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Artificial intelligence is revolutionizing protein structure prediction, providing unprecedented opportunities for drug design. To assess the potential impact on ligand discovery, we compared virtual screens using protein structures generated by the AlphaFold machine learning method and traditional homology modeling. More than 16 million compounds were docked to models of the trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor of unknown structure and target for treating neuropsychiatric disorders. Sets of 30 and 32 highly ranked compounds from the AlphaFold and homology model screens, respectively, were experimentally evaluated. Of these, 25 were TAAR1 agonists with potencies ranging from 12 to 0.03 μM. The AlphaFold screen yielded a more than twofold higher hit rate (60%) than the homology model and discovered the most potent agonists. A TAAR1 agonist with a promising selectivity profile and drug-like properties showed physiological and antipsychotic-like effects in wild-type but not in TAAR1 knockout mice. These results demonstrate that AlphaFold structures can accelerate drug discovery.

Structure-based virtual screening aims to find molecules forming favorable interactions with a biological macromolecule using computational models of complexes. The recent surge of commercially available chemical space provides the opportunity to search for ligands of therapeutic targets among billions of compounds. This review offers a compact overview of structure-based virtual screens of vast chemical spaces, highlighting successful applications in early drug discovery for therapeutically important targets such as G protein-coupled receptors and viral enzymes. Emphasis is placed on strategies to explore ultra-large chemical libraries and synergies with emerging machine learning techniques. The current opportunities and future challenges of virtual screening are discussed, indicating that this approach will play an important role in the next-generation drug discovery pipeline., Competing Interests: Declaration of competing interest J.C. is a founder of DareMe Drug Discovery Consulting., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A set of 2-aryl-9-H or methyl-6-morpholinopurine derivatives were synthesized and assayed through radioligand binding tests at human A 1 , A 2A , A 2B , and A 3 adenosine receptor subtypes. Eleven purines showed potent antagonism at A 1 , A 3 , dual A 1 /A 2A , A 1 /A 2B , or A 1 /A 3 adenosine receptors. Additionally, three compounds showed high affinity without selectivity for any specific adenosine receptor. The structure-activity relationships were made for this group of new compounds. The 9-methylpurine derivatives were generally less potent but more selective, and the 9H-purine derivatives were more potent but less selective. These compounds can be an important source of new biochemical tools and/or pharmacological drugs.

Three Odontaspis ferox (confirmed by mtDNA barcoding) were found in the English Channel and Celtic Sea in 2023 at Lepe, UK (50.7846, -1.3508), Kilmore Quay, Ireland (52.1714, -6.5937), and Lyme Bay, UK (50.6448, -2.9302). These are the first records of O. ferox in either country, and extend the species' range by over three degrees of latitude, to >52° N. They were ~275 (female), 433 (female), and 293 cm (male) total length, respectively. These continue a series of new records, possibly indicative of a climate change-induced shift in the species' range., (© 2023 Fisheries Society of the British Isles.)

Development of subtype-selective leads is essential in drug discovery campaigns targeting G protein-coupled receptors (GPCRs). Herein, a structure-based virtual screening approach to rationally design subtype-selective ligands was applied to the A 1 and A 2A adenosine receptors (A 1 R and A 2A R). Crystal structures of these closely related subtypes revealed a non-conserved subpocket in the binding sites that could be exploited to identify A 1 R selective ligands. A library of 4.6 million compounds was screened computationally against both receptors using molecular docking and 20 A 1 R selective ligands were predicted. Of these, seven antagonized the A 1 R with micromolar activities and several compounds displayed slight selectivity for this subtype. Twenty-seven analogs of two discovered scaffolds were designed, resulting in antagonists with nanomolar potency and up to 76-fold A 1 R-selectivity. Our results show the potential of structure-based virtual screening to guide discovery and optimization of subtype-selective ligands, which could facilitate the development of safer drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K M, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake., (© 2023. The Author(s).)

