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β-arrestins are multifunctional intracellular proteins that regulate the desensitization, internalization and signaling of over 800 different G protein-coupled receptors (GPCRs) and interact with a diverse array of cellular partners 1,2 . Beyond the plasma membrane, GPCRs can initiate unique signaling cascades from various subcellular locations, a phenomenon known as "location bias" 3,4 . Here, we investigate how β-arrestins direct location-biased signaling of the angiotensin II type I receptor (AT1R). Using novel bioluminescence resonance energy transfer (BRET) conformational biosensors and extracellular signal-regulated kinase (ERK) activity reporters, we reveal that in response to the endogenous agonist Angiotensin II and the β-arrestin-biased agonist TRV023, β-arrestin 1 and β-arrestin 2 adopt distinct conformations across different subcellular locations, which are intricately linked to differential ERK activation profiles. We also uncover a population of receptor-free catalytically activated β-arrestins in the plasma membrane that exhibits insensitivity to different agonists and promotes ERK activation on the plasma membrane independent of G proteins. These findings deepen our understanding of GPCR signaling complexity and also highlight the nuanced roles of β-arrestins beyond traditional G protein pathways., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Benzosiloxaboroles are an emerging class of medicinal agents possessing promising antimicrobial activity. Herein, the expedient synthesis of two novel thiol-functionalized benzosiloxaboroles 1e and 2e is reported. The presence of the SH group allowed for diverse structural modifications involving the thiol-Michael addition, oxidation, as well as nucleophilic substitution giving rise to a series of 27 new benzosiloxaboroles containing various polar functional groups, e.g. , carbonyl, ester, amide, imide, nitrile, sulfonyl and sulfonamide, and pendant heterocyclic rings. The activity of the obtained compounds against selected bacterial and yeast strains, including multidrug-resistant clinical strains, was investigated. Compounds 6, 12, 20 and 22-24 show high activity against Staphylococcus aureus , including both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains, with MIC values in the range of 1.56-12.5 μg mL -1 , while their cytotoxicity is relatively low. The in vitro assay performed with 2-(phenylsulfonyl)ethylthio derivative 20 revealed that, in contrast to the majority of known antibacterial oxaboroles, the plausible mechanism of antibacterial action, involving inhibition of the leucyl-tRNA synthetase enzyme, is not responsible for the antibacterial activity. Structural bioinformatic analysis involving molecular dynamics simulations provided a possible explanation for this finding., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Lipases are versatile catalysts widely used in industrial biotransformations and laboratory-scale developed reactions with industrial potential. Despite the fact that lipase B from Candida antarctica (CALB) is one of the most widely used lipolytic enzymes, its substrate specificity is still poorly understood. One observed trend is that reactions carried out with carboxylic acids containing a double bond are less efficient on average. Here, we have utilized a combination of in vitro and in silico techniques, to better understand the negative impact of a double bond on CALB-mediated esterification. Then through extensive molecular dynamics (MD) simulations, we were able to map the entry pathway of cinnamic acid and its derivative into the CALB active site, and their interactions with catalytic residues. We observed a 2 step binding mechanism of studied compounds, where they first penetrate the enzyme pocket in a conformation where their carboxylic groups are extended towards the solvent. This is followed by further penetration of the acid into the enzymatic active pocket, and a full rotation within the active site, which orients the acid in a conformation that allows further steps of the esterification reaction. As acids containing a double bond are more rigid, their mobility and thus ability to rotate in the narrow CALB active site is hampered, which provides a structural explanation for the decreased efficiency of such acids. Our data provide insight into the substrate specificity of CALB-mediated esterification, providing important structural guidelines to better understand and potentially improve the efficiency of such reactions., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The G protein-coupled receptor GPR183 is a chemotactic receptor with an important function in the immune system and association with a variety of diseases. It recognizes ligands with diverse physicochemical properties as both the endogenous oxysterol ligand 7α,25-OHC and synthetic molecules can activate the G protein pathway of the receptor. To better understand the ligand promiscuity of GPR183, we utilized both molecular dynamics simulations and cell-based validation experiments. Our work reveals that the receptor possesses two ligand entry channels: one lateral between transmembrane helices 4 and 5 facing the membrane, and one facing the extracellular environment. Using enhanced sampling, we provide a detailed structural model of 7α,25-OHC entry through the lateral membrane channel. Importantly, the first ligand recognition point at the receptor surface has been captured in diverse experimentally solved structures of different GPCRs. The proposed ligand binding pathway is supported by in vitro data employing GPR183 mutants with a sterically blocked lateral entrance, which display diminished binding and signaling. In addition, computer simulations and experimental validation confirm the existence of a polar water channel which might serve as an alternative entrance gate for less lipophilic ligands from the extracellular milieu. Our study reveals knowledge to understand GPR183 functionality and ligand recognition with implications for the development of drugs for this receptor. Beyond, our work provides insights into a general mechanism GPCRs may use to respond to chemically diverse ligands., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D 2 receptor (D 2 R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D 2 R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D 2 R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D 2 R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

Protein-protein interactions (PPIs) are responsible for a number of key physiological processes in the living cells and underlie the pathomechanism of many diseases. Nowadays, along with the concept of so-called "hot spots" in protein-protein interactions, which are well-defined interface regions responsible for most of the binding energy, these interfaces can be targeted with modulators. In order to apply structure-based design techniques to design PPIs modulators, a three-dimensional structure of protein complex has to be available. In this context in silico approaches, in particular protein-protein docking, are a valuable complement to experimental methods for elucidating 3D structure of protein complexes. Protein-protein docking is easy to use and does not require significant computer resources and time (in contrast to molecular dynamics) and it results in 3D structure of a protein complex (in contrast to sequence-based methods of predicting binding interfaces). However, protein-protein docking cannot address all the aspects of protein dynamics, in particular the global conformational changes during protein complex formation. In spite of this fact, protein-protein docking is widely used to model complexes of water-soluble proteins and less commonly to predict structures of transmembrane protein assemblies, including dimers and oligomers of G protein-coupled receptors (GPCRs). In this chapter we review the principles of protein-protein docking, available algorithms and software and discuss the recent examples, benefits, and drawbacks of protein-protein docking application to water-soluble proteins, membrane anchoring and transmembrane proteins, including GPCRs.

