Evaluation of Synthetic Cytochrome P450-Mimetic Metalloporphyrins To Facilitate "Biomimetic" Biotransformation of a Series of mGlu5 Allosteric Ligands.
ABSTRACT: Allosteric ligands within a given chemotype can have the propensity to display a wide range of pharmacology, as well as unexpected changes in GPCR subtype selectivity, typically mediated by single-atom modifications to the ligand. Due to the unexpected nature of these "molecular switches", chemotypes with this property are typically abandoned in lead optimization. Recently, we have found that in vivo oxidative metabolism by CYP450s can also engender molecular switches within allosteric ligands, changing the mode of pharmacology and leading to unwanted toxicity. We required a higher-throughput approach to assess in vivo metabolic molecular switches, and we turned to a "synthetic liver", a 96 well kit of biomimetic catalysts (e.g., metalloporphyrins) to rapidly survey a broad panel of synthetic CYP450s' ability to oxidize/"metabolize" an mGlu5 PAM (VU0403602) known to undergo an in vivo CYP450-mediated molecular switch. While the synthetic CYP450s did generate a number of oxidative "metabolites" at known "hot spots", several of which proved to be pure mGlu5 PAMs comparable in potency to the parent, the known CYP450-mediated in vivo ago-PAM metabolite, namely, VU0453103, was not formed. Thus, this technology platform has potential to identify hot spots for oxidative metabolism and produce active metabolites of small-molecule ligands in a high-throughput, scalable manner.
Project description:Allosteric modulation of GPCRs represents an increasingly explored approach in drug development. Due to complex pharmacology, however, the relationship(s) between modulator properties determined in vitro with in vivo concentration-effect phenomena is frequently unclear. We investigated key pharmacological properties of a set of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) and their relevance to in vivo concentration-response relationships. These studies identified a significant relationship between in vitro PAM cooperativity (??), as well as the maximal response obtained from a simple in vitro PAM concentration-response experiment, with in vivo efficacy for reversal of amphetamine-induced hyperlocomotion. This correlation did not exist with PAM potency or affinity. Data across PAMs were then converged to calculate an in vivo concentration of glutamate putatively relevant to the mGlu5 PAM mechanism of action. This work demonstrates the ability to merge in vitro pharmacology profiles with relevant behavioral outcomes and also provides a novel method to estimate neurotransmitter concentrations in vivo.
Project description:BACKGROUND:Metabotropic glutamate receptor subtype 5 (mGlu5) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu5 have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu5 PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu5 PAMs do not activate mGlu5 directly but selectively potentiate activation of mGlu5 by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu5 activation and achieving optimal in vivo effects. METHODS:Using specially engineered mGlu5 cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu5 PAMs have intrinsic allosteric agonist activity in the absence of glutamate. RESULTS:Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu5 PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations. CONCLUSIONS:Loss of the absolute dependence of mGlu5 PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu5 PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia.
Project description:Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system disorders, including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 were 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, that was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by coadministration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine. 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 cytochrome P450 (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 proconvulsant 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 after in vivo administration of this class of allosteric receptor modulators.
Project description: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 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, 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 noncompetitive. 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.
Project description:Allosteric modulators, that exhibit no intrinsic agonist activity, offer the advantage of spatial and temporal fine-tuning of endogenous agonist activity, allowing the potential for increased selectivity, reduced adverse effects and improved clinical outcomes. Some allosteric ligands can differentially activate and/or modulate distinct signaling pathways arising from the same receptor, phenomena referred to as 'biased agonism' and 'biased modulation'. Emerging evidence for CNS disorders with glutamatergic dysfunction suggests the metabotropic glutamate receptor subtype 5 (mGlu5) is a promising target. Current mGlu5 allosteric modulators have largely been classified based on modulation of intracellular calcium (iCa2+) responses to orthosteric agonists alone. We assessed eight mGlu5 allosteric modulators previously classified as mGlu5 PAMs or PAM-agonists representing four distinct chemotypes across multiple measures of receptor activity, to explore their potential for engendering biased agonism and/or modulation. Relative to the reference orthosteric agonist, DHPG, the eight allosteric ligands exhibited distinct biased agonism fingerprints for iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation in HEK293A cells stably expressing mGlu5 and in cortical neuron cultures. VU0424465, DPFE and VU0409551 displayed the most disparate biased signaling fingerprints in both HEK293A cells and cortical neurons that may account for the marked differences observed previously for these ligands in vivo. Select mGlu5 allosteric ligands also showed 'probe dependence' with respect to their cooperativity with different orthosteric agonists, as well as biased modulation for the magnitude of positive cooperativity observed. Unappreciated biased agonism and modulation may contribute to unanticipated effects (both therapeutic and adverse) when translating from recombinant systems to preclinical models. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.
