Publications by authors named "Leonardo Pardo"

96 Publications

Structure and function of adenosine receptor heteromers.

Cell Mol Life Sci 2021 Feb 12. Epub 2021 Feb 12.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Campus Universitari, 08193, Bellaterra (Barcelona), Spain.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A, A, A, and A, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A and A, A and A, and A and A receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-021-03761-6DOI Listing
February 2021

Discovery of a macromolecular complex mediating the hunger suppressive actions of cocaine: Structural and functional properties.

Addict Biol 2021 Feb 8:e13017. Epub 2021 Feb 8.

Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Barcelona, Spain.

Cocaine not only increases brain dopamine levels but also activates the sigma receptor (σ R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D (D R), ghrelin (GHS-R ), and σ receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ R, D R, and GHS-R is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ R, as confirmed using samples from σ R mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ R, D R, and GHS-R complex constitutes a functional unit that is altered upon cocaine binding to the σ R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/adb.13017DOI Listing
February 2021

The mutational landscape of human olfactory G protein-coupled receptors.

BMC Biol 2021 Feb 5;19(1):21. Epub 2021 Feb 5.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Spain.

Background: Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain.

Results: OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset.

Conclusion: We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-021-00962-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866472PMC
February 2021

Experimental and computational analysis of biased agonism on full-length and a C-terminally truncated adenosine A receptor.

Comput Struct Biotechnol J 2020 24;18:2723-2732. Epub 2020 Sep 24.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. Instituto de Salud Carlos III, Madrid, Spain.

Biased agonism, the ability of agonists to differentially activate downstream signaling pathways by stabilizing specific receptor conformations, is a key issue for G protein-coupled receptor (GPCR) signaling. The C-terminal domain might influence this functional selectivity of GPCRs as it engages G proteins, GPCR kinases, β-arrestins, and several other proteins. Thus, the aim of this paper is to compare the agonist-dependent selectivity for intracellular pathways in a heterologous system expressing the full-length (AR) and a C-tail truncated (AR lacking the last 40 amino acids) adenosine A receptor, a GPCR that is already targeted in Parkinson's disease using a first-in-class drug. Experimental data such as ligand binding, cAMP production, β-arrestin recruitment, ERK1/2 phosphorylation and dynamic mass redistribution assays, which correspond to different aspects of signal transduction, were measured upon the action of structurally diverse compounds (the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and the SCH-58261 antagonist) in cells expressing AR and AR. The results show that taking cAMP levels and the endogenous adenosine agonist as references, the main difference in bias was obtained with PSB-0777 and LUF-5834. The C-terminus is dispensable for both G-protein and β-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the μs timescale, were used to understand the structural arrangements of the binding cavity, triggered by these chemically different agonists, facilitating G protein binding with different efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.csbj.2020.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550916PMC
September 2020

Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB Receptor*.

Chemistry 2020 Dec 9;26(68):15839-15842. Epub 2020 Nov 9.

Medicinal Chemistry Institute, Spanish Research Council, Madrid, Spain.

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB receptor. These compounds selectively target CB versus CB receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202003389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756656PMC
December 2020

Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity.

Cell Metab 2020 Jul 25;32(1):56-70.e7. Epub 2020 Jun 25.

Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, 53127 Bonn, Germany. Electronic address:

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437516PMC
July 2020

The computational modeling of allosteric modulation of metabotropic glutamate receptors.

Adv Pharmacol 2020 20;88:1-33. Epub 2020 Mar 20.