G-protein-coupled receptors (GPCRs) play important roles in physiological processes and are modulated by drugs that either activate or block signaling. Rational design of the pharmacological efficacy profiles of GPCR ligands could enable the development of more efficient drugs, but is challenging even if high-resolution receptor structures are available. We performed molecular dynamics simulations of the β 2 adrenergic receptor in active and inactive conformations to assess if binding free energy calculations can predict differences in ligand efficacy for closely related compounds. Previously identified ligands were successfully classified into groups with comparable efficacy profiles based on the calculated shift in ligand affinity upon activation. A series of ligands were then predicted and synthesized, leading to the discovery of partial agonists with nanomolar potencies and novel scaffolds. Our results demonstrate that free energy simulations enable design of ligand efficacy and the same approach can be applied to other GPCR drug targets., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The dopamine D 2 receptor, which belongs to the family of G protein-coupled receptors (GPCR), is an important and well-validated drug target in the field of medicinal chemistry due to its wide distribution, particularly in the central nervous system, and involvement in the pathomechanism of many disorders thereof. Schizophrenia is one of the most frequent diseases associated with disorders in dopaminergic neurotransmission, and in which the D 2 receptor is the main target for the drugs used. In this work, we aimed at discovering new selective D 2 receptor antagonists with potential antipsychotic activity. Twenty-three compounds were synthesized, based on the scaffold represented by the D2AAK2 compound, which was discovered by our group. This compound is an interesting example of a D 2 receptor ligand because of its non-classical binding to this target. Radioligand binding assays and SAR analysis indicated structural modifications of D2AAK2 that are possible to maintain its activity. These findings were further rationalized using molecular modeling. Three active derivatives were identified as D 2 receptor antagonists in cAMP signaling assays, and the selected most active compound 17 was subjected to X-ray studies to investigate its stable conformation in the solid state. Finally, effects of 17 assessed in animal models confirmed its antipsychotic activity in vivo.

Recently determined structures of class C G protein-coupled receptors (GPCRs) revealed the location of allosteric binding sites and opened new opportunities for the discovery of novel modulators. In this work, molecular docking screens for allosteric modulators targeting the metabotropic glutamate receptor 5 (mGlu 5 ) were performed. The mGlu 5 receptor is activated by the main excitatory neurotransmitter of the nervous central system, L-glutamate, and mGlu 5 receptor activity can be allosterically modulated by negative or positive allosteric modulators. The mGlu 5 receptor is a promising target for the treatment of psychiatric and neurodegenerative diseases, and several allosteric modulators of this GPCR have been evaluated in clinical trials. Chemical libraries containing fragment- (1.6 million molecules) and lead-like (4.6 million molecules) compounds were docked to an allosteric binding site of mGlu 5 identified in X-ray crystal structures. Among the top-ranked compounds, 59 fragments and 59 lead-like compounds were selected for experimental evaluation. Of these, four fragment- and seven lead-like compounds were confirmed to bind to the allosteric site with affinities ranging from 0.43 to 8.6 μM, corresponding to a hit rate of 9%. The four compounds with the highest affinities were demonstrated to be negative allosteric modulators of mGlu 5 signaling in functional assays. The results demonstrate that virtual screens of fragment- and lead-like chemical libraries have complementary advantages and illustrate how access to high-resolution structures of GPCRs in complex with allosteric modulators can accelerate lead discovery.

The adenosine A 2A receptor (A 2A R), dopamine D 2 receptor (D 2 R) and metabotropic glutamate receptor type 5 (mGluR 5 ) form A 2A R-D 2 R-mGluR 5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A 2A R protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D 2 R-mGluR 5 heteromeric component of the A 2A R-D 2 R-mGluR 5 complex in vitro and in vivo. The A 2A R and mGluR 5 protomers interact and modulate D 2 R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A 2A R in HEK293T cells co-expressing D 2 R and mGluR 5 resulted in a significant and marked increase in the formation of the D 2 R-mGluR 5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D 2 R (D2R Ki High) values was found upon cotreatment with the mGluR 5 and A 2A R agonists in the cells expressing A 2A R, D 2 R and mGluR 5 with a significant effect observed also with the mGluR 5 agonist alone compared to cells expressing only D 2 R and mGluR 5 . In cells co-expressing A 2A R, D 2 R and mGluR 5 , stimulation of the cells with an mGluR 5 agonist like or D 2 R antagonist fully counteracted the D 2 R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR 5 and D 2 R. In agreement, the mGluR 5 -negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A 2A R knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR 5 and A 2A R in enhancing D 2 R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes., (© 2022. The Author(s).)

