Methods for automating the analysis of live-cell single-molecule FRET data

Single-molecule FRET (smFRET) is a powerful imaging platform capable of revealing dynamic changes in the conformation and proximity of biological molecules. The expansion of smFRET imaging into living cells creates both numerous new research opportunities and new challenges. Automating dataset curation processes is critical to providing consistent, repeatable analysis in an efficient manner, freeing experimentalists to advance the technical boundaries and throughput of what is possible in imaging living cells. Here, we devise an automated solution to the problem of multiple particles entering a region of interest, an otherwise labor-intensive and subjective process that had been performed manually in our previous work. The resolution of these two issues increases the quantity of FRET data and improves the accuracy with which FRET distributions are generated, increasing knowledge about the biological functions of the molecules under study. Our automated approach is straightforward, interpretable, and requires only localization and intensity values for donor and acceptor channel signals, which we compute through our previously published smCellFRET pipeline. The development of our automated approach is informed by the insights of expert experimentalists with extensive experience inspecting smFRET trajectories (displacement and intensity traces) from live cells. We test our automated approach against our recently published research on the metabotropic glutamate receptor 2 (mGluR2) and reveal substantial similarities, as well as potential shortcomings in the manual curation process that are addressable using the algorithms we developed here

Discovery of a Novel Selective Kappa-Opioid Receptor Agonist Using Crystal Structure-Based Virtual Screening

Kappa-opioid (KOP) receptor agonists exhibit analgesic effects without activating reward pathways. In the search for non-addictive opioid therapeutics and novel chemical tools to study physiological functions regulated by the KOP receptor, we screened in silico its recently released inactive crystal structure. A selective novel KOP receptor agonist emerged as a notable result, and is proposed as a new chemotype for the study of the KOP receptor in the etiology of drug addiction, depression, and/or pain

GPCR-OKB: the G protein coupled receptor oligomer knowledge base

Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers

The respiratory depressant effects of mitragynine are limited by its conversion to 7-OH mitragynine

Background and Purpose: Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), is a partial agonist at the μ opioid receptor. CYP3A-dependent oxidation of mitragynine yields the metabolite 7-OH mitragynine, a more efficacious μ receptor agonist. While both mitragynine and 7-OH mitragynine can induce anti-nociception in mice, recent evidence suggests that 7-OH mitragynine formed as a metabolite is sufficient to explain the anti-nociceptive effects of mitragynine. However, the ability of 7-OH mitragynine to induce μ receptor-dependent respiratory depression has not yet been studied. Experimental Approach: Respiration was measured in awake, freely moving, male CD-1 mice, using whole body plethysmography. Anti-nociception was measured using the hot plate assay. Morphine, mitragynine, 7-OH mitragynine and the CYP3A inhibitor ketoconazole were administered orally. Key Results: The respiratory depressant effects of mitragynine showed a ceiling effect, whereby doses higher than 10 mg·kg−1 produced the same level of effect. In contrast, 7-OH mitragynine induced a dose-dependent effect on mouse respiration. At equi-depressant doses, both mitragynine and 7-OH mitragynine induced prolonged anti-nociception. Inhibition of CYP3A reduced mitragynine-induced respiratory depression and anti-nociception without affecting the effects of 7-OH mitragynine. Conclusions and Implications: Both the anti-nociceptive effects and the respiratory depressant effects of mitragynine are partly due to its metabolic conversion to 7-OH mitragynine. The limiting rate of conversion of mitragynine into its active metabolite results in a built-in ceiling effect of the mitragynine-induced respiratory depression. These data suggest that such ‘metabolic saturation’ at high doses may underlie the improved safety profile of mitragynine as an opioid analgesic

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole

Partial agonists of the dopamine D2 receptor (D2R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments and molecular dynamics simulations to identify the D2R-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2 and 7 determine the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism through which to fine-tune the intrinsic efficacy of D2R agonists

Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor's downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in "indirect pathway" medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics

Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

By analyzing and simulating inactive conformations of the highly-homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands

Extrapyramidal side effects of antipsychotics are linked to their association kinetics at dopamine D2 receptors

Atypical antipsychotic drugs (APDs) have been hypothesized to show reduced extrapyramidal side effects (EPS) due to their rapid dissociation from the dopamine D2 receptor. However, support for this hypothesis is limited to a relatively small number of observations made across several decades and under different experimental conditions. Here we show that association rates, but not dissociation rates, correlate with EPS. We measured the kinetic binding properties of a series of typical and atypical APDs in a novel time-resolved fluorescence resonance energy transfer assay, and correlated these properties with their EPS and prolactin-elevating liabilities at therapeutic doses. EPS are robustly predicted by a rebinding model that considers the microenvironment of postsynaptic D2 receptors and integrates association and dissociation rates to calculate the net rate of reversal of receptor blockade. Thus, optimizing binding kinetics at the D2 receptor may result in APDs with improved therapeutic profile

Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier

Despite the controversy regarding the existence and physiological relevance of class A G protein-coupled receptor dimerization, there is substantial evidence for functional interactions between the dopamine D2 receptor (D2R) and the adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by proximity ligation assay; however, their existence in the human brain has not been demonstrated. In this study, we used Brightfield proximity ligation assay, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated proximity between the D2R and the A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the relative quantification of proximity of two proteins, as well as the expression levels of individual proteins