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Molecular Modeling and Enhanced Molecular Dynamics Simulations of GPCRs (Especially Opioid Receptors) and Other Membrane Proteins

Among the milestones that we were able to accomplish under the auspices of continued NIH funding over the past 12 years are the design, testing, and implementation of innovative computational strategies to build improved molecular models of G Protein-Coupled Receptors (GPCRs) and to study, more efficiently, the conformational plasticity and dynamical nature of liganded or unliganded, single or interacting, receptors within their natural lipid environment. In particular, we pioneered the use of enhanced, molecular dynamics (MD)-based computational strategies in combination with either atomistic or coarse-grained (CG) system representations to improve dynamic molecular models of GPCR molecular recognition, activation, and oligomerization, with the ultimate goal of elucidating receptor allostery and functional selectivity for successful use in rational drug design. In particular, we were able: (i) to obtain reliable models of ligand-bound conformations of GPCRs that do not require very long and computationally inefficient standard MD simulations, (ii) to establish a possible molecular basis for the functional selectivity of GPCRs through the prediction of ligand-specific conformations, and (iii) to advance current understanding of the role of oligomerization in receptor function through the generation of novel testable hypotheses of specific mutations that could eventually be used to modulate receptor function (see section 2, below). While the specific computational methods we explored are not new from an algorithmic standpoint, we developed some distinctive combinations of such methods, and showed in recent publications that they are indeed able to generate ligand-specific conformations of both isolated  and interacting, inactive and active, GPCRs that are consistent with experimental data. Notably, the methodologies we developed represent fundamental tools that can be generalized to other transmembrane receptors, as well as broadly to other proteins.

Among the various GPCRs, we have devoted special attention to opioid receptors, which are important drug targets for pain management, drug abuse/addiction, and mood disorders. We have a long history of work on these GPCR subtypes, having contributed over the past 20 years to several structural and mechanistic insights into their pharmacology and signaling. Most recently, we have focused on their dynamics, seeking answers to questions like: How does an opioid drug bind to his receptor? How can sodium ions modulate opioid receptor activity? What are the likely interfaces of opioid receptor homodimers? What are the thermodynamic and kinetic elements of ligand binding and functional selectivity?


Representative Publications

Provasi, D., Bortolato A., Filizola, M. “Exploring Molecular Mechanisms of Ligand Recognition by Opioid Receptors with Metadynamics.” Biochemistry (2009) 48 (42): 10020-10029.


Filizola M. & Devi, L.A. “How opioid drugs bind to receptors” Nature (2012) 485, 314-7.


Johnston, J.M., Aburi, M., Provasi, D., Bortolato, A., Urizar, E., Lambert, N.A., Javitch, J.A., Filizola, M. “Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers”, Biochemistry (2011) 50(10):1682-1690


Shang, Y., LeRouzic, V., Schneider, S., Bisignano, P., Pasternak, G.W., Filizola, M. “Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions” Biochemistry (2014) 53(31):5140-9.


Mobarec, J.C., Sanchez, R., and Filizola, M. “Modern Homology Modeling of G-Protein Coupled Receptors: Which Structural Template to Use?” Journal of Medicinal Chemistry (2009) 52 (16), 5207-5216.


Provasi, D., Camacho-Artacho, M., Negri, A., Mobarec, J.C., Filizola, M. “Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques.” PLOS Computational Biology (2011) 7(10):e1002193.


Provasi, D. & Filizola, M. “Putative Active States of a Prototypic G-Protein Coupled Receptor from Biased Molecular Dynamics.”Biophysical Journal (2010) 19(10):2347-2355.


Schneider, S., Provasi, D. & Filizola, M. “The Dynamic Process of Drug-GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics” Methods in Molecular Biology (2015) 1335:277-294.