Atlantic herring in International Council for Exploration of the Sea (ICES) Divisions 6.a, 7.b-c comprises at least three populations, distinguished by temporal and spatial differences in spawning, which have until recently been managed as two stocks defined by geographical delineators. Outside of spawning the populations form mixed aggregations, which are the subject of acoustic surveys. The inability to distinguish the populations has prevented the development of separate survey indices and separate stock assessments. A panel of 45 single-nucleotide polymorphisms, derived from whole-genome sequencing, were used to genotype 3480 baseline spawning samples (2014-2021). A temporally stable baseline comprising 2316 herring from populations known to inhabit Division 6.a was used to develop a genetic assignment method, with a self-assignment accuracy greater than 90%. The long-term temporal stability of the assignment model was validated by assigning archive (2003-2004) baseline samples (270 individuals) with a high level of accuracy. Assignment of non-baseline samples (1514 individuals) from Divisions 6.a, 7.b-c indicated previously unrecognized levels of mixing of populations outside of the spawning season. The genetic markers and assignment models presented constitute a 'toolbox' that can be used for the assignment of herring caught in mixed survey and commercial catches in Division 6.a into their population of origin with a high level of accuracy., (© 2022 The Authors.)

The virus family Totiviridae had originally been considered to include only viruses which infected fungal and protist hosts, but since 2006 a growing number of viruses found in invertebrates and fish have been shown to cluster phylogenetically within this family. These Totiviridae- like, or toti-like, viruses do not appear to belong within any existing genera of Totiviridae , and whilst a number of new genus names have been suggested, none has yet been universally accepted. Within this growing number of toti-like viruses from animal hosts, there exists emerging viral threats particularly to aquaculture, namely Infectious myonecrosis virus in whiteleg shrimp and Piscine myocarditis virus (PMCV) in Atlantic salmon ( Salmo salar ). PMCV in particular continues to be an issue in salmon aquaculture as a number of questions remain unanswered about how the virus is transmitted and the route of entry into host fish. Using a phylogenetic approach, this study shows how PMCV and the other fish toti-like viruses probably have deeper origins in an arthropod host. Based on this, it is hypothesized that sea lice could be acting as a vector for PMCV, as seen with other RNA viruses in Atlantic salmon aquaculture and in the toti-like Cucurbit yellows-associated virus which is spread by the greenhouse whitefly Trialeurodes vaporariorum .

Bacteria in the Shigella genus remain a major cause of dysentery in sub-Saharan Africa, and annually cause an estimated 600,000 deaths worldwide. Being spread by contaminated food and water, this study highlights how wild caught food, in the form of freshwater catfish, can act as vectors for Shigella flexneri in Southern Kenya. A metatranscriptomic approach was used to identify the presence of Shigella flexneri in the catfish which had been caught for consumption from the Galana river. The use of nanopore sequencing was shown to be a simple and effective method to highlight the presence of Shigella flexneri and could represent a potential new tool in the detection and prevention of this deadly pathogen. Rather than the presence/absence results of more traditional testing methods, the use of metatranscriptomics highlighted how primarily one SOS response gene was being transcribed, suggesting the bacteria may be dormant in the catfish. Additionally, COI sequencing of the vector catfish revealed they likely represent a cryptic species. Morphological assignment suggested the fish were widehead catfish Clarotes laticeps, which range across Africa, but the COI sequences from the Kenyan fish are distinctly different from C. laticeps sequenced in West Africa., (© 2022. The Author(s).)

A long evolution of knowledge of the psychostimulant caffeine led in the 1960s to another purine natural product, adenosine and its A 2A receptor. Adenosine is a short-lived autocrine/paracrine mediator that acts pharmacologically at four different adenosine receptors in a manner opposite to the pan-antagonist caffeine and serves as an endogenous allostatic regulator. Although detrimental in the developing brain, caffeine appears to be cerebroprotective in aging. Moderate caffeine consumption in adults, except in pregnancy, may also provide benefit in pain, diabetes, and kidney and liver disorders. Inhibition of A 2A receptors is one of caffeine's principal effects and we now understand this interaction at the atomic level. The A 2A receptor has become a prototypical example of utilizing high-resolution structures of GPCRs for the rational design of chemically diverse drug molecules. The previous focus on discovery of selective A 2A receptor antagonists for neurodegenerative diseases has expanded to include immunotherapy for cancer, and clinical trials have ensued. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc., (© 2020 The British Pharmacological Society.)

