Dr. Juan Carlos Mobarec, PhD - Heptares Therapeutics

Dr. Juan Carlos Mobarec

PhD

Heptares Therapeutics

United Kingdom

Additional Specialties: GPCRs, drug discovery, molecular modeling and simulations


Top Author

Dr. Juan Carlos Mobarec, PhD - Heptares Therapeutics

Dr. Juan Carlos Mobarec

PhD

Introduction

Primary Affiliation: Heptares Therapeutics - United Kingdom

Additional Specialties:

Research Interests:

Education

Mount Sinai School Of Medicine
Ph.D.
(USA)

Publications

24Publications

300Reads

3057Profile Views

310PubMed Central Citations

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach.

Mol Cell Endocrinol 2017 10 30;454:39-49. Epub 2017 May 30.

Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.mce.2017.05.034DOI Listing
October 2017
36 Reads
1 Citation
4.410 Impact Factor

High affinity binding of the peptide agonist TIP-39 to the parathyroid hormone 2 (PTH) receptor requires the hydroxyl group of Tyr-318 on transmembrane helix 5.

Biochem Pharmacol 2017 03 22;127:71-81. Epub 2016 Dec 22.

School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bcp.2016.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303546PMC
March 2017
22 Reads
1 Citation
5.010 Impact Factor

Genetically-Encoded Photocrosslinkers Determine the Biological Binding Site of Exendin-4 in the N-Terminal Domain of the Intact Human Glucagon-Like Peptide-1 Receptor (GLP-1R)

jbc.M117.779496. doi: 10.1074/jbc.M117.779496

The Journal of Biological Chemistry

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, functional receptor-ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-L-phenylalanine (azF) into N-terminal residues of a full-length, functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocrosslinking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Crosslinking data were compared directly to the crystal structure of the isolated N-terminal extracellular domain (ECD) of the GLP-1R in complex with exendin(9-39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocrosslinking constraints highlights the potential influence of environmental conditions on the conformation of the receptor-peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide-receptor interactions, and should be combined with additional experimental constraints to reveal peptide-receptor interactions occurring in the dynamic, native, full-length receptor state.

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March 2017
13 Reads

Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor.

Biochem Pharmacol 2016 Oct 26;118:68-87. Epub 2016 Aug 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.bcp.2016.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063953PMC
October 2016
24 Reads
5 Citations
5.010 Impact Factor

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.

Cell 2016 Jun;165(7):1632-1643

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.cell.2016.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912689PMC
June 2016
18 Reads
20 Citations
32.242 Impact Factor

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism

Cell 2016 165(7):1632-43

Cell

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

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June 2016
11 Reads

The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs.

Nucleic Acids Res 2016 Mar 17;44(5):2160-72. Epub 2015 Nov 17.

Center for Molecular Biology (ZMBH) and Center for the Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

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http://dx.doi.org/10.1093/nar/gkv1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797271PMC
March 2016
16 Reads
2 Citations
9.112 Impact Factor

A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures.

Mol Pharmacol 2016 Mar 23;89(3):335-47. Epub 2015 Dec 23.

Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (D.W., C.K., T.Q., T.C., S.G.B.F., A.C., P.M.S.); School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK (C.A.R., K.J.S., J.C.M.); School of Life and Health Sciences, Aston University, Birmingham, UK (J.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ (L.J.M.).

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http://molpharm.aspetjournals.org/content/89/3/335.full.pdf
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http://molpharm.aspetjournals.org/cgi/doi/10.1124/mol.115.10
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http://dx.doi.org/10.1124/mol.115.101246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767408PMC
March 2016
26 Reads
14 Citations
4.130 Impact Factor

The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling

Sci. Signal. 2015 Nov 03;401(8):ra110

Science Signaling

Most G protein–coupled receptors (GPCRs) are activated by ligand binding, but some are also affected by changes in plasma membrane potential, which can either enhance or inhibit GPCR-mediated signaling. Through FRET-based experiments in single cells, Rinne et al. found that depolarization enhanced signaling by the M3 muscarinic acetylcholine receptor when the receptor was bound to the agonists choline or pilocarpine; however, depolarization attenuated M3 receptor signaling when either carbachol or acetylcholine was bound. Molecular docking simulations showed that each group of agonists adopted a distinct binding position. Mutation of a critical residue in the binding pocket changed the binding position of carbachol and switched the response of the carbachol-bound receptor so that signaling was enhanced by membrane depolarization. Together, these data suggest that the binding mode of the agonist determines whether membrane potential changes will enhance or attenuate GPCR signals. These results provide a potential molecular mechanism for drugs that are agonists of specific GPCRs, yet have distinct effects.

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November 2015
10 Reads

Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant.

Nature 2015 Mar 22;519(7542):247-50. Epub 2014 Dec 22.

Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

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http://dx.doi.org/10.1038/nature14035DOI Listing
March 2015
22 Reads
31 Citations
42.351 Impact Factor

Computational modeling, docking and molecular dynamics of the transcriptional activator ComA bound to a newly-identified functional DNA binding site

J Cheminform. 6(Suppl 1): P30.

J Cheminform.

