112.083949
Source: PubMed


Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu(5)) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand-(orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit, while positive allosteric modulators (PAMs) enhance, the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu(5) modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu(5) served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a prototypical NAM. Three conserved amino acids (Y658, T780, S808) and two non-conserved residues (P654, A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TM6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs.

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    • "Thus, rigorous molecular pharmacological analysis was successful in accurately predicting utility of a given PET ligand for assessing occupancy of a test compound. These data are consistent with recent molecular modeling and docking studies that suggest that VU0360172 binds to the allosteric site on mGlu 5 with a pose that is distinct from that of MPEP (Gregory et al, 2013). In contrast, docking studies predicted that VU0409106, while structurally distinct, binds to this site in a manner that is overlapping with the binding of MPEP or FPEB (Gregory et al, 2014). "
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    ABSTRACT: Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) have exciting potential as therapeutic agents for multiple brain disorders. Translational studies with mGlu5 modulators have relied on mGlu5 allosteric site positron emission tomography (PET) radioligands to assess receptor occupancy in the brain. However, recent structural and modeling studies suggest that closely related mGlu5 allosteric modulators can bind to overlapping but not identical sites, which could complicate interpretation of in vivo occupancy data, even when PET ligands and drug leads are developed from the same chemical scaffold. We now report that systemic administration of the novel mGlu5 positive allosteric modulator (PAM) VU0092273 displaced the structurally related mGlu5 PET ligand, [(18)F]FPEB, with measures of in vivo occupancy that closely aligned with its in vivo efficacy. In contrast, a close analog of VU0092273 and [(18)F]FPEB, VU0360172, provided robust efficacy in rodent models in the absence of detectable occupancy. Furthermore, a structurally unrelated mGlu5 negative allosteric modulator (NAM), VU0409106 displayed measures of in vivo occupancy that correlated well with behavioral effects, despite the fact that VU0409106 is structurally unrelated to [(18)F]FPEB. Interestingly, all three compounds inhibit radioligand binding to the prototypical MPEP/FPEB allosteric site in vitro. However, VU0092273 and VU0409106 bind to this site in a fully competitive manner, whereas the interaction of VU0360172 is non-competitive. Thus, while close structural similarity between PET ligands and drug leads does not circumvent issues associated with differential binding to a given target, detailed molecular pharmacology analysis accurately predicts utility of ligand pairs for in vivo occupancy studies.Neuropsychopharmacology accepted article preview online, 22 September 2014. doi:10.1038/npp.2014.245.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 09/2014; 40(3). DOI:10.1038/npp.2014.245 · 7.05 Impact Factor
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    • "y " molecular switches " in which subtle changes to the structure of a compound changes the mode of pharmacology of allosteric modulators ( i . e . mode switching with PAM to NAM / SAM conversion with subtle structural changes ) and / or changes the receptor subtype se - lectivity can confound lead compound development ( Bhagwanth et al . , 2012 ; Gregory et al . , 2013 ; Melancon et al . , 2012 ; Utley et al . , 2011 ; Wood et al . , 2011"
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    ABSTRACT: The discovery of allosteric modulators of G protein-coupled receptors (GPCRs) provides a promising new strategy with potential for developing novel treatments for a variety of central nervous system (CNS) disorders. Traditional drug discovery efforts targeting GPCRs have focused on developing ligands for orthosteric sites which bind endogenous ligands. Allosteric modulators target a site separate from the orthosteric site to modulate receptor function. These allosteric agents can either potentiate (positive allosteric modulator, PAM) or inhibit (negative allosteric modulator, NAM) the receptor response and often provide much greater subtype selectivity than do orthosteric ligands for the same receptors. Experimental evidence has revealed more nuanced pharmacological modes of action of allosteric modulators, with some PAMs showing allosteric agonism in combination with positive allosteric modulation in response to endogenous ligand (ago-potentiators) as well as "bitopic" ligands that interact with both the allosteric and orthosteric sites. Drugs targeting the allosteric site allow for increased drug selectivity and potentially decreased adverse side effects. Promising evidence has demonstrated potential utility of a number of allosteric modulators of GPCRs in multiple CNS disorders, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as psychiatric or neurobehavioral diseases such as anxiety, schizophrenia, and addiction.
    Neurobiology of Disease 09/2013; 61. DOI:10.1016/j.nbd.2013.09.013 · 5.08 Impact Factor
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    ABSTRACT: Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system (CNS) disorders including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 have been discovered to exhibit in vivo efficacy in rodent models of psychosis, including PAMs possessing varying degrees of agonist activity (ago-PAMs) as well as PAMs devoid of agonist activity. However, previous studies revealed that ago-PAMs can induce seizure activity and behavioral convulsions, whereas pure mGlu5 PAMs do not induce these adverse effects. We recently identified a potent and selective mGlu5 PAM, VU0403602, which was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by co-administration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine (MPEP). Consistent with potential adverse effects induced by ago-PAMs, we found that VU0403602 had significant allosteric agonist activity. Interestingly, inhibition of VU0403602 metabolism in vivo by a pan P450-inactivator completely protected rats from induction of seizures. P450-mediated biotransformation of VU0403602 was discovered to produce another potent ago-PAM metabolite-ligand (M1) of mGlu5. Electrophysiological studies in rat hippocampal slices confirmed agonist activity of both M1 and VU0403602 and revealed that M1 can induce epileptiform activity in a manner consistent with its pro-convulsant behavioral effects. Furthermore, unbound brain exposure of M1 was similar to that of the parent compound VU0403602. These findings indicate that biotransformation of mGlu5 PAMs to active metabolite-ligands may contribute to the epileptogenesis observed following in vivo administration of this class of allosteric receptor modulators.
    Drug metabolism and disposition: the biological fate of chemicals 07/2013; 41(9). DOI:10.1124/dmd.113.052084 · 3.25 Impact Factor
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