Computational Chemistry, Janssen Research & Development, Janssen Pharmaceutica N. V., Beerse, Belgium. Electronic address:

Allosteric modulation of GPCRs, especially metabotropic glutamate (mGlu) receptors, has become an important strategy for drug discovery. Positive and negative allosteric modulators (PAM, NAM) are widely reported for the mGlu receptor family with leads mostly originating by high-throughput screening followed by iterative medicinal chemistry. The progression of the field from mutagenesis and homology modeling to elaborate structure-enabled drug discovery is described. We detail how computational methods have delivered new chemical matter and revealed the functional details of PAM and NAM activity. The breakthrough in mGlu receptor 7-transmembrane (7TM) crystal structures enabled recent combined modeling and experimental studies to confirm common binding sites, interactions and the origins of ligand effect on functional activity. Focusing on allosteric modulation of the mGlu and mGlu receptors, similarities are seen that still accommodate the known differences in binding sites and SAR. This work reveals the promise of a methodical computational approach built upon deep analysis of 7TM receptor simulations and interpretation of results in the context of our current understanding of receptor function. A crucial aspect was the close collaboration between modeling and experiment necessary to build and interrogate the hypotheses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.apha.2020.02.004DOI Listing
September 2020

Altered Signaling in CB1R-5-HT2AR Heteromers in Olfactory Neuroepithelium Cells of Schizophrenia Patients is Modulated by Cannabis Use.

Schizophr Bull 2020 12;46(6):1547-1557

Integrative Pharmacology and Systems Neuroscience, IMIM-Hospital del Mar Research Institute, Barcelona, Spain.

Schizophrenia (SCZ) has been associated with serotonergic and endocannabinoid systems dysregulation, but difficulty in obtaining in vivo neurological tissue has limited its exploration. We investigated CB1R-5-HT2AR heteromer expression and functionality via intracellular pERK and cAMP quantification in olfactory neuroepithelium (ON) cells of SCZ patients non-cannabis users (SCZ/nc), and evaluated whether cannabis modulated these parameters in patients using cannabis (SCZ/c). Results were compared vs healthy controls non-cannabis users (HC/nc) and healthy controls cannabis users (HC/c). Further, antipsychotic effects on heteromer signaling were tested in vitro in HC/nc and HC/c. Results indicated that heteromer expression was enhanced in both SCZ groups vs HC/nc. Additionally, pooling all 4 groups together, heteromer expression correlated with worse attentional performance and more neurological soft signs (NSS), indicating that these changes may be useful markers for neurocognitive impairment. Remarkably, the previously reported signaling properties of CB1R-5-HT2AR heteromers in ON cells were absent, specifically in SCZ/nc treated with clozapine. These findings were mimicked in cells from HC/nc exposed to clozapine, suggesting a major role of this antipsychotic in altering the quaternary structure of the CB1R-5-HT2AR heteromer in SCZ/nc patients. In contrast, cells from SCZ/c showed enhanced heteromer functionality similar to HC/c. Our data highlight a molecular marker of the interaction between antipsychotic medication and cannabis use in SCZ with relevance for future studies evaluating its association with specific neuropsychiatric alterations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/schbul/sbaa038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846100PMC
December 2020

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BMC Biol 2020 01 23;18(1). Epub 2020 Jan 23.

Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, 21224, USA.

Background: It has been hypothesized that heteromers of adenosine A receptors (A2AR) and cannabinoid CB receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals.

Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A receptors or dopamine D receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level.

Conclusions: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-020-0739-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979073PMC
January 2020

Oligomerization of G protein-coupled receptors: Still doubted?

Prog Mol Biol Transl Sci 2020 12;169:297-321. Epub 2019 Dec 12.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.

Experimental data accumulated during the last 10 years strongly support the existence and functional significance of oligomerization of G protein-coupled receptors (GPCRs). In this essay, we discuss the unique biochemical properties of GPCR oligomers in the frame of "allosterism" and how these properties can be used to identify GPCR oligomers in artificial systems and in native tissues. We also address how the significant advances in biophysical, computational and crystallization techniques have provided significant structural insights about the mechanisms behind allosterism in GPCRs, giving distinct clues about the mechanisms of allosteric interactions in GPCR homomers and heteromers. Finally, we elaborate on the emerging picture of the role of GPCR oligomers as components of pre-coupled macromolecular complexes that include different G proteins and effectors, such as adenylyl cyclase. Allosteric properties of GPCR oligomers can therefore extend to ligand interactions through the different components of the macromolecular complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.pmbts.2019.11.006DOI Listing
December 2020

Inhibition of the Alanine-Serine-Cysteine-1 Transporter by BMS-466442.

