Entrectinib—A SARS-CoV-2 Inhibitor in Human Lung Tissue (HLT) Cells

COVID-19; Drug repurposing; Viral cell entry assaysCOVID-19; Reutilización de medicamentos; Ensayos de entrada de células viralesCOVID-19; Reutilització de medicaments; Assajos d'entrada de cèl·lules viralsSince the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.This research was funded by the Spanish Ministry of Science, Innovation, and Universities (FPU16/01209 to M.T.-F.); the Health department of the Government of Catalonia (DGRIS 3_9 to A.P.G. and J.S. and DGRIS 1_5 to M.J.B. and M.G.)

Unraveling the complex signaling behavior of neurotransmitters and their receptors in the brain

G protein-coupled receptors (GPCRs) are the main acceptors of neurotransmitters, and thus play an important role in communication between neurons. Because of this they are an attractive drug target for multiple neurodegenerative and neuropsychiatric disorders. The primary function of GPCRs is to initiate diverse intracellular signaling cascades in response to extracellular events, like neurotransmitter binding. Despite the wealth of available biochemical data, the structural foundations of GPCR activity remain poorly understood. Such insights would not only expand our knowledge of those receptors, but also facilitate the design of safer and more efficient drugs. Here, using several computational approaches, we propose structural mechanisms that explain GPCR in vitro data. We unravel features GPCR functionality at multiple levels of action including ligandreceptor interactions, allosteric signal transmission as well as posttranslational modifications. Our results identify phenomena potentially conserved among GPCRs that advance our understanding of this relevant receptor family.Los receptores acoplados a proteína G (en inglés GPCRs) son los principales receptores de neurotransmisores, teniendo un papel importante en la comunicación neuronal. Por esto, se los considera una atractiva diana farmacológica en múltiples trastornos neurodegenerativos y neuropsiquiátricos. El rol primario de los GPCRs es iniciar múltiples cascadas de señalización intracelular en respuesta a eventos extracelulares. A pesar de la vasta información bioquímica disponible, los fundamentos estructurales de la actividad de GPCRs no se comprenden totalmente. Estos fundamentos, no sólo podrían expandir nuestro conocimiento de los receptores, sino que podrían facilitar el diseño de farmacos más seguros y eficaces. Utilizando varios enfoques computacionales, proponemos mecanismos estructurales que explican los resultados in vitro de los GPCRs. Desciframos elementos de la funcionalidad de GPCRs en múltiples niveles, incluyendo interacciones ligando-receptor, transmisión de señales alostérica y modificaciones post-traduccionales. Nuestros resultados identifican fenómenos potencialmente conservados entre los GPCRs, ampliando nuestro conocimiento de esta relevante familia de receptores

Unraveling the complex signaling behavior of neurotransmitters and their receptors in the brain

G protein-coupled receptors (GPCRs) are the main acceptors of neurotransmitters, and thus play an important role in communication between neurons. Because of this they are an attractive drug target for multiple neurodegenerative and neuropsychiatric disorders. The primary function of GPCRs is to initiate diverse intracellular signaling cascades in response to extracellular events, like neurotransmitter binding. Despite the wealth of available biochemical data, the structural foundations of GPCR activity remain poorly understood. Such insights would not only expand our knowledge of those receptors, but also facilitate the design of safer and more efficient drugs. Here, using several computational approaches, we propose structural mechanisms that explain GPCR in vitro data. We unravel features GPCR functionality at multiple levels of action including ligandreceptor interactions, allosteric signal transmission as well as posttranslational modifications. Our results identify phenomena potentially conserved among GPCRs that advance our understanding of this relevant receptor family.Los receptores acoplados a proteína G (en inglés GPCRs) son los principales receptores de neurotransmisores, teniendo un papel importante en la comunicación neuronal. Por esto, se los considera una atractiva diana farmacológica en múltiples trastornos neurodegenerativos y neuropsiquiátricos. El rol primario de los GPCRs es iniciar múltiples cascadas de señalización intracelular en respuesta a eventos extracelulares. A pesar de la vasta información bioquímica disponible, los fundamentos estructurales de la actividad de GPCRs no se comprenden totalmente. Estos fundamentos, no sólo podrían expandir nuestro conocimiento de los receptores, sino que podrían facilitar el diseño de farmacos más seguros y eficaces. Utilizando varios enfoques computacionales, proponemos mecanismos estructurales que explican los resultados in vitro de los GPCRs. Desciframos elementos de la funcionalidad de GPCRs en múltiples niveles, incluyendo interacciones ligando-receptor, transmisión de señales alostérica y modificaciones post-traduccionales. Nuestros resultados identifican fenómenos potencialmente conservados entre los GPCRs, ampliando nuestro conocimiento de esta relevante familia de receptores

Dopamine D 2 receptor agonist binding kinetics-role of a conserved serine residue

The forward (kon) and reverse (koff) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D2R agonist candidate drugs