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Fragment-based drug discovery relies on successful optimization of weakly binding ligands for affinity and selectivity. Herein, we explored strategies for structure-based evolution of fragments binding to a G protein-coupled receptor. Molecular dynamics simulations combined with rigorous free energy calculations guided synthesis of nanomolar ligands with up to >1000-fold improvements of binding affinity and close to 40-fold subtype selectivity.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways., (© 2021. The Author(s).)

Structure-based docking screens of large compound libraries have become common in early drug and probe discovery. As computer efficiency has improved and compound libraries have grown, the ability to screen hundreds of millions, and even billions, of compounds has become feasible for modest-sized computer clusters. This allows the rapid and cost-effective exploration and categorization of vast chemical space into a subset enriched with potential hits for a given target. To accomplish this goal at speed, approximations are used that result in undersampling of possible configurations and inaccurate predictions of absolute binding energies. Accordingly, it is important to establish controls, as are common in other fields, to enhance the likelihood of success in spite of these challenges. Here we outline best practices and control docking calculations that help evaluate docking parameters for a given target prior to undertaking a large-scale prospective screen, with exemplification in one particular target, the melatonin receptor, where following this procedure led to direct docking hits with activities in the subnanomolar range. Additional controls are suggested to ensure specific activity for experimentally validated hit compounds. These guidelines should be useful regardless of the docking software used. Docking software described in the outlined protocol (DOCK3.7) is made freely available for academic research to explore new hits for a range of targets., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome and are important therapeutic targets. During the last decade, the number of atomic-resolution structures of GPCRs has increased rapidly, providing insights into drug binding at the molecular level. These breakthroughs have created excitement regarding the potential of using structural information in ligand design and initiated a new era of rational drug discovery for GPCRs. The molecular docking method is now widely applied to model the three-dimensional structures of GPCR-ligand complexes and screen for chemical probes in large compound libraries. In this review article, we first summarize the current structural coverage of the GPCR superfamily and the understanding of receptor-ligand interactions at atomic resolution. We then present the general workflow of structure-based virtual screening and strategies to discover GPCR ligands in chemical libraries. We assess the state of the art of this research field by summarizing prospective applications of virtual screening based on experimental structures. Strategies to identify compounds with specific efficacy and selectivity profiles are discussed, illustrating the opportunities and limitations of the molecular docking method. Our overview shows that structure-based virtual screening can discover novel leads and will be essential in pursuing the next generation of GPCR drugs. SIGNIFICANCE STATEMENT: Extraordinary advances in the structural biology of G protein-coupled receptors have revealed the molecular details of ligand recognition by this large family of therapeutic targets, providing novel avenues for rational drug design. Structure-based docking is an efficient computational approach to identify novel chemical probes from large compound libraries, which has the potential to accelerate the development of drug candidates., (Copyright © 2021 by The Author(s).)

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1,2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Many diseases are polygenic and can only be treated efficiently with drugs that modulate multiple targets. However, rational design of compounds with multi-target profiles is rarely pursued because it is considered too difficult, in particular if the drug must enter the central nervous system. Here, a structure-based strategy to identify dual-target ligands of G-protein-coupled receptors is presented. We use this approach to design compounds that both antagonize the A 2A adenosine receptor and activate the D 2 dopamine receptor, which have excellent potential as antiparkinson drugs. Atomic resolution models of the receptors guided generation of a chemical library with compounds designed to occupy orthosteric and secondary binding pockets in both targets. Structure-based virtual screens identified ten compounds, of which three had affinity for both targets. One of these scaffolds was optimized to nanomolar dual-target activity and showed the predicted pharmacodynamic effect in a rat model of Parkinsonism., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D3 dopamine receptor (D3R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D3R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development., Competing Interests: The authors have declared that no competing interests exist.

Ligand binding stabilizes different G protein-coupled receptor states via a complex allosteric process that is not completely understood. Here, we have derived free energy landscapes describing activation of the β 2 adrenergic receptor bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics simulations. These reveal shifts toward active-like states at the Gprotein-binding site for receptors bound to partial and full agonists, and that the ligands modulate the conformational ensemble of the receptor by tuning protein microswitches. We indeed find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cyclic adenosine monophosphate signaling responses. Dimensionality reduction further reveals the similarity between the unique conformational states induced by different ligands, and examining the output of classifiers highlights two distant hotspots governing agonism on transmembrane helices 5 and 7., Competing Interests: OF, JC No competing interests declared, LD Reviewing editor, eLife, (© 2021, Fleetwood et al.)