Quorum sensing is the mechanism by which bacterial cells communicate to each other in response to changes in cell density. Secreted signaling molecules reach other bacteria and trigger an internal physiological response, like production of degradative enzymes and antibiotics, competence development, sporulation and pathogenesis. In Bacillus subtilis, the transcriptional activator ComA regulates several genes of the quorum sensing response. ComA binds to recognition elements (RE) in bacterial promoters, activating transcription. The DNA binding domain of ComA has four α-helices, which contain a helix-turn-helix (HTH) motif. Several promoters in B. subtilis have known inverted repeat motifs (RE1 and RE2), where a ComA dimer can bind. However, a third and fourth recognition-like element (RE3 and RE4) in a direct repeat (DR) arrangement have recently been identified downstream of the known ComA box. Currently there is no structural information on how a DR would bind to ComA. Here, we present computational results supported by experiments that the DR (RE3 and RE4) form a functional domain for recognition of a ComA dimer. Flexible protein-DNA docking was used to get insight into the putative binding mode of ComA bound to the new direct repeat. The lowest-energy docked conformations of the ComA-DNA complex were tested for dynamic stability with explicit molecular dynamics simulations. Clustering of the sampled conformations was used to select a representative structure of the ComA-DR- DNA complex. In our results, the second α-helix of the HTH contributes most of the DNA recognition, binding to the major grooves of the DR-DNA, and interacting mostly with the DNA bases. We pinpoint specific ComA-DNA interactions that may have a key role for recognition and affinity. Furthermore, the physical interaction between ComA and the new DR was demonstrated in vitro, and its functionality was confirmed in vivo. Our results strongly support the hypothesis that ComA dimers can bind to direct repeats, and provide an atomistic model for its recognition. Additionally, we suggest specific interactions for fine-tuning of transcription which will be tested experimentally.

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March 2014
11 Reads

Voltage Dependence of Muscarinic M1-,M3- and M5 Receptors

Biophysical Journal 106 (2) Supplement 1, p105a

Biophysical Journal

Muscarinic receptors (MR) are G protein-coupled receptors (GPCRs). Localized to the plasma membrane, they sense changes of the membrane potential and exhibit voltage-dependent signaling. Because voltage activates Gq-coupled receptors (e.g. M1R) but deactivates Gi-coupled receptors (e.g. M2R), it was proposed that GPCRs which couple to the same G protein class share common voltage-dependent properties. In this study we analyzed signaling of the Gq protein-coupled M1R, M3R and M5R with fluorescence resonance energy transfer (FRET) microscopy and voltage clamp. To test whether Gq protein coupling influences voltage sensitivity, we expressed a biosensor that reports conformations of M1R in HEK 293 cells and analyzed Carbachol (CCh)-induced receptor activation at −90 mV and at +60 mV. Depolarization caused activation of M1R, which was not abolished after uncoupling of Gq proteins with GTPγS. A FRET assay that reports activation of Gq was then used to compare wild type M1R, M3R and M5R. Application of CCh induced robust activation of Gq at −90 mV for all three receptors. Subsequent depolarization revealed subtype-specific phenotypes: at +60 mV the Gq protein was either activated (M1R) or deactivated (M3R or M5R). Voltage sensitivity of MR was also influenced by the nature of the activating agonist: M3R displayed deactivation (CCh) or activation (Pilocarpine) at +60 mV. Computational docking studies predicted different molecular binding modes for each agonist and guided us to select key point mutations in the orthosteric binding site of muscarinic receptors. Further docking of mutant M3R predicted a novel “Pilocarpine-like” binding mode for CCh. Subsequent experiments confirmed that mutant M3R were indeed activated by voltage in the presence of CCh. Together, these results imply that the molecular mechanism by which GqPCRs sense voltage resides in the receptor protein with negligible contribution of G protein coupling.

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January 2014
35 Reads

Ligand-induced modulation of the free-energy landscape of G protein-coupled receptors explored by adaptive biasing techniques.

PLoS Comput Biol 2011 Oct 13;7(10):e1002193. Epub 2011 Oct 13.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.

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http://dx.doi.org/10.1371/journal.pcbi.1002193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192824PMC
October 2011
20 Reads
25 Citations

Life in blue: copper resistance mechanisms of bacteria and archaea used in industrial biomining of minerals.

Biotechnol Adv 2010 Nov-Dec;28(6):839-48. Epub 2010 Jul 11.

Laboratory of Molecular Microbiology and Biotechnology, Department of Biology, and Millennium Institute for Cell Dynamics and Biotechnology, Faculty of Sciences, University of Chile, Santiago, Chile.

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http://dx.doi.org/10.1016/j.biotechadv.2010.07.003DOI Listing
January 2011
14 Reads
19 Citations
9.020 Impact Factor

Progress in elucidating the structural and dynamic character of G Protein-Coupled Receptor oligomers for use in drug discovery.