Provasi, D., Boz, M.B., Johnston, J.M., Filizola, M. “Preferred Supramolecular Organization and Dimer Interfaces of Opioid Receptors from Simulated Self-Association” PLOS Computational Biology (2015) Mar 30;11(3):e1004148


Shang, Y., Yeatman, H.R., Provasi, D., Alt, A., Christopoulos, A.,  Canals, M., and Filizola, M. “Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors” (2016) ACS Chemical Biology11(5):1220-9


Schneider, S., Provasi, D., Filizola, M. “How Oliceridine (TRV-130) Binds and Stabilizes a μ-Opioid Receptor Conformational State that Selectively Triggers G Protein-Signaling Pathways.” (2016) Biochemistry55(46):6456-6466


Marino, K., Prada-Gracia, D., Provasi, D., Filizola, M. “Impact of Lipid Composition and Receptor Conformation on the Spatio-Temporal Organization of mu-Opioid Receptors in a Multi-component Plasma Membrane Model” (2016) PLOS Computational Biology 12(12):e1005240


Kapoor, A., Martinez-Rosell, Provasi, D., de Fabritiis, G., and Filizola, M. “Dynamic and Kinetic Elements of µ-Opioid Receptor Functional Selectivity” Nature – Scientific Reports, (2017) 7(1):11255.


Marino, K.A. & Filizola, M. “Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations” (2018) “Methods in Molecular Biology”, 1705: 351-364.

Mechanistic Insights into GPCR Dimerization/Oligomerization

Compelling evidence that models of GPCR signaling must consider oligomeric assemblies rather than isolated monomers started to appear in the literature during my postdoctoral training. Many of the published studies described (i) effects resulting from activating one GPCR in the presence of another and (ii) modulation of the activity of one receptor using ligands targeting another one. Whether these effects resulted from downstream crosstalk or from differential signaling of a receptor complex has so far been difficult to ascertain for the largest subfamily A of GPCRs. My lab has been hard at work in the area of GPCR oligomerization, contributing to their recognition, nomenclature, storing, structural models, and estimates of relative stability.  We are committed to providing a rigorous mechanistic insight into the spatio-temporal organization of GPCRs in living cells at a level of molecular detail that is unattainable using current experimental techniques alone, but is required for an ultimate understanding of the role of GPCR oligomerization in receptor function.


 Representative Publications

Pin, J.-P., Neubig, R., Bouvier, M., Devi, L., Filizola, M., Javitch, J.A., Lohse, M.J., Milligan, G., Palczewski, K., Parmentier, M., Spedding, M. “International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the Recognition and Nomenclature of G Protein-Coupled Receptor Heteromultimers.” Pharmacological Reviews (2007) 59:1-9.


Khelashvili, G., Dorff, K., Shan, J., Camacho-Artacho, M., Skrabanek, L., Vroling, B., Bouvier, M., Devi, L., George, S.R., Javitch, J.A.,Lohse, M.J., Milligan, G., Neubig, R., Palczewski, K., Parmentier, M., Pin, J.-P., Vriend, G., Campagne, F., Filizola, M.GPCR-OKB: A database for G protein-coupled receptor oligomers.” Bioinformatics (2010) 26(14):1804-1805.


Johnston, J.M., Wang, H., Provasi, D., Filizola, M. “Assessing the Relative Stability of Dimer Interfaces in G Protein-Coupled Receptors.” PLOS Computational Biology (2012) 8(8): e1002649.


Provasi, D., Boz, M.B., Johnston, J.M., Filizola, M. “Preferred Supramolecular Organization and Dimer Interfaces of Opioid Receptors from Simulated Self-Association” (2015) PLOS Computational Biology Mar 30;11(3):e1004148.


Marino, K., Prada-Gracia, D., Provasi, D., Filizola, M. “Impact of Lipid Composition and Receptor Conformation on the Spatio-Temporal Organization of mu-Opioid Receptors in a Multi-component Plasma Membrane Model” (2016) PLOS Computational Biology 12(12):e1005240


Structure-Function Relationships of beta3 Integrins

A close and productive collaboration with the laboratory of Dr. Barry Coller at Rockefeller University has kept part of our research interests focused on the study of structure-function relationships of both aIIbb3 and aVb3 integrins. The lab’s interest in these systems stems from the exciting computational challenges they pose, and the opportunity to develop and/or apply cutting-edge computational approaches that will render accurate representations of the dynamic allosteric mechanisms regulating integrin function.