ACS Chem Neurosci 2019 05 14;10(5):2510-2517. Epub 2019 Mar 14.

Laboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina , Universitat Autonoma de Barcelona , 08193 , Bellaterra , Spain.

Experiment and modeling were combined to understand inhibition of the alanine-serine-cysteine-1 (asc1) transporter. The structure-activity relationship (SAR) was explored with synthesis of analogues of BMS-466442. Direct target interaction and binding site location between TM helices 6 and 10 were confirmed via site directed mutagenesis. Computational modeling suggested the inhibitor binds via competitive occupation of the orthosteric site while also blocking the movement of TM helices that are required for transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschemneuro.9b00019DOI Listing
May 2019

Mechanisms Underlying Allosteric Molecular Switches of Metabotropic Glutamate Receptor 5.

J Chem Inf Model 2019 05 12;59(5):2456-2466. Epub 2019 Mar 12.

Laboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina , Universitat Autonoma de Barcelona , 08193 Bellaterra , Spain.

The metabotropic glutamate 5 (mGlu) receptor is a class C G protein-coupled receptor (GPCR) that is implicated in several CNS disorders making it a popular drug discovery target. Years of research have revealed allosteric mGlu ligands showing an unexpected complete switch in functional activity despite only small changes in their chemical structure, resulting in positive allosteric modulators (PAM) or negative allosteric modulators (NAM) for the same scaffold. Up to now, the origins of this effect are not understood, causing difficulties in a drug discovery context. In this work, experimental data was gathered and analyzed alongside docking and Molecular Dynamics (MD) calculations for three sets of PAM and NAM pairs. The results consistently show the role of specific interactions formed between ligand substituents and amino acid side chains that block or promote local movements associated with receptor activation. The work provides an explanation for how such small structural changes lead to remarkable differences in functional activity. While this work can greatly help drug discovery programs avoid these switches, it also provides valuable insight into the mechanisms of class C GPCR allosteric activation. Furthermore, the approach shows the value of applying MD to understand functional activity in drug design programs, even for such close structural analogues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jcim.8b00924DOI Listing
May 2019

Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer.

J Med Chem 2018 10 11;61(20):9335-9346. Epub 2018 Oct 11.

Biomaterials and Nanomedicine , Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , Barcelona Science Park , 08028 Barcelona , Spain.

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity ( K = 21 pM) for the dopamine D receptor (DR) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of DR, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the DR homodimer through TM6 ex vivo. In conclusion, by using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the DR homodimer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01249DOI Listing
October 2018

Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors.

Mol Neurobiol 2019 Feb 7;56(2):1196-1210. Epub 2018 Jun 7.

Centro de Investigación en Red, Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain.

Despite ancient knowledge on cocaine appetite-suppressant action, the molecular basis of such fact remains unknown. Addiction/eating disorders (e.g., binge eating, anorexia, bulimia) share a central control involving reward circuits. However, we here show that the sigma-1 receptor (σR) mediates cocaine anorectic effects by interacting in neurons with growth/hormone/secretagogue (ghrelin) receptors. Cocaine increases colocalization of σR and GHS-R1a at the cell surface. Moreover, in transfected HEK-293T and neuroblastoma SH-SY5Y cells, and in primary neuronal cultures, pretreatment with cocaine or a σR agonist inhibited ghrelin-mediated signaling, in a similar manner as the GHS-R1a antagonist YIL-781. Results were similar in G protein-dependent (cAMP accumulation and calcium release) and in partly dependent or independent (ERK1/2 phosphorylation and label-free) assays. We provide solid evidence for direct interaction between receptors and the functional consequences, as well as a reliable structural model of the macromolecular σR-GHS-R1a complex, which arises as a key piece in the puzzle of the events linking cocaine consumption and appetitive/consummatory behaviors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-018-1140-7DOI Listing
February 2019

The size matters? A computational tool to design bivalent ligands.

