Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and are implicated in various neuropsychological conditions, ranging from addiction to psychosis 1 . GABAB belongs to G protein-coupled receptor class C, and its functional entity consists of an obligate heterodimer composed of GB1 and GB2 2 .Each subunit possesses an extracellular Venus flytrap domain, connected to a canonical seventransmembrane domain. Here, we present four cryo-EM structures of the human full-length GB1-GB2 heterodimer in its inactive apo, two intermediate agonist-bound, and active agonist/positive allosteric modulator bound forms. The structures reveal startling differences, shedding light onto the complex motions underlying the unique activation mechanism of GABAB. Our results show that agonist binding in the GB1 Venus flytrap domain triggers a series of transitions, first bringing the two transmembrane domains into contact and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism, potentiated by a positive allosteric modulator binding at the dimerization interface, to initiate downstream signaling.

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Metabotropic γ-aminobutyric acid G protein–coupled receptors (GABAB) represent one of the two main types of inhibitory neurotransmitter receptors in the brain. These receptors act both pre- and postsynaptically by modulating the transmission of neuronal signals and are involved in a range of neurological diseases, from alcohol addiction to epilepsy. A series of recent cryo-EM studies revealed critical details of the activation mechanism of GABAB. Structures are now available for the receptor bound to ligands with different modes of action, including antagonists, agonists, and positive allosteric modulators, and captured in different conformational states from the inactive apo to the fully active state bound to a G protein. These discoveries provide comprehensive insights into the activation of the GABAB receptor, which not only broaden our understanding of its structure, pharmacology, and physiological effects but also will ultimately facilitate the discovery of new therapeutic drugs and neuromodulators.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT 2 R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT 2 R. We repurposed EMA401, an AT 2 R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT 2 R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT 2 R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or β-arrestin recruitment. The architecture and interactions of EMA401 in AT 2 R differ drastically from complexes of AT 2 R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2–tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

Mutating the side‐chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half‐life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6‐Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4‐disubstituted 1,2,3‐triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2R/AT1R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross‐peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

25 The potential of partial least squares regression (PLSR) and multivariate curve resolution 26 alternating least squares (MCR-ALS) is evaluated for simultaneous determination of 27 diclofenac (DCF), naproxen (NAP), mefenamic acid (MEF) and carbamazepine (CBZ) 28 as target analytes and gemfibrozil (GEM) as interference in synthetic and real 29 environmental samples. The analysis of first-order UV-Vis spectra is performed using 30 PLSR with different variable selection methods including variable importance in 31 projection (VIP), recursive partial least squares (rPLS), regression coefficient (RV) and 32 uninformative variable elimination (UVE) and using MCR-ALS with correlation 33 constraint (MCR-ALS-CC). The obtained statistical parameters in terms of relative error 34 (RE), regression coefficient (R 2 ) and root mean square error (RMSE) were satisfactory 35 for calibration and validation sets. Furthermore, in real environmental samples, the 36 obtained statistical parameters of PLSR using VIP and rPLS and also MCR-ALS-CC 37 were reasonable by considering the heavy overlap of target analytes and complex 38 samples matrices. In general, PLSR showed better performance for determination of 39 analytes in samples which are free of interference or contains calibrated interference(s). 40On the other hand, MCR-ALS-CC allowed for the accurate determination of analytes in 41 the presence of unknown interference and more complex sample matrices. 42

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