Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein-coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor mGlu5 and enables the optical control of endogenous mGlu5 receptors.

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.

There is evidence that some heavy users of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) show signs of neurotoxicity (a cognitive dysfunction, a larger incidence of psychopathology). It has been postulated that the catechol intermediates of methylenedioxyamphetamines such as 3,4-dihydroxymethamphetamine (HHMA), a metabolite of MDMA, may play a role in their neurotoxicity by formation of thioether adducts. This study describes the first validated method for HHMA determination in plasma and urine by strong cation-exchange solid-phase extraction high-performance liquid chromatography/electrochemical detection (HPLC/ED) analysis. The method has been applied for the determination of HHMA in plasma and urine samples from a clinical study in healthy volunteers of MDMA and provides preliminary kinetic data on this metabolite. HHMA appeared to be a major MDMA metabolite with plasma concentrations as high as the parent compound. Thus, HHMA C(max) (154.5 microg/L) and AUC(0-24h)(1990.9 microg/L h) were similar to those obtained in previously published reports for MDMA (181.6 microg/L and 1465.9 microg/L h, respectively). The 24-h urinary recovery of HHMA accounted for 17.7% of the MDMA dose administered and increases the total 24 h recovery of MDMA and metabolites to 58% of the 100 mg dose administered. The determination of HHMA in plasma and urine samples is of interest in order to establish its relevance in MDMA metabolism and its possible contribution to MDMA neurotoxicity in humans. Its validation showed appropriate accuracy and precision for its use in pharmacokinetic studies.

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.

Contrary to acute pain, chronic pain does not serve as a warning signal and must be considered as a disease per se. This pathology presents a sensory and psychological dimension at the origin of affective and cognitive disorders. Being largely refractory to current pharmacotherapies, identification of endogenous systems involved in persistent and chronic pain is crucial. The amygdala is a key brain region linking pain sensation with negative emotions. Here, we show that activation of a specific intrinsic neuromodulatory system within the amygdala associated with type 4 metabotropic glutamate receptors (mGlu) abolishes sensory and affective symptoms of persistent pain such as hypersensitivity to pain, anxiety- and depression-related behaviors, and fear extinction impairment. Interestingly, neuroanatomical and synaptic analysis of the amygdala circuitry suggests that the effects of mGlu activation occur outside the central nucleus via modulation of multisensory thalamic inputs to lateral amygdala principal neurons and dorso-medial intercalated cells. Furthermore, we developed optogluram, a small diffusible photoswitchable positive allosteric modulator of mGlu. This ligand allows the control of endogenous mGlu activity with light. Using this photopharmacological approach, we rapidly and reversibly inhibited behavioral symptoms associated with persistent pain through optical control of optogluram in the amygdala of freely behaving animals. Altogether, our data identify amygdala mGlu signaling as a mechanism that bypasses central sensitization processes to dynamically modulate persistent pain symptoms. Our findings help to define novel and more precise therapeutic interventions for chronic pain, and exemplify the potential of optopharmacology to study the dynamic activity of endogenous neuromodulatory mechanisms in vivo.

A gas chromatography-mass spectrometry (GC-MS) method was used for the simultaneous quantitation of 3,4-methylenedioxymethamphetamine (MDMA) and the 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) metabolites in plasma and urine samples after the administration of 100 mg MDMA to healthy volunteers. Samples were hydrolyzed prior to a solid-phase extraction with Bond Elut Certify columns. Analytes were eluted with ethyl acetate (2% ammonium hydroxide) and analyzed as their trifluoroacyl derivatives. Linear calibration curves were obtained at plasma and urine concentration ranges of 25-400 ng/mL and 250-2000 ng/mL for MDMA and HMMA, and of 2.5-40 ng/mL and 100-1000 ng/mL for MDA and HMA. Following the same urine preparation procedure but without the derivatization step, a capillary electrophoresis (CE) method for enantiomerical resolution of compounds was developed using (2-hydroxy)propyl-beta-cyclodextrin at two different concentrations (10 and 50mM in 50mM H3PO4, pH 2.5) as chiral selector. Calibration curves for the CE method were prepared with the corresponding racemic mixture and were linear between 125 and 2000 ng/mL, 50 and 1000 ng/mL, and 125 and 1500 ng/mL for each enantiomer of MDMA, MDA, and HMMA, respectively. Stereoselective disposition of MDMA and MDA was confirmed. HMMA disposition seems to be in apparent contradiction with MDMA findings as the enantiomer ratio is close to 1 and constant over the time.

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.

Type 4 metabotropic glutamate (mGlu 4 ) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu 4 -positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu 4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu 4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(t B ) = 1.15 6 0.38 and 1.25 6 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na + binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu 4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes.-Rovira, X., Malhaire, F., Scholler, P., Rodrigo, J., Gonzalez-Bulnes, P., Llebaria, A., Pin, J.-P., Giraldo, J., Goudet, C. Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors. FASEB J. 29, 116-130 (2015). www.fasebj.org