Bioinformatics 2018 11;34(22):3857-3863

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.

Motivation: Bivalent ligands are increasingly important such as for targeting G protein-coupled receptor (GPCR) dimers or proteolysis targeting chimeras (PROTACs). They contain two pharmacophoric units that simultaneously bind in their corresponding binding sites, connected with a spacer chain. Here, we report a molecular modelling tool that links the pharmacophore units via the shortest pathway along the receptors van der Waals surface and then scores the solutions providing prioritization for the design of new bivalent ligands.

Results: Bivalent ligands of known dimers of GPCRs, PROTACs and a model bivalent antibody/antigen system were analysed. The tool could rapidly assess the preferred linker length for the different systems and recapitulated the best reported results. In the case of GPCR dimers the results suggest that in some cases these ligands might bind to a secondary binding site at the extracellular entrance (vestibule or allosteric site) instead of the orthosteric binding site.

Availability And Implementation: Freely accessible from the Molecular Operating Environment svl exchange server (https://svl.chemcomp.com/).

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/bty422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223368PMC
November 2018

Essential Control of the Function of the Striatopallidal Neuron by Pre-coupled Complexes of Adenosine A-Dopamine D Receptor Heterotetramers and Adenylyl Cyclase.

Front Pharmacol 2018 9;9:243. Epub 2018 Apr 9.

Circuit Therapeutics, Inc., Menlo Park, CA, United States.

The central adenosine system and adenosine receptors play a fundamental role in the modulation of dopaminergic neurotransmission. This is mostly achieved by the strategic co-localization of different adenosine and dopamine receptor subtypes in the two populations of striatal efferent neurons, striatonigral and striatopallidal, that give rise to the direct and indirect striatal efferent pathways, respectively. With optogenetic techniques it has been possible to dissect a differential role of the direct and indirect pathways in mediating "Go" responses upon exposure to reward-related stimuli and "NoGo" responses upon exposure to non-rewarded or aversive-related stimuli, respectively, which depends on their different connecting output structures and their differential expression of dopamine and adenosine receptor subtypes. The striatopallidal neuron selectively expresses dopamine D receptors (D2R) and adenosine A receptors (A2AR), and numerous experiments using multiple genetic and pharmacological and approaches, demonstrate they can form A2AR-D2R heteromers. It was initially assumed that different pharmacological interactions between dopamine and adenosine receptor ligands indicated the existence of different subpopulations of A2AR and D2R in the striatopallidal neuron. However, as elaborated in the present essay, most evidence now indicates that all interactions can be explained with a predominant population of striatal A2AR-D2R heteromers forming complexes with adenylyl cyclase subtype 5 (AC5). The A2AR-D2R heteromer has a tetrameric structure, with two homodimers, which allows not only multiple allosteric interactions between different orthosteric ligands, agonists, and antagonists, but also the canonical Gs-Gi antagonistic interaction at the level of AC5. We present a model of the function of the A2AR-D2R heterotetramer-AC5 complex, which acts as an integrative device of adenosine and dopamine signals that determine the excitability and gene expression of the striatopallidal neurons. The model can explain most behavioral effects of A2AR and D2R ligands, including the psychostimulant effects of caffeine. The model is also discussed in the context of different functional striatal compartments, mainly the dorsal and the ventral striatum. The current accumulated knowledge of the biochemical properties of the A2AR-D2R heterotetramer-AC5 complex offers new therapeutic possibilities for Parkinson's disease, schizophrenia, SUD and other neuropsychiatric disorders with dysfunction of dorsal or ventral striatopallidal neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2018.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900444PMC
April 2018

Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

Nat Commun 2018 03 28;9(1):1242. Epub 2018 Mar 28.

Integrative Neurobiology Section, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.