Salmon pancreas disease virus, more commonly known as salmonid alphavirus (SAV), is a single-stranded positive sense RNA virus and the causative agent of pancreas disease and sleeping disease in salmonids. In this study, a unique strain of SAV previously isolated from ballan wrasse was subjected to whole genome sequencing using nanopore sequencing. In order to accurately examine the evolutionary history of this strain in comparison to other SAV strains, a partitioned phylogenetic analysis was performed to account for variation in the rate of evolution for both individual genes and codon positions. Partitioning the genome alignments almost doubled the observed branch lengths in the phylogenetic tree when compared to the more common approach of applying one model of substitution across the genome and significantly increased the statistical fit of the best-fitting models of nucleotide substitution. Based on the genomic data, a valid case can be made for the viral strain examined in this study to be considered a new SAV genotype. In addition, this study adds to a growing number of studies in which SAV has been found to infect non-salmonid fish, and as such we have suggested that the viral species name be amended to the more inclusive 'piscine alphavirus'.

Solvent reorganization is a major driving force of protein-ligand association, but the contribution of binding site waters to ligand affinity is poorly understood. We investigated how altered interactions with a water network can influence ligand binding to a receptor. A series of ligands of the A 2A adenosine receptor, which either interacted with or displaced an ordered binding site water, were studied experimentally and by molecular dynamics simulations. An analog of the endogenous ligand that was unable to hydrogen bond to the ordered water lost affinity and this activity cliff was captured by molecular dynamics simulations. Two compounds designed to displace the ordered water from the binding site were then synthesized and evaluated experimentally, leading to the discovery of an A 2A agonist with nanomolar activity. Calculation of the thermodynamic profiles resulting from introducing substituents that interacted with or displaced the ordered water showed that the gain of binding affinity was enthalpy driven. Detailed analysis of the energetics and binding site hydration networks revealed that the enthalpy change was governed by contributions that are commonly neglected in structure-based drug optimization. In particular, simulations suggested that displacement of water from a binding site to the bulk solvent can lead to large energy contributions. Our findings provide insights into the molecular driving forces of protein-ligand binding and strategies for rational drug design., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

Knowledge of the location, physical attributes and impacts of obstacles on river connectivity is a requirement for any mitigating action aimed at restoring the connectivity of a river system. Here, we present a study that recorded the numbers and physical diversity of obstacles in 10 river catchments in Ireland, together with the impact these structures had on overall river connectivity. A total of 372 obstacles were recorded, 3 of these were dams, and the remainder were low-head weirs/sluices, obstacles associated with road or rail crossings of rivers and natural structures. The degree of fragmentation was estimated in each catchment by calculating obstacle density and the Dendritic Connectivity Index (DCI). DCI scores were calculated for 4 native Irish fish species with different life-histories, namely diadromous (Atlantic salmon, sea trout, European eel, sea lamprey) and potamodromous (brown trout). Obstacle density ranged between 1.2 and 0.02 obstacles/km of river. Six of the 10 catchments had at least one obstacle located on the mainstem river at least 5 km from its mouth/confluence. These 6 catchments typically had the lowest connectivity scores for diadromous species and ranged between 0.6 and 44.1 (a fully connected river would receive a maximum score of 100). While there was no significant correlation between obstacle density and the DCI score for diadromous fish, a significant negative correlation was detected between obstacle density and the DCI score for potamodromous brown trout. Here, we highlight the merit of these obstacle assessments and associated challenges for decision-making relating to prioritisation of obstacles for removal or modification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Rational drug design for G protein-coupled receptors (GPCRs) is limited by the small number of available atomic resolution structures. We assessed the use of homology modeling to predict the structures of two therapeutically relevant GPCRs and strategies to improve the performance of virtual screening against modeled binding sites. Homology models of the D2 dopamine (D2R) and serotonin 5-HT2A receptors (5-HT2AR) were generated based on crystal structures of 16 different GPCRs. Comparison of the homology models to D2R and 5-HT2AR crystal structures showed that accurate predictions could be obtained, but not necessarily using the most closely related template. Assessment of virtual screening performance was based on molecular docking of ligands and decoys. The results demonstrated that several templates and multiple models based on each of these must be evaluated to identify the optimal binding site structure. Models based on aminergic GPCRs showed substantial ligand enrichment and there was a trend toward improved virtual screening performance with increasing binding site accuracy. The best models even yielded ligand enrichment comparable to or better than that of the D2R and 5-HT2AR crystal structures. Methods to consider binding site plasticity were explored to further improve predictions. Molecular docking to ensembles of structures did not outperform the best individual binding site models, but could increase the diversity of hits from virtual screens and be advantageous for GPCR targets with few known ligands. Molecular dynamics refinement resulted in moderate improvements of structural accuracy and the virtual screening performance of snapshots was either comparable to or worse than that of the raw homology models. These results provide guidelines for successful application of structure-based ligand discovery using GPCR homology models., Competing Interests: The authors have declared that no competing interests exist.