During the past 17 years we have been able to provide an innovative structure-guided approach to the functional studies carried out in the lab of our experimental collaborator. Specifically, we have contributed i) Early three-dimensional molecular models of aIIbb3 using aVb3 as a template; ii) Important structural insights into the ligand-associated metal binding site (LIMBS, later re-termed SyMBS) of β3 integrins from molecular dynamics (MD) simulations; iii) Specific molecular determinants for limiting extension at the αIIb genu or ligand binding to integrin αIIbβ3; iv) Information about the overall common conformational changes occurring in β3 integrins upon hybrid domain swing-out using targeted MD simulations; v) Mechanistic information about αIIbβ3- and αVβ3-specific antagonists that stabilize the receptors in their resting states, vi) Structure-guided design of potent pure antagonist of β3 receptors; vii) Identification of novel antagonists by virtual screening, viii) Multimicrosecond, all-atom MD simulations of the talin-driven inside-out activation mechanism of αIIbβ3 integrin, and ix) Predictions of the functional effect of αIIbβ3 variants defined by next-generation sequencing.


Representative Publications

Zhu, J., Zhu, J., Negri, A., Provasi, D., Filizola, M., Coller, B.S., Springer, T.A. “Closed headpiece of integrin αIIbβ3 and its complex with an αIIbβ3-specific antagonist that does not induce opening” Blood (2010) 116 (23):5050-5059.


Negri, A., Li, J., Naini, S., Coller, B.S., Filizola, M. “Structure-Based Virtual Screening of Small-Molecule Antagonists of Platelet Integrin αIIbβ3that Do Not Prime the Receptor to Bind Ligand” Journal of Computer-Aided Molecular Design (2012) 26 (9): 1005-1015


Zhu, J., Choi, W.-S., McCoy, J. G., Negri, A., Zhu, J., Naini, S., Li, J., Shen, M., Huang, W., Bougie, D., Rasmussen, M., Aster, R., Thomas, C. J.,  Filizola, M.,  Springer, T. A., Coller, B. S. “Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS.” Science Translational Medicine (2012) 4: 125ra32


Buitrago, L., Rendon, A., Liang, Y., Turro, E., Simeoni, I., Negri, A., ThromboGenomics Consortium, Filizola, M., Ouwehand, W.H., Coller, B.S. “αIIbβ3 Variants Defined by Next Generation Sequencing: Predicting Variants Likely to Cause Glanzmann Thrombasthenia” (2015)Proceedings of the National Academy of Science USA, 112(15):E1898-907.


Zafar, H., Shang, Y., Li, J., David, G.A. III, Fernandez, J.P., Molina, H., Filizola, M., Coller, B.S. “αIIbβ3-Binding to a Fibrinogen Fragment Lacking the g-chain Dodecapeptide is Activation Dependent and EDTA-Inducible (2017) Blood Advances 1:417-428.