G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A receptor and dopamine D receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-03522-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871782PMC
March 2018

Cross-communication between G and G in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain.

BMC Biol 2018 02 28;16(1):24. Epub 2018 Feb 28.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, University of Barcelona, 08028, Barcelona, Spain.

Background: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (AR or AR) can form AR-AR heteromers (A-AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for AR and AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream G- and G-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of AR impedes signaling via AR.

Results: We examined the mechanism by which A-AHet integrates G- and G-dependent signals. AR blockade by AR in the A-AHet is not observed in the absence of AR activation by agonists, in the absence of the C-terminal domain of AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A-AHet, indicating that signaling mediated by AR and AR is controlled by both G and G proteins.

Conclusions: We identified a new mechanism of signal transduction that implies a cross-communication between G and G proteins guided by the C-terminal tail of the AR. This mechanism provides the molecular basis for the operation of the A-AHet as an adenosine concentration-sensing device that modulates the signals originating at both AR and AR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-018-0491-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389107PMC
February 2018

Identifying human diamine sensors for death related putrescine and cadaverine molecules.

PLoS Comput Biol 2018 01 11;14(1):e1005945. Epub 2018 Jan 11.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

Pungent chemical compounds originating from decaying tissue are strong drivers of animal behavior. Two of the best-characterized death smell components are putrescine (PUT) and cadaverine (CAD), foul-smelling molecules produced by decarboxylation of amino acids during decomposition. These volatile polyamines act as 'necromones', triggering avoidance or attractive responses, which are fundamental for the survival of a wide range of species. The few studies that have attempted to identify the cognate receptors for these molecules have suggested the involvement of the seven-helix trace amine-associated receptors (TAARs), localized in the olfactory epithelium. However, very little is known about the precise chemosensory receptors that sense these compounds in the majority of organisms and the molecular basis of their interactions. In this work, we have used computational strategies to characterize the binding between PUT and CAD with the TAAR6 and TAAR8 human receptors. Sequence analysis, homology modeling, docking and molecular dynamics studies suggest a tandem of negatively charged aspartates in the binding pocket of these receptors which are likely to be involved in the recognition of these small biogenic diamines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1005945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783396PMC
January 2018

Molecular Switches of Allosteric Modulation of the Metabotropic Glutamate 2 Receptor.

Structure 2017 07 22;25(7):1153-1162.e4. Epub 2017 Jun 22.

Computational Chemistry, Janssen Research and Development, Calle Jarama 75A, 45007 Toledo, Spain; Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium. Electronic address:

Metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) crucial for CNS function and important drug discovery targets. Glutamate triggers receptor activation from an extracellular domain binding site while allosteric modulators bind in the seven-transmembrane domain. Little is known about how allosteric modulators produce their functional effects at the molecular level. Here we address this topic with combined experimental and computational approaches and reveal that mGlu receptor allosteric modulators interact with the homologous "trigger switch" and "transmission switch" amino acids as seen in class A GPCRs, in short, the characteristic hallmarks of class A agonist activation translate to the mGlu allosteric modulator. The proposed "trigger switch" for the mGlu involves the side chains of F643, N735, and W773, whereas the "transmission switch" involves the Y647, L738, and T769 amino acids. The work has wide impact on understanding mGlu GPCR function and for future allosteric modulator drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2017.05.021DOI Listing
July 2017

Ligand chain length drives activation of lipid G protein-coupled receptors.

Sci Rep 2017 05 17;7(1):2020. Epub 2017 May 17.

Laboratoire de Structure et Fonction des Membranes Biologiques, Université Libre de Bruxelles, Brussels, Belgium.