Swabbing scat has proved to be an effective noninvasive method to collect DNA from mammals in the field. Previously, this method has relied on preservative liquids or freezing to preserve the DNA collected on swabs. In this study, we determine the effectiveness of using silica to simply dry the swab in field as an alternative way to prevent DNA degredation. Four species were included in the study; reticulated giraffe, impala, fringe-eared oryx, and lion. Swabs were taken at multiple time points for giraffe and impala scat samples, with the lion and oryx sampled opportunistically. Mitochondrial DNA was successfully amplified and sequenced from scat swabs from all species; however, effectiveness varied between species, with 81.8% amplification success rate from swabs taken from impala scat compared to 25% amplification success rate in giraffe. This variation in success rate was overcome by taking multiple swabs, thus increasing the probability of a successful amplification. The true merit of this method is in its simplicity and cheapness; no preservative liquids were required to be brought into the field, at no stage in the 2 weeks of field sampling were samples frozen, and no commercial kits were used for DNA extraction., Competing Interests: The authors declare no competing interests., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Siboglinids were sampled from four mud volcanoes in the Gulf of Cádiz (El Cid MV, Bonjardim MV, Al Gacel MV, and Anastasya MV). These invertebrates are characteristic to cold seeps and are known to host chemosynthetic endosymbionts in a dedicated trophosome organ. However, little is known about their tube as a potential niche for other microorganisms. Analyses by scanning and transmission electron microscopy showed dense biofilms on the tube in Al Gacel MV and Anastasya MV specimens by prokaryotic cells. Methanotrophic bacteria were the most abundant forming these biofilms as further supported by 16S rRNA sequence analysis. Furthermore, elemental analyses with electron microscopy and energy-dispersive X-ray spectroscopy point to the mineralization and silicification of the tube, most likely induced by the microbial metabolisms. Bacterial and archaeal 16S rRNA sequence libraries revealed abundant microorganisms related to these siboglinid specimens and certain variations in microbial communities among samples. Thus, the tube remarkably increases the microbial biomass related to the worms and provides an additional microbial niche in deep-sea ecosystems.

Steppe-pastoralist-related ancestry reached Central Europe by at least 2500 BC, whereas Iranian farmer-related ancestry was present in Aegean Europe by at least 1900 BC. However, the spread of these ancestries into the western Mediterranean, where they have contributed to many populations that live today, remains poorly understood. Here, we generated genome-wide ancient-DNA data from the Balearic Islands, Sicily and Sardinia, increasing the number of individuals with reported data from 5 to 66. The oldest individual from the Balearic Islands (~2400 BC) carried ancestry from steppe pastoralists that probably derived from west-to-east migration from Iberia, although two later Balearic individuals had less ancestry from steppe pastoralists. In Sicily, steppe pastoralist ancestry arrived by ~2200 BC, in part from Iberia; Iranian-related ancestry arrived by the mid-second millennium BC, contemporary to its previously documented spread to the Aegean; and there was large-scale population replacement after the Bronze Age. In Sardinia, nearly all ancestry derived from the island's early farmers until the first millennium BC, with the exception of an outlier from the third millennium BC, who had primarily North African ancestry and who-along with an approximately contemporary Iberian-documents widespread Africa-to-Europe gene flow in the Chalcolithic. Major immigration into Sardinia began in the first millennium BC and, at present, no more than 56-62% of Sardinian ancestry is from its first farmers. This value is lower than previous estimates, highlighting that Sardinia, similar to every other region in Europe, has been a stage for major movement and mixtures of people.