Some 5-HT 2B fluorescent probes were obtained by tagging 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (DOI) with a subset of fluorescent amines. Some of the resulting fluorescent ligands showed excellent affinity and selectivity profiles at the 5-HT 2B receptors (e.g. 12b), while retain the agonistic functional behaviour of the model ligand (DOI). The study highlighted the most salient features of the structure-activity relationship in this series and these were substantiated by a molecular modelling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human 5-HT 2B receptor. One of the fluorescent ligands developed in this work, compound 12i, specifically labelled CHO-K1 cells expressing 5-HT 2B receptors and not parental CHO-K1 cells in a concentration-dependent manner. 12i enables imaging and quantification of specific 5-HT 2B receptor labelling in live cells by automated fluorescence microscopy as well as quantification by measurements of fluorescence intensity using a fluorescence plate reader.

All possible twenty-three prototropic tautomers of neutral and redox adenine (nine amine and fourteen imine forms, including geometric isomerism of the exo ═NH group) were examined in vacuo {DFT(B3LYP)/6-311+G(d,p)}. The NH → NH conversions as well as those usually omitted, NH → CH and CH → CH, were considered. An interesting change of the tautomeric preference occurs when proceeding from neutral to reduced adenine. One-electron reduction favors the nonaromatic amine C8H-N10H tautomer. This tautomeric preference is similar to that (C2H) for reduced imidazole. Water molecules (PCM model) seem to not change this trend. They influence solely the relative energies. The DFT vertical detachment energy in the gas phase is positive for each tautomer, e.g., 0.03 eV for N9H-N10H and 1.84 eV for C8H-N10H. The DFT adiabatic electron affinity for the favored process, neutral N9H-N10H → reduced C8H-N10H (ground states), is equal to 0.18 eV at 0 K (ZPE included). One-electron oxidation does not change the tautomeric preference in the gas phase. The aromatic amine N9H-N10H tautomer is favored for the oxidized molecule similarly as for the neutral one. The DFT adiabatic ionization potential for the favored process, neutral N9H-N10H → oxidized N9H-N10H (ground states), is equal to 8.12 eV at 0 K (ZPE included). Water molecules (PCM model) seem to influence solely the composition of the tautomeric mixture and the relative energies. They change the energies of the oxidation and reduction processes by ca. 2 eV.

Quantum-chemical calculations {DFT(B3LYP)/6-311+G(d,p)} were performed for all possible tautomers (aromatic and nonaromatic) of neutral 2- and 4-aminopyridines and their oxidized and reduced forms. One-electron oxidation has no important effect on the tautomeric preference for 2-aminopyridine. The amine tautomer is favored. However, oxidation increases the stability of the imine NH tautomer, and its contribution in the tautomeric mixture cannot be neglected. In the case of 4-aminopyridine, one-electron oxidation increases the stability of both the amine and imine NH tautomers. Consequently, they possess very close energies. As major tautomers, they dictate the composition of the tautomeric mixture. The CH tautomers may be considered as very rare forms for both neutral and oxidized aminopyridines. A reverse situation takes place for the reduced forms of aminopyridines. One-electron reduction favors the C3 atom for the labile proton for both aminopyridines. This may partially explain the origin of the CH tautomers for the anionic states of nucleobases containing the exo NH(2) group.

The consequences of one-electron oxidation and one-electron reduction were studied for 4-aminopyrimidine (4APM), which displays prototropic tautomerism. Since experimental techniques are incapable of detecting less than 0.1% of minor tautomers, quantum-chemical calculations [DFT(B3LYP)/6-311+G(d,p)] were carried out for all possible tautomers of neutral 4AMP and its redox forms, 4APM (+ •) and 4APM (- •). Four tautomers were considered: one amine and three imine tautomers (two NH and one CH form). Geometric isomerism of the exo = NH group was also taken into account. One-electron oxidation (4APM - e → 4APM  (+ •)) has no significant effect on the tautomeric preferences; it influences solely the composition of the tautomeric mixture. The amine tautomer is favored for both 4APM  (+ •) and 4APM. An interesting change in the tautomeric preference occurs for 4APM (- •). One-electron reduction (4APM + e → 4APM (- •)) favors the C5 atom for the labile proton. The preference of the imine CH tautomer in the tautomeric mixture of 4APM (- •) may partially explain the origin of CH tautomers in nucleobases.

Quantum-chemical calculations were performed for all possible isomers of neutral aniline and its redox forms, and intramolecular proton-transfer (prototropy) accompanied by π-electron delocalization was analyzed. One-electron oxidation (PhNH(2) - e → [PhNH(2)](+•)) has no important effect on tautomeric preferences. The enamine tautomer is preferred for oxidized aniline similarly as for the neutral molecule. Dramatical changes take place when proceeding from neutral to reduced aniline. One-electron reduction (PhNH(2) + e → [PhNH(2)](-•)) favors the imine tautomer. Independently on the state of oxidation, π- and n-electrons are more delocalized for the enamine than imine tautomers. The change of the tautomeric preferences for reduced aniline may partially explain the origin of the CH tautomers for reduced nucleobases (cytosine, adenine, and guanine).