Sphingosine-1-phosphate (S1P) is a lipid mediator that can activate five cell membrane G protein-coupled receptors (GPCRs) which carry a variety of essential functions and are promising drug targets. S1P is composed of a polar zwitterionic head-group and a hydrophobic alkyl chain. This implies an activation mechanism of its cognate receptor that must be significantly different from what is known for prototypical GPCRs (ie receptor to small hydrophilic ligands). Here we aim to identify the structural features responsible for S1P agonism by combining molecular dynamics simulations and functional assays using S1P analogs of different alkyl chain lengths. We propose that high affinity binding involves polar interactions between the lipid head-group and receptor side chains while activation is due to hydrophobic interactions between the lipid tail and residues in a distinct binding site. We observe that ligand efficacy is directly related to alkyl chain length but also varies with receptor subtypes in correlation with the size of this binding pocket. Integrating experimental and computational data, we propose an activation mechanism for the S1P receptors involving agonist-induced conformational events that are conserved throughout class A GPCRs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-02104-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435731PMC
May 2017

Central Hypothyroidism Due to a TRHR Mutation Causing Impaired Ligand Affinity and Transactivation of Gq.

J Clin Endocrinol Metab 2017 07;102(7):2433-2442

Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, La Paz University Hospital, Autonomous University of Madrid, 28046 Madrid, Spain.

Context: Central congenital hypothyroidism (CCH) is an underdiagnosed disorder characterized by deficient production and bioactivity of thyroid-stimulating hormone (TSH) leading to low thyroid hormone synthesis. Thyrotropin-releasing hormone (TRH) receptor (TRHR) defects are rare recessive disorders usually associated with incidentally identified CCH and short stature in childhood.

Objectives: Clinical and genetic characterization of a consanguineous family of Roma origin with central hypothyroidism and identification of underlying molecular mechanisms.

Design: All family members were phenotyped with thyroid hormone profiles, pituitary magnetic resonance imaging, TRH tests, and dynamic tests for other pituitary hormones. Candidate TRH, TRHR, TSHB, and IGSF1 genes were screened for mutations. A mutant TRHR was characterized in vitro and by molecular modeling.

Results: A homozygous missense mutation in TRHR (c.392T > C; p.I131T) was identified in an 8-year-old boy with moderate hypothyroidism (TSH: 2.61 mIU/L, Normal: 0.27 to 4.2; free thyroxine: 9.52 pmol/L, Normal: 10.9 to 25.7) who was overweight (body mass index: 20.4 kg/m2, p91) but had normal stature (122 cm; -0.58 standard deviation). His mother, two brothers, and grandmother were heterozygous for the mutation with isolated hyperthyrotropinemia (TSH: 4.3 to 8 mIU/L). The I131T mutation, in TRHR intracellular loop 2, decreases TRH affinity and increases the half-maximal effective concentration for signaling. Modeling of TRHR-Gq complexes predicts that the mutation disrupts the interaction between receptor and a hydrophobic pocket formed by Gq.

Conclusions: A unique missense TRHR defect identified in a consanguineous family is associated with central hypothyroidism in homozygotes and hyperthyrotropinemia in heterozygotes, suggesting compensatory elevation of TSH with reduced biopotency. The I131T mutation decreases TRH binding and TRHR-Gq coupling and signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/jc.2016-3977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505191PMC
July 2017

A new serotonin 5-HT receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding.

Sci Rep 2017 01 24;7:41293. Epub 2017 Jan 24.

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain.

Serotonin 5-HT receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep41293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259792PMC
January 2017

Development of a Nucleotide Exchange Inhibitor That Impairs Ras Oncogenic Signaling.

Chemistry 2017 Jan 27;23(7):1676-1685. Epub 2016 Dec 27.

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040, Madrid, Spain.

Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras-guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switch II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201604905DOI Listing
January 2017

Analysis of the interactions of sulfur-containing amino acids in membrane proteins.

Protein Sci 2016 08 8;25(8):1517-24. Epub 2016 Jun 8.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, E-08193, Bellaterra, Spain.