A molecular sensor for cholesterol in the human serotonin 1A receptor

The function of several G protein-coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs

Mechanistic insights into dopaminergic and serotonergic neurotransmission - concerted interactions with helices 5 and 6 drive the functional outcome

Brain functions rely on neurotransmitters that mediate communication between billions of neurons. Disruption of this communication can result in a plethora of psychiatric and neurological disorders. In this work, we combine molecular dynamics simulations, live-cell biosensor and electrophysiological assays to investigate the action of the neurotransmitter dopamine at the dopaminergic D2 receptor (D2R). The study of dopamine and closely related chemical probes reveals how neurotransmitter binding translates into the activation of distinct subsets of D2R effectors (i.e.: Gi2, GoB, Gz and β-arrestin 2). Ligand interactions with key residues in TM5 (S5.42) and TM6 (H6.55) in the D2R binding pocket yield a dopamine-like coupling signature, whereas exclusive TM5 interaction is typically linked to preferential G protein coupling (in particular GoB) over β-arrestin. Further experiments for serotonin receptors indicate that the reported molecular mechanism is shared by other monoaminergic neurotransmitter receptors. Ultimately, our study highlights how sequence variation in position 6.55 is used by nature to fine-tune β-arrestin recruitment and in turn receptor signaling and internalization of neurotransmitter receptors

How do molecular dynamics data complement static structural data of GPCRs

G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling partners. However, GPCR functionality is largely determined by their flexibility and ability to transition between distinct structural conformations. Due to this dynamic nature, a static snapshot does not fully explain the complexity of GPCR signal transduction. Molecular dynamics (MD) simulations offer the opportunity to simulate the structural motions of biological processes at atomic resolution. Thus, this technique can incorporate the missing information on protein flexibility into experimentally solved structures. Here, we review the contribution of MD simulations to complement static structural data and to improve our understanding of GPCR physiology and pharmacology, as well as the challenges that still need to be overcome to reach the full potential of this technique

Oxazoline scaffold in synthesis of benzosiloxaboroles and related ring-expanded heterocycles: diverse reactivity, structural peculiarities and antimicrobial activity

Two isomeric benzosiloxaborole derivatives 3a and 5a bearing fluorine and 4,4-dimethyl-2-oxazolin-2-yl substituents attached to the aromatic rings were obtained. Both compounds were prone to hydrolytic cleavage of the oxazoline ring after initial protonation or methylation of the nitrogen atom. The derivative 3c featuring N-methylammoniumalkyl ester functionality was successfully subjected to N-sulfonylation and N-acylation reactions to give respective derivatives which demonstrates its potential for modular synthesis of structurally extended benzosiloxaboroles. Compound 5c bearing N-ammoniumalkyl ester underwent conversion to a unique macrocyclic dimer due to siloxaborole ring opening. Furthermore, an unexpected 4-electron reduction of the oxazoline ring occurred during an attempted synthesis of 5a. The reaction gave rise to an unprecedented 7-membered heterocyclic system 4a comprising a relatively stable B-O-B-O-Si linkage and stabilized by an intramolecular N-B coordination. It could be cleaved to derivative 4c bearing BOH and SiMe2OH groups which acts as a pseudo-diol as demonstrated by formation of an adduct with Tavaborole. Apart from the multinuclear NMR spectroscopy characterization, crystal structures of the obtained products were determined in many cases by X-ray diffraction. Investigation of biological activity of the obtained compounds revealed that derivatives 3e and 3f with pendant N-methyl arylsulfonamide groups exhibit high activity against Gram-positive cocci such as methicillin-sensitive Staphylococcus aureus ATCC 6538P, methicillin-resistant S. aureus (MRSA) ATCC 43300 as well as the MRSA clinical strains, with MIC values in the range of 3.12-6.25 mg L-1. These two compounds also showed activity against Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 6057 (with MICs of 25-50 mg L-1). The results of the antimicrobial activity and cytotoxicity studies indicate that 3e and 3f can be considered as potential antibacterial agents, especially against S. aureus MRSA.This research was financially supported by National Science Centre (Poland) in the framework of the project UMO-2018/31/B/ST5/00210. Work implemented as a part of Operational Project Knowledge Education Development 2014–2020 co-financed by the European Social Fund (the TRIBIOCHEM interdisciplinary PhD programme for P.H.M.-U.). The authors thank Wroclaw Centre for Networking and Supercomputing (http://www.wcss.pl), grant no. 285, for providing computer facilities (Gaussian16). The molecular docking studies were performed under the Project HPC-EUROPA3 (INFRAIA-2016-1-730897), with the support of the EC Research Innovation Action under the H2020 Programme; in particular, the authors gratefully acknowledge the support by Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University (Barcelona, Spain) and the computer resources and technical support provided by Barcelona Supercomputing Center (BSC); the authors thank Dr Jana Selent for her assistance in this matter. The work was supported by the Warsaw University of Technology

Ligand entry pathways control the chemical space recognized by GPR183

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