Project description:Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.
Project description:As part of our efforts to identify a suitable back-up compound to our recently disclosed mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212, this letter details the investigation and challenges of a novel series of 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one derivatives. From these efforts, compound 4k emerged as a potent and selective mGlu5 PAM displaying overall attractive in vitro (pharmacological and ADMET) and PK profiles combined with in vivo efficacy in preclinical models of schizophrenia. However, further advancement of the compound was precluded due to severely limiting CNS-related side-effects confirming the previously reported association between excessive mGlu5 activation and target-related toxicities.
Project description:Exemestane (EXE) is an endocrine therapy commonly used by postmenopausal women with hormone-responsive breast cancer due to its potency in inhibiting aromatase-catalyzed estrogen synthesis. Preliminary in vitro studies sought to identify phase I EXE metabolites and hepatic cytochrome P450s (CYP450s) that participate in EXE biotransformation. Phase I metabolites were identified by incubating EXE with HEK293-overexpressed CYP450s. CYP450s 1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5 produce 17β-dihydroexemestane (17β-DHE), an active major metabolite, as well as two inactive metabolites. 17β-DHE formation in pooled human liver microsomes subjected to isoform-specific CYP450 inhibition was also monitored using tandem mass spectrometry. 17β-DHE production in human liver microsomes was unaffected by isoform-specific inhibition of CYP450s 2A6, 2B6, and 2E1 but decreased 12-39% following inhibition of drug-metabolizing enzymes from CYP450 subfamilies 1A, 2C, 2D, and 3A. These results suggest that redundancy exists in the EXE metabolic pathway with multiple hepatic CYP450s catalyzing 17β-DHE formation in vitro. To further expand the knowledge of phase I EXE metabolism, the impact of CYP450 genetic variation on 17β-DHE formation was assessed via enzyme kinetic parameters. Affinity for EXE substrate and enzyme catalytic velocity were calculated for hepatic wild-type CYP450s and their common nonsynonymous variants by monitoring the reduction of EXE to 17β-DHE. Several functional polymorphisms in xenobiotic-metabolizing CYP450s 1A2, 2C8, 2C9, and 2D6 resulted in deviant enzymatic activity relative to wild-type enzyme. Thus, it is possible that functional polymorphisms in EXE-metabolizing CYP450s contribute to inter-individual variability in patient outcomes by mediating overall exposure to the drug and its active metabolite, 17β-DHE.
Project description:BACKGROUND:Members of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. RESULTS:In the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding >?300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum. CONCLUSIONS:This study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.
Project description:Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) have been proposed as potential therapies for various CNS disorders. These ligands bind to sites distinct from the orthosteric (or endogenous) ligand, often with improved subtype selectivity and spatio-temporal control over receptor responses. We recently revealed that mGlu5 allosteric agonists and positive allosteric modulators exhibit biased agonism and/or modulation. To establish whether negative allosteric modulators (NAMs) engender similar bias, we rigorously characterized the pharmacology of eight diverse mGlu5 NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu5 for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [3H]methoxy-PEPy. We assessed mGlu5-mediated intracellular Ca2+ (iCa2+) mobilization and inositol phosphate (IP1) accumulation in HEK293A cells stably expressing low levels of mGlu5 (HEK293A-rat mGlu5-low) and mouse embryonic cortical neurons. The apparent affinity of acetylenic NAMs, MPEP, MTEP and dipraglurant, was dependent on the signaling pathway measured, agonist used, and cell type (HEK293A-rat mGlu5-low versus mouse cortical neurons). In contrast, the acetylenic partial NAM, M-5MPEP, and structurally distinct NAMs (VU0366248, VU0366058, fenobam), had similar affinity estimates irrespective of the assay or cellular background. Biased modulation was evident for VU0366248 in mouse cortical neurons where it was a NAM for DHPG-mediated iCa2+ mobilization, but neutral with DHPG in IP1 accumulation assays. Overall, this study highlights the inherent complexity in mGlu5 NAM pharmacology that we hypothesize may influence interpretation when translating into preclinical models and beyond in the design and development of novel therapeutics for neuropsychiatric and neurological disorders.