Agonist binding to G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signaling pathways. Although high-resolution structures of different receptor states are available, atomistic details of allosteric signaling across the membrane remain elusive. We calculated free energy landscapes of β 2 adrenergic receptor activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which shed new light on how microswitches govern the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of the agonist BI-167107 is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. The connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y-Y motif), which lead to a larger population of active-like states at the G protein binding site. This coupling is augmented by protonation of the strongly conserved Asp79 2.50 . The agonist binding site hence communicates with the intracellular region via a cascade of locally connected microswitches. Characterization of these can be used to understand how ligands stabilize distinct receptor states and contribute to development drugs with specific signaling properties. The developed simulation protocol can likely be transferred to other class A GPCRs.

High-throughput screening has revealed dark chemical matter, a set of drug-like compounds that has never shown bioactivity despite being extensively assayed. If dark molecules are found active at a therapeutic target, their extraordinary selectivity profiles make excellent starting points for drug development. We explored if ligands of therapeutically relevant G-protein-coupled receptors could be discovered by structure-based virtual screening of the dark chemical matter. Molecular docking screens against crystal structures of the A 2A adenosine and the D 4 dopamine receptors were carried out, and 53 top-ranked molecules were evaluated experimentally. Two ligands of each receptor were discovered, and the most potent had sub-micromolar affinities. Analysis of bioactivity data showed that the ligands lacked activity at hundreds of off-targets, including several that are associated with adverse effects. Our results demonstrate that virtual screening provides an efficient means to mine the dark chemical space, which could contribute to development of drugs with improved safety profiles.

Globally, aquaculture products are expected to account for >60% of total seafood produce by 2030. In the European Union, the seafood sector is of considerable economic importance to member states with household spending on seafood produce totalling €54.8 billion in 2016. Within the EU, shellfish aquaculture supports livelihoods and employment in many rural communities throughout the region. Harmful algal blooms pose considerable risk to consumer safety and in turn, stability of the shellfish market. If contaminated produce was to make it to the market the health risk to the public could be considerable, but the damage to the sector through loss of trust in producers would also be significant. Mytilus edulis account for a considerable portion of the aquaculture sector in the Northeast Atlantic. At present, no scientific tool is available to industry regulators, to allow them to trace mussel produce to its source, uphold food safety standards and ensure consumer confidence. The present study uses chemical analysis of shells and soft tissues to classify individual M. edulis to their site of harvest. The use of random forest classification of trace element composition has revealed location specific elemental signatures for all examined sites. This led to the correct classification of 100% of individuals sampled to their respective harvesting locations, including two sites located just 6 km apart within the same bay. The protocol demonstrated here provides the basis for a scientifically driven traceability framework for shellfish produce., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We report the first application of CRISPR-Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR-based techniques to detect a given species from eDNA samples, posing a logistical challenge for on-site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR-Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof-of-concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity., (© 2019 John Wiley & Sons Ltd.)

Piscine myocarditis virus (PMCV) is a double-stranded RNA virus which has been linked to cardiomyopathy syndrome (CMS) in Atlantic salmon (Salmo salar L.). The first recorded outbreak of CMS in Ireland occurred in 2012. Heart tissue samples were collected in the current study from farmed Atlantic salmon from various marine sites around Ireland, and the open reading frames (ORFs) 1 and 3 were amplified and sequenced in order to examine the genetic diversity of PMCV. Results showed PMCV to be largely homogenous in Irish samples, showing little genetic diversity. However, several amino acid positions within both ORF1 and ORF3 showed consistent variations unique to the Irish PMCV strains when compared with previously published Norwegian strains. The phylogeny generated in the present study suggests that PMCV may have been introduced into Ireland in two waves, both coming from the southern part of PMCV's range in Norway. In addition, over three-quarters of the PMCV strains which were sequenced came from fish not exhibiting any clinical signs of CMS, suggesting that either PMCV is evolving to become less virulent in Ireland or Irish Atlantic salmon are developing immunity to the disease., (© 2019 John Wiley & Sons Ltd.)