The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic-aromatic, aromatic-aliphatic or/and aliphatic-aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small-molecule model systems at the ab-initio level, predicts that Met-Met interactions are stronger than Met-Cys ≈ Met-Phe ≈ Cys-Phe interactions, stronger than Phe-Phe ≈ Phe-Leu interactions, stronger than the Met-Leu interaction, and stronger than Leu-Leu ≈ Cys-Leu interactions. These results show that sulfur-containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.2955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972207PMC
August 2016

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

BMC Biol 2016 Apr 5;14:26. Epub 2016 Apr 5.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown.

Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins.

Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-016-0247-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822319PMC
April 2016

The pathway of ligand entry from the membrane bilayer to a lipid G protein-coupled receptor.

Sci Rep 2016 Mar 4;6:22639. Epub 2016 Mar 4.

Computational Biophysics Laboratory (GRIB-IMIM), Universitat Pompeu Fabra, Barcelona Biomedical Research Park (PRBB), C/Doctor Aiguader 88, 08003 Barcelona, Spain.

The binding process through the membrane bilayer of lipid-like ligands to a protein target is an important but poorly explored recognition process at the atomic level. In this work we succeeded in resolving the binding of the lipid inhibitor ML056 to the sphingosine-1-phosphate receptor 1 (S1P1R) using unbiased molecular dynamics simulations with an aggregate sampling of over 800 μs. The binding pathway is a multi-stage process consisting of the ligand diffusing in the bilayer leaflet to contact a "membrane vestibule" at the top of TM 7, subsequently moving from this lipid-facing vestibule to the orthosteric binding cavity through a channel formed by TMs 1 and 7 and the N-terminal of the receptor. Unfolding of the N-terminal alpha-helix increases the volume of the channel upon ligand entry, helping to reach the crystallographic pose that also corresponds to the predicted favorable pose. The relaxation timescales of the binding process show that the binding of the ligand to the "membrane vestibule" is the rate-limiting step in the multi microseconds timescale. We comment on the significance and parallels of the binding process in the context of other binding studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep22639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778059PMC
March 2016

TMalphaDB and TMbetaDB: web servers to study the structural role of sequence motifs in α-helix and β-barrel domains of membrane proteins.

BMC Bioinformatics 2015 Aug 20;16:266. Epub 2015 Aug 20.

Department de Biologia de Sistemes, Universitat de Vic, Vic, Barcelona, Spain.

Background: Membrane proteins represent over 25 % of human protein genes and account for more than 60 % of drug targets due to their accessibility from the extracellular environment. The increasing number of available crystal structures of these proteins in the Protein Data Bank permits an initial estimation of their structural properties.

Description: We have developed two web servers-TMalphaDB for α-helix bundles and TMbetaDB for β-barrels-to analyse the growing repertoire of available crystal structures of membrane proteins. TMalphaDB and TMbetaDB permit to search for these specific sequence motifs in a non-redundant structure database of transmembrane segments and quantify structural parameters such as ϕ and ψ backbone dihedral angles, χ1 side chain torsion angle, unit bend and unit twist.

Conclusions: The structural information offered by TMalphaDB and TMbetaDB permits to quantify structural distortions induced by specific sequence motifs, and to elucidate their role in the 3D structure. This specific structural information has direct implications in homology modeling of the growing sequences of membrane proteins lacking experimental structure. TMalphaDB and TMbetaDB are freely available at http://lmc.uab.cat/TMalphaDB and http://lmc.uab.cat/TMbetaDB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12859-015-0699-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546024PMC
August 2015

Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms.

ACS Med Chem Lett 2015 Aug 16;6(8):872-6. Epub 2015 Jul 16.

Institut de Química Avançada de Catalunya (I.Q.A.C.-C.S.I.C.) , E-08034 Barcelona, Spain.

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide-receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.5b00126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538429PMC
August 2015

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

PLoS Biol 2015 Jul 9;13(7):e1002194. Epub 2015 Jul 9.

Neuropharmacology Laboratory, University Pompeu Fabra, Barcelona, Spain; Integrative Pharmacology and Systems Neuroscience, IMIM-Hospital del Mar Medical Research Institute, Barcelona, Spain.

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.1002194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497644PMC
July 2015