Curr Pharm Des 2009 ;15(35):4017-25

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332530PMC
March 2010
11 Reads
6 Citations
3.452 Impact Factor

Computational Insight into the Ligand-Induced Conformational Specificity of G-Protein Coupled Receptors

Biophysical Journal 98, (3), S1, p290a,

Biophysical Journal

Several observations in the G-protein coupled receptor (GPCR) literature support the existence of ligand-specific intermediate conformational states that are likely to be involved in differential activation of signaling pathways. Fluorescence spectroscopy studies provide direct evidence for ligand-specific receptor conformations of the β2-adrenergic receptor, making this system an attractive target to test the ability of computational methodologies to predict different activated states of GPCRs. To this end, we designed a computational strategy that combines adiabatic biased molecular dynamics (ABMD) and metadynamics simulations. Firstly, ABMD is used to generate transition paths between the experimental inactive crystal structure of the β2-adrenergic receptor and a conformation containing established features of activated states of GPCRs (modeled using the opsin crystal structures). Secondly, metadynamics is applied to study how ligands with different efficacies affect the free-energy of different metastable states identified along these putative activation pathways. The calculated free-energy profiles of the different ligand-β2 adrenoceptor complexes help rationalize the published experimental results, including the different kinetics of catecholaminergic agonists such as epinephrine, norepinephrine, dopamine, and isoproterenol. Representative structures of the identified energy basins suggest specific residues and contacts that may help stabilize different activated states of the receptor. This information holds promise for the crystallization of different GPCR conformations.

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January 2010
12 Reads

Modern homology modeling of G-protein coupled receptors: which structural template to use?

J Med Chem 2009 Aug;52(16):5207-16

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1677, New York, NY 10029-6574, USA.

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http://dx.doi.org/10.1021/jm9005252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891345PMC
August 2009
11 Reads
37 Citations
5.450 Impact Factor

On Template Selection for Homology Modeling of G-Protein Coupled Receptors

Biophysical Journal 96, (3) S1 p653a

Biophysical Journal

G-Protein Coupled Receptors (GPCRs) are a family of structurally similar integral membrane proteins that bind diverse ligands, from the size of a photon to small peptides. For several years the inactive conformation of Bos taurus rhodopsin has been the only GPCR crystal structure available at atomic resolution, thus serving as the most reliable template for homology modeling of other GPCRs. Over the past year, the atomic coordinates of several different new crystal structures of GPCRs (two of them encompassing some of the characteristic structural features that have often been attributed to GPCR activated states) have become available. Considering that acceptable models of the transmembrane (TM) regions of membrane proteins may be obtained for template sequence identities of 30% or higher, we investigated the extent to which current crystal structures of GPCRs are valuable templates for homology modeling of the TM regions of a dataset of non-redundant non-orphan non-olfactory Class A GPCRs from the human genome aligned using conserved functional residues in their TMs. While the recently solved crystal structures of beta-2 adrenergic receptor and mutant m23 beta-1 adrenergic receptor are calculated to be valuable templates for 16% and 18% of class A human GPCRs, respectively, our results indicate that the majority of GPCRs in the human genome needs better templates for their accurate homology modeling. Thus, our calculations point to specific GPCR targets whose crystal structures would be most beneficial to the majority of human GPCRs. Moreover, we suggest specific ways to improve GPCR modeling, including the use of hybrid templates.

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February 2009
11 Reads

Dopamine D2 receptors form higher order oligomers at physiological expression levels.

EMBO J 2008 Sep;27(17):2293-304

Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.

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http://dx.doi.org/10.1038/emboj.2008.153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2529367PMC
September 2008
15 Reads
123 Citations
10.434 Impact Factor

Advances in the Development and Application of Computational Methodologies for Structural Modeling of G-Protein Coupled Receptors

Expert Opin Drug Discov. 2008 Mar;3(3):343-355.

Expert Opin Drug Discov.

BACKGROUND:Despite the large amount of experimental data accumulated in the past decade on G-protein coupled receptor (GPCR) structure and function, understanding of the molecular mechanisms underlying GPCR signaling is still far from being complete, thus impairing the design of effective and selective pharmaceuticals. OBJECTIVE:Understanding of GPCR function has been challenged even further by more recent experimental evidence that several of these receptors are organized in the cell membrane as homo- or hetero-oligomers, and that they may exhibit unique pharmacological properties. Given the complexity of these new signaling systems, researcher's efforts are turning increasingly to molecular modeling, bioinformatics and computational simulations for mechanistic insights of GPCR functional plasticity. METHODS:We review here current advances in the development and application of computational approaches to improve prediction of GPCR structure and dynamics, thus enhancing current understanding of GPCR signaling. RESULTS/CONCLUSIONS:Models resulting from use of these computational approaches further supported by experiments are expected to help elucidate the complex allosterism that propagates through GPCR complexes, ultimately aiming at successful structure-based rational drug design.

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April 2008
10 Reads

Identification of putative sulfurtransferase genes in the extremophilic Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins.

OMICS 2005 ;9(1):13-29

Laboratory of Molecular Microbiology and Biotechnology, and Millennium Institute for Advanced Studies in Cell Biology and Biotechnology (CBB), Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile.

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http://dx.doi.org/10.1089/omi.2005.9.13DOI Listing
September 2005
13 Reads
11 Citations
2.362 Impact Factor