Structure-Guided Drug Discovery and Chemotype Optimization

The recent high-resolution crystal structures of several GPCR or integrin types offer tremendous opportunities for computer-aided drug discovery/optimization approaches to discover novel and selective binders as some of our recent publications demonstrate. By combining our virtual screening, cheminformatics, and molecular dynamics simulations, with collaborative functional and structural studies, as well as chemical synthesis, we have recently contributed to the discovery of (i) a novel agonist of the kappa-opioid receptor (in collaboration with Jonathan A. Javitch at Columbia U. and Thomas Prisinzano at  Kansas U.); (ii) novel positive allosteric modulators of the delta-opioid receptor (in collaboration with Neil Burford, Andy Alt, and Samuel Gerritz at BMS, Merixtell Canals and Arthur Christopoulos at Monash U., and John Traynor at U. of Michigan); (iii) a μOR-δOR heteromer-biased agonist with antinociceptive activity (in collaboration with Lakshmi Devi at Mount Sinai and Peter Hodder at Scripps), and (iv) novel antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor, thus resulting in improved anti-platelets agents (in collaboration with Barry S. Coller at Rockefeller U. and Craig Thomas at the NIH). In particular, this latter discovery was protected by a patent application entitled: “Organic Compounds (Anti-platelet agents)” (Serial number: PCT/US2013/021749).


Representative Publications

Negri, A., Rives, M.L., Caspers, M.J., Prisinzano, T.E., Javitch, J.A., and Filizola, M. “Discovery of a Novel Selective Kappa-Opioid Receptor Agonist Using Crystal Structure-Based Virtual Screening” Journal of Chemical Information and Modeling (2013) 53: 521-526


Burford, N., Livingston, K., Canals, M., Ryan, M., Budenholzer, L., Han, Y., Shang, Y., Herbst, J.J., O’Connell, J., Banks, M., Zhang, L., Filizola, M., Bassoni, D., Wehrman, T., Christopoulos, A., Traynor, J., Gerritz, S., Alt, A. “Discovery, Synthesis and Molecular Pharmacology of Selective Positive Allosteric Modulators of the δ-Opioid Receptor” (2015) Journal of Medicinal Chemistry Apr 22. [Epub ahead of print]


Zhu, J., Choi, W.-S., McCoy, J.G., Negri, A., Zhu, J., Naini, S., Li, J., Shen, M., Huang, W., Bougie, D., Rasmussen, M., Aster, R., Thomas, C.J., Filizola, M., Springer, T.A., and Coller, B.S. “Structure-Guided Design of a High Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS” Science Translational Medicine (2012) 4(125):1-13.


Gomes, I., Fujita, W., Gupta, A., Saldanha, A.S., Negri, A., Pinello, C.E., Roberts, E., Filizola, M., Hodder, P., and Devi, L.A. “Identification of a μOR-δOR heteromer-biased agonist with antinociceptive activity” Natl. Acad. Sci. USA  (2013) 110(29):12072-7.


Jiang, J.-K. McCoy, J.G., Shen, M., LeClair, C., Huang, W., Negri, A., Li, J., Blue, R., Harrington, A., Naini,  S., David, G. III, Choi, W.-S., Volpi, E., Fernandez, J.,  Babayeva, M., Nedelman, M.A., Filizola, M., Coller, B.S., Thomas, C.G. “A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor” Bioorganic & Medicinal Chemistry Letters (2014) 24(4):1148-53


Li, J., Vootukuri, S., Shang, Y., Negri, A., Jiang, J.-K., Nedelman, M., Diacovo, T.G., Filizola, M., Thomas, C.J., Coller, B.S. “RUC-4: A Novel αIIbβ3 Antagonist for Pre-hospital Therapy of Myocardial Infarction” Arteriosclerosis, Thrombosis, and Vascular Biology (2014) Aug 21. pii: ATVBAHA.114.303724.


Burford, N., Livingston, K., Canals, M., Ryan, M., Budenholzer, L., Han, Y., Shang, Y., Herbst, J., O’Connell, J., Banks, M., Zhang, L., Filizola, M., Bassoni, D., Wehrman, T., Christopoulos, A., Traynor, J., Gerritz, S., Alt, A. “Discovery, Synthesis and Molecular Pharmacology of Selective Positive Allosteric Modulators of the δ-Opioid Receptor” (2015) Journal of Medicinal Chemistry 58(10):4220-9.


