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Beta-adrenoceptors (ß-AR) are prototypical G proteincoupled receptors and important pharmacological targets for many diseases. Indeed, a number of approved drugs target these receptors due to their key role on many physiological functions. Among other examples, we encounter ß1-AR antagonists (ß- Blockers), which constitute the first-line therapy for the treatment of heart diseases, and ß2-AR agonists, which act as bronchodilators for the treatment of breathing pathologies. Considering the relevance of these receptors, achieving a reversible and localised control of their activity would provide a powerful tool, both for its research applications and its clinical potential. In this context, photopharmacology arises as a potent approach. Photopharmacology is an emerging field based on the use of synthetic light-regulated molecules to allow reversible spatiotemporal control of target receptors in native tissues. These ligands have the potential to provide a precise and controllable therapeutic action with increased efficacy and reduced side effects. Moreover, the fine regulation on demand of the receptor activation state is of great interest for their study in non-modified cells, tissues and organisms. The present project provides the first proof of concept for beta-adrenoceptor photopharmacology. We first designed and synthesised libraries of lightregulated compounds in order to regulate ß-AR activity with spatiotemporal precision. Subsequent testing highlighted the successful development of compounds with promising pharmacological properties which can be reversibly and irreversibly controlled by light. The discovered molecules enable a fine control of ß-AR in their native environment that will certainly open the door to innovative research procedures and may inspire future personalized therapies targeting these receptors.

Beta-adrenoceptors (ß-AR) are prototypical G protein-coupled receptors (GPCR) and important pharmacological targets for numerous diseases. Indeed, a number of approved drugs target ß-AR, which are key regulators of many physiological functions. Among other examples, ß1-AR antagonists (known as ß-Blockers) are first-line therapies for the treatment of heart failure, and ß2-AR agonists, which act as bronchodilators, are widely used for the treatment of breathing pathologies. Considering the medical relevance of these receptors, achieving a reversible and localized control of their activity would provide a powerful research and clinical tool. GPCR signaling is currently recognized as a multidimensional process governed by molecular, spatial and temporal components. Uncovering the role of each of these dimensions is crucial to improve our knowledge on cell communication, to understand how different pathways give rise to cellular and physiological effects, and to know how can we interact with biological systems with precision using drugs. Photopharmacology is an emerging field in which light-sensitive molecules are used to control the function of a given target protein in native tissues. The modulation of the target activity is achieved by small, drug-like, photoregulated ligands. By the use of light, both spatial and temporal control of the compound activity can be achieved in unprecedented manners compared to conventional pharmacology. These ligands have the potential to provide highly precise and controllable therapeutic actions that may result in increased efficacies and reduced side effects. Importantly, photopharmacology may allow to gain mechanistic insight on the interplay between the activation time and the receptor location during signaling processes in non-modified cells, tissues and whole organisms. Our research focused on the generation of new molecular tools for beta-adrenoceptors photopharmacology will be presented in this communication. First, several libraries of light-sensitive compounds with the aim to regulate ß-AR activity with spatiotemporal precision were designed and synthesized. Subsequent testing in cell preparations demonstrated the successful development of compounds with promising pharmacological properties, which can be reversibly and irreversibly controlled by light. Among those, several hit compounds were identified as ligands for beta-1 and beta-2 adrenoceptors with low nanomolar activities. These libraries compounds were found to be active enough to become useful photopharmacological tools, so we also performed in vivo experiments to determine their research potential in physiological environments. Indeed, the discovered molecules enabled a fine control of ß-AR in their native environment. We believe that the results of these studies will certainly open the door to innovative research procedures and may inspire future therapies targeting ß-AR.

New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment., Funding Agence Nationale de la Recherche ANR-17-CE11-0046 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe CTQ2017-89222-R Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PCI2018-093047 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PID2020-120499RB-I00 Catalan government 2017 SGR 1604 the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 801342 (TecniospringINDUSTRY) and the Government of Catalonia's Agency for Business Competitive-ness (ACCIÓ). TECSPR19-1-0062

Breakthrough cancer pain (BTcP) refers to a sudden and transient exacerbation of pain, which develops in patients treated with opioid analgesics. Fast-onset analgesia is required for the treatment of BTcP. Light-activated drugs offer a novel potential strategy for the rapid control of pain without the typical adverse effects of systemic analgesic drugs. mGlu5 metabotropic glutamate receptor antagonists display potent analgesic activity, and light-induced activation of one of these compounds (JF-NP-26) in the thalamus was found to induce analgesia in models of inflammatory and neuropathic pain. We used an established mouse model of BTcP based on the injection of cancer cells into the femur, followed, 16 days later, by systemic administration of morphine. BTcP was induced by injection of endothelin-1 (ET-1) into the tumor, 20 min after morphine administration. Mice were implanted with optic fibers delivering light in the visible spectrum (405 nm) in the thalamus or prelimbic cortex to locally activate systemically injected JF-NP-26. Light delivery in the thalamus caused rapid and substantial analgesia, and this effect was specific because light delivery in the prelimbic cortex did not relieve BTcP. This finding lays the groundwork for the use of optopharmacology in the treatment of BTcP., This work was supported by the Italian Ministry of Health (Project code: GR-2016-02362046).