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD 4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

Many aquatic species of conservation concern exist at low densities and are inherently difficult to detect or monitor using conventional methods. However, the introduction of environmental (e)DNA has recently transformed our ability to detect these species and enables effective deployment of limited conservation resources. Identifying areas for breeding, as well as the ecological distribution of species, is vital to the survival or recovery of a conservation species (i.e., areas of critical habitat). In many species, spawning events are associated with a higher relative abundance of DNA released within an aquatic system (i.e., gametes, skin cells etc.), making this the ideal time to monitor these species using eDNA techniques. This study aims to examine whether a "snapshot" eDNA sampling approach (i.e., samples taken at fixed points in chronological time) could reveal areas of critical habitat including spawning sites for our target species Petromyzon marinus . We utilized a species-specific qPCR assay to monitor spatial and temporal patterns in eDNA concentration within two river catchments in Ireland over three consecutive years. We found that eDNA concentration increased at the onset of observed spawning activity and patterns of concentration increased from downstream to upstream over time, suggesting dispersal into the higher reaches as the spawning season progressed. We found P. marinus to be present upstream of several potential barriers to migration, sometimes in significant numbers. Our results also show that the addition of a lamprey-specific fish pass at an "impassable" weir, although assisting in ascent, did not have any significant impact on eDNA concentration upstream after the pass had been installed. eDNA concentration was also found to be significantly correlated with both the number of fish and the number of nests encountered. The application of snapshot sampling techniques for species monitoring therefore has substantial potential for the management of low-density species in fast-moving aquatic systems.

Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD 5 ) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD 5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD 5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD 5 activated Gα q and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD 5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications, and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1 H [1,2,3]triazole-4-yl-LIGKV) with functional potency higher than that of SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel agonists and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins., Competing Interests: The authors declare no competing financial interest., (Copyright © 2018 American Chemical Society.)

The A 2A adenosine (A 2A R) and D 2 dopamine (D 2 R) receptors form oligomers in the cell membrane and allosteric interactions across the A 2A R-D 2 R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A 2A R-D 2 R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A 2A R-D 2 R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A 2A R-D 2 R receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A 2A R blocked heterodimer interactions and disrupted the allosteric effect of A 2A R activation on D 2 R agonist binding. Protein-protein docking was used to construct a model of the A 2A R-D 2 R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A 2A R-D 2 R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A 2A R-D 2 R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

Antagonistic allosteric A2AR-D2R receptor-receptor interactions in heteroreceptor complexes counteract cocaine self-administration and cocaine seeking in rats as seen in biochemical and behavioral experiments. It was shown that the human A2AR transmembrane five (TM5) was part of the interface of the human A2AR-D2R receptor heteromer. In the current paper, the rat A2AR synthetic TM5 (synthTM5) peptide disrupts the A2AR-D2R heteroreceptor complex in HEK293 cells as shown by the bioluminescence resonance energy transfer method. Rat A2AR synthTM5 peptide, microinjected into the nucleus accumbens, produced a complete counteraction of the inhibitory effects of the A2AR agonist CGS21680 on cocaine self-administration. It was linked to a disappearance of the accumbal A2AR-D2R heteroreceptor complexes and the A2AR agonist induced inhibition of D2R recognition using proximity ligation assay and biochemical binding techniques. However, possible effects of the A2AR synthTM5 peptide on accumbal A2AR-D3R and A2AR-D4R heteroreceptor complexes remain to be excluded. Evidence is provided that accumbal A2AR-D2R-like heteroreceptor complexes with their antagonistic receptor-receptor interactions can be major targets for treatment of cocaine use disorder.

Modulation of multiple biological targets with a single drug can lead to synergistic therapeutic effects and has been demonstrated to be essential for efficient treatment of CNS disorders. However, rational design of compounds that interact with several targets is very challenging. Here, we demonstrate that structure-based virtual screening can guide the discovery of multi-target ligands of unrelated proteins relevant for Parkinson's disease. A library with 5.4 million molecules was docked to crystal structures of the A 2A adenosine receptor (A 2A AR) and monoamine oxidase B (MAO-B). Twenty-four compounds that were among the highest ranked for both binding sites were evaluated experimentally, resulting in the discovery of four dual-target ligands. The most potent compound was an A 2A AR antagonist with nanomolar affinity ( K i = 19 nM) and inhibited MAO-B with an IC 50 of 100 nM. Optimization guided by the predicted binding modes led to the identification of a second potent dual-target scaffold. The two discovered scaffolds were shown to counteract 6-hydroxydopamine-induced neurotoxicity in dopaminergic neuronal-like SH-SY5Y cells. Structure-based screening can hence be used to identify ligands with specific polypharmacological profiles, providing new avenues for drug development against complex diseases.