Bisignano, P., Burford, N.T., Shang, Y., Marlow, B., Livingston, K.E., Fenton, A.M., Rockwell,K., Budenholzer, L., Traynor, J., Gerritz, S.W., Alt, A., and Filizola, M. “Ligand-Based Discovery of a New Scaffold for Allosteric Modulation of the mu-Opioid Receptor” (2015) Journal of Chemical Information & Modeling 55(9):1836-43


Kruegel, A.C., Gassaway, M.M., Kapoor, A., Varadi, A., Majumdar, S., Filizola, M., Javitch, J.A., Sames, D. “Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators” (2016) Journal of the American Chemical Society 138(21):6754-64


Wardman, J.H., Gomes, I., Bobeck, E.N., Stockert, J., Kapoor, A., Bisignano, P., Gupta, A., Mezei, M., Kumar, S., Filizola, M., Devi, L.A. “Identification of a small molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake” (2016) Science Signaling 9(430):ra55.


Crowley, R.S., Riley, A.P., Sherwood, A.M., Groer, C.E., Shivaperumal, N., Biscaia, M., Paton, K., Schneider, S., Provasi, D., Kivell, B.M., Filizola, M., and Prisinzano, T.E. “Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Identification of a Potent and Centrally Acting μ Opioid Analgesic with Reduced Abuse Liability” (2016) Journal of Medicinal Chemistry, 59(24):11027-11038

Contributions to Team-Science Projects

Through application and implementation of cutting-edge developments in theory and computer simulations, my lab contributes a level of molecular detail to biological and biomedical problems that is impossible or difficult to obtain experimentally. This information lays the foundation for novel experimental studies aimed at furthering our understanding of physiological functions, and at developing new therapeutic strategies.


Representative Publications

González-Maeso, J., Ang, R., Yuen,T, Chan, P., Weisstaub, N.V., López-Giménez, J., Zhou, M., Okawa, Y., Callado, L.F., Milligan, G., Gingrich, J.A., Filizola, M., Meana, J.J., Sealfon, S.C. “Identification of a Novel Serotonin/Glutamate Receptor Complex Implicated in Psychosis” Nature (2008) 452(7183):93-97.


Guo, W., Urizar, E., Kralikova,M., Mobarec, J.C., Shi, L., Filizola, M., Javitch, J.A. “Dopamine D2 Receptors Form Higher Order Oligomers at Physiological Expression Levels” The EMBO Journal, (2008) Sep 3;27(17):2293-304.


Fribourg, M., Moreno, J.L., Holloway, T., Provasi, D., Baki, L., Mahajan, R., Park, G., Adney, S.K., Hatcher, C., Eltit, J.M., Ruta, J.D., Albizu, L., Li, Z., Umali, A., Shim, J., Fabiato, A., MacKerell, A.D. Jr., Brezina, V., Sealfon, S.C., Filizola, M., Gonzalez-Maeso, J., Logothetis, D.E. “Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs” Cell (2011) 147 (5):1011-102.


Coudray, N., Valvo, S., Hu, M., Lasala, R., Kim, C., Vink, M., Zhou, M., Provasi, D., Filizola, M., Tao, J., Fang, J., Penczek, P.A., Ubarretxena-Belandia, I., Stokes, D.L. “Inward-Facing Conformation of the Zinc Transporter YiiP revealed by Cryo-electron Microscopy” Proc. Natl. Acad. Sci. USA (2013) 110(6):2140-2145.


Yano, H., Provasi, D., Sheng Cai, N., Filizola, M., Ferré, S., Javitch, J.A., “Development of novel biosensors to study receptor-mediated activation of Gs and Golf” (2017) Journal of Biological Chemistry Dec 8;292(49):19989-19998.


Hughes, T.E.T., Lodowski, D.T., Huynh, K.W., Yazici, A., Del Rosario, J., Kapoor, A., Bashak, S., Samanta, A., Han, X., Chakrapani, S., Zhou, Z.H., Filizola, M., Rohacs, T., Han, S., Moiseenkova-Bell, V.Y. “Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM ” (2018) Nature Structural & Molecular Biology, 25(1):53-60