Metabotropic glutamate receptors (mGlu) are a family of class C G protein-coupled receptors (GPCRs) with important biological functions and widespread expression. The mechanisms of mGlu activation and the development of allosteric modulators for these dimeric proteins have attracted singular attention including the use of light regulated ligands. Photopharmacology involves the integration of a photoactive moiety into the ligand structure that following specific illumination undergoes a structural rearrangement and changes its biological activity. The use of light-regulated allosteric ligands offers the opportunity to manipulate mGlu signalling with spatiotemporal precision, unattainable with classical pharmacological approaches. In this review, we will discuss some of the innovations that have been made in the allosteric photopharmacology of mGlu receptors to date. We discuss the prospects of these molecular tools in the control of mGluRs and the new perspectives in understanding mGlu mechanisms, pharmacology and (patho)physiology that can ultimately result in innovative drug discovery concepts., The project on which these results are based has received funding from Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación 10.13039/501100011033 and ERDF A way of making Europe (projects I+D+i CTQ2017-89222-R, PCI2018-093047 and PID2020-120499RB-I00), the Neuron-ERANET program (MAGNOLIA project), by the Catalan government (2017 SGR 1604) to AL and XR, and the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 801342 (Tecniospring INDUSTRY, TECSPR19-1-0062) and the Government of Catalonia's Agency for Business Competitiveness (ACCIÓ) to XG-S.

Lepidopteran pests are major factors limiting soybean productivity in South America. In some cases, the control of these species requires the use of foliar insecticides. For a sustainable use of these insecticides, they should be sprayed when insect population sizes reach an economic threshold. Since this estimation requires to determine the consumption of different species, this work aimed to integrate all the main factors, studying the consumption of small-and medium-size larvae of major lepidopteran pests to vegetative and reproductive tissues on Bt and non-Bt soybeans. The damage to vegetative tissues was tested in detached-leaf assays in grow chambers, and to reproductive structures was measured in greenhouse with infestation at early (flowering) and mid reproductive (mid grain filling) stages. Based on the feeding behavior of the species tested, they were cast in four groups: a) A. gemmatalis and C. includens, defoliating only the RR variety with the lowest consumption of foliar area; b) S. eridania, defoliating both RR and IPRO varieties, consuming twice than the species mentioned above; c) H. armigera, defoliating and being the most damaging species to pods in the RR variety; d) S. cosmioides and S. frugiperda, defoliating and damaging pods in both varieties. The species differed in their ability to feed on IPRO varieties, so a different economic threshould could be considered. This clasification contributes to a recommendation of insecticide use sustainable, taking into account the behavior of these species that are major soybeans pests in South America.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl., This work was supported by “Agencia Estatal de Investigación” from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21), IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S), which financed M.F. with Severo Ochoa funds. Juliette Bedrossiantz was supported by a PhD grant (PRE2018-083513) co-financied by the Spanish Government and the European Social Fund (ESF). This work was supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF-FEDER European Fund (projects CTQ2017-89222-R and PID2020-120499RB-I00) and by the Catalan Government (2017 SGR 1604 and 2017-SGR-1807) to AL. XR research was financed by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-74132-JIN). We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor and Dr. Karen Martinez (University of Copenhagen, Copenhagen, Denmark) for providing the HEK 293 SNAP-β1AR). The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

Although learning second language phonology is a difficult task, orthographic input may support the learning of difficult sound contrasts through a process known as orthographic facilitation. We extended this research by examining the effects of orthographic input together with individual differences in three different phonological learning processes, namely, the production of, perception of, and memorization of words containing three Marathi phonemic contrasts (i.e., [k-kh], [], and []) by native English speakers. Moreover, because the [] and [] contrasts were particularly challenging in previous auditory training studies (e.g., Polka, 1991), we used cross-modal training in order to enhance learning by pairing auditory perception tasks with visual orthographic information, the amplification of relevant acoustic cues, and proprioceptive descriptions to the articulation of target phonemes. Results showed significant learning from the pre- to the posttest across tasks and contrasts, supporting the effectiveness of cross-modal training. Furthermore, incongruent orthographic input could inhibit perception, and orthographic input generally supported memory for word pronunciations. Moreover, individual differences regarding phonological skills and nonspeech auditory discrimination predicted participants’ success in different phonological learning processes. These results provide a detailed picture of the complexity between different aspects of second language phonological learning and cross-modal training.

Catecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β

SummaryCatecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β1- and β2-AR subtypes are expressed in cardiomyocytes, drugs selectively targeting β1-AR have proven this receptor as the main target for the therapeutic effects of beta blockers in heart. Here, we report a new strategy for the spatiotemporal control of β1-AR activation by means of light-regulated drugs with a high level of β1-/β2-AR selectivity. All reported molecules allow for an efficient real time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on alive zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target β1-AR in native environments using light.

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues. Ligand directed approaches, may offer a solution to this problem. Such approaches consist in molecular probes that include a selective ligand moiety and a reactive moiety. Upon ligand binding, the labelling is directed to a nucleophilic amino acid in the vicinity of the binding pocket. However, this requires the use of non-nucleophilic ligand moieties, which is particularly difficult since many GPCR ligands contain amines or other nucleophilic functional groups. In the present work, we developed an innovative ligand-directed toolbox based on a novel approach. The later uses molecular modules to build fluorescent ligand-directed probes to label an archetypical aminergic GPCR (D1R). Our molecular probes can be readily prepared before the labelling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific chemical reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labelling reaction occurs. Subsequently, the ligand will unbind the GPCR pocket, leaving the receptor fluorescently labelled and fully functional. This novel labelling approach allowed us to label endogenous D1 receptor both in transfected cells and primary cultures of neurons and will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution and activity in their native environment.

Positive allosteric modulators (PAMs) for metabotropic glutamate receptors have been postulated to treat neuropsychiatric diseases. Besides, obtaining a reversible and efficient spatiotemporal control of mGlu activity would be therapeutically advantageous. Photopharmacology may provide a solution on this topic, since it is based on the use of light and photoswitchable ligands to modulate a protein activity. This approach offers new perspectives for drug discovery and promises a better drug action control reducing side effects to unattained levels.