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Project Number16-14-10273

Project titleStructural and Functinal Study of Human Endothelin Receptors

Project LeadCherezov Vadim

AffiliationMoscow Institute of Physics and Technology,

Implementation period2016 - 2018

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-202 - Proteomics; structure and functions of proteins

KeywordsStructural biology, molecular mechanisms of function, G protein coupled receptors (GPCR), lipidic cubic mesophase, drug design, protein expression, X ray crystallography, optical spectroscopy, superresolution microscopy, endothelin receptors, endothelin



The proposed project is focused on structural and functional studies of the human peptide-binding G protein-coupled receptor (GPCR) from the Endothelin family, ETB, and on the structure-based development of early drug-like candidates for this receptor. Endothelin receptors respond to 21-mer endogenous cyclic peptides and control various physiological processes including vasoconstriction, vasodilation, and stimulation of cell proliferation. They are implicated in hypertension, atherosclerosis, heart failure, renal disease and various forms of cancer. Despite over 25 years of extensive studies and generation of about 1000 small molecule ligands targeting endothelin receptors, there have been only two drugs approved, while many others have failed in clinical trials, mostly due to their poorly understood and complex biological action. Proposed in this project structural studies combined with extensive biophysical, biochemical, and pharmacological characterization should help to develop a better understanding of these receptors and answer a number of important questions, such as (i) what is the size, shape and location of the orthosteric binding site and what is the nature of ligand-protein interactions, (ii) are there any allosteric binding sites and what are their properties, (iii) what is the structural basis for long-lasting agonism, (iv) do these receptors form homo or hetero dimers and what is their biological significance, and (v) are there any specific structural signatures of functionally biased states. The knowledge obtained during this project will be used to design, synthesize, and characterize novel small molecule ligands, which will serve as initial candidates for further drug development. Along with tackling very important and difficult problems aimed at improving human health, this research project will help fostering a close international collaboration between recently established Laboratory for Structural and Functional Studies of GPCRs at the Moscow Institute of Physics and Technology and the GPCR Network center (, based at the University of Southern California, USA. The GPCR Network is a world leading research community focused on understanding human GPCR biology and using this information for designing efficient drug candidates with reduced side-effects. Through this project Russian scientists will obtain a rare and unique opportunity to engage in the cutting-edge research and to have access to the expertise, protocols and tools developed by the GPCR Network.

Expected results
The project will generate new scientific knowledge about the fundamental structural and functional properties of human endothelin receptor B. The location and the shape of the key ligand-binding sites will be identified, and potential drug candidates aimed at them will be proposed. The molecular mechanisms of these receptors will be established. The data obtained will serve as a basis for the rational design and development of new pharmaceutical products aimed at treating such diseases as hypertension, chronic renal failure, autoimmune diseases and cancer. For this reason, the work will be of great importance for medicine and pharmacology, as well as for the whole society. Furthermore, the obtained structural information will be used to develop new strategies to minimize side effects of drugs acting on the endothelin receptors and other GPCRs, and, thus, will lead to significant improvements in the quality of patients lives. In addition, the development and optimization of our integrated methodological platform will enable future projects in the field of structural and functional studies of other GPCR receptors. The obtained results will be presented in at least eight (8) publications in peer-reviewed journals indexed in the Web of Science or Scopus. In addition, we plan to submit at least six (6) abstracts to international conferences.



Annotation of the results obtained in 2018
During 2018 year in the frame of this project we conducted comprehensive studies of GPCR receptors associated with the pathogenesis of inflammatory and autoimmune diseases: 1. Processed and systematic experimental X-ray structural data obtained. Data processing was carried out: scaling, integration, search for isomorphic datasets, association. This phase included additional rounds of sample preparation, functional testing, crystallization, and data collection. 2. The structure of target receptors was deciphered, on the basis of diffraction data: the search for initial phases was carried out using the molecular replacement method, the structure was refined, and a high-resolution model was constructed. 3. A number of single mutant receptors have been prepared based on the structures obtained for validating the structures and for obtaining additional information about the ligand binding mechanisms and signal transduction. 4. Analysis and comparison of structures with different ligands was carried out. The mechanism of ligand recognition and receptor activation has been determined. 5. A virtual screening of ligands was carried out on the basis of the obtained structures for the discovery of new basic chemical compounds for the development of new generation drugs. Based on the results of the work, 4 publications were prepared.



1. Андрей Ищенко, Корнелиус Гати, Вадим Черезов Structural biology of G protein-coupled receptors: new opportunities from XFELs and cryoEM Current Opinion in Structural Biology, Volume 51, August 2018, Pages 44-52, Current Opinion in Structural Biology, Volume 51, August 2018, Pages 44-52 (year - 2018).

2. Попов П., Пенг Я., Шен Л., Стивенс СР., Черезов В., Лиу ЖЖ., Катрич В. Computational design of thermostabilizing point mutations for G protein-coupled receptors eLife Sciences Publications, Ltd, eLife 2018;7:e34729 (year - 2018).

3. Мартин Одэ, Кэйт Л. Вайт .... Петр Попов.... Всеволод Катрич, Вадим Черезов... Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor Nature Chemical Biology (2018), (year - 2018).

4. - Учёные выявили особенности белков, позволяющие точно нацеливать лекарства пресс-релиз МФТИ, - (year - ).

5. - «Карман» для мизопростола: ученые установили 3D структуру комплекса лекарства с рецептором пресс-релиз МФТИ, - (year - ).

6. Эмили Неве, Петр Попов, Александр Гофман, Анджело Миглиози, Ксавье Бессерон, Грегуар Даной, Паскаль Буври, Сергей Грудинин RapidRMSD: Rapid determination of RMSDs corresponding to motions of flexible molecules Bioinformatics, Oxford University Press (OUP), - (year - 2018).

7. Гусач A., Лугинина A., Ляпина E., Шевцов M., Сафронова N., Хорн P. , Борщевский V., Мишин A., Черезов V. Optimization of G protein-coupled receptor expression for functional studies Journal of Bioenergetics and Biomembranes, - (year - 2018).

8. Марьин E., Гусач A., Лугинина A., Ковалев K., Лиу В, Виершталь У., Hyun Nam K., Cho Y., Мишин A., Борщевский В., Черезов В. Successful GPCR structure determination using PAL XFEL Journal of Bioenergetics and Biomembranes, - (year - 2018).

Annotation of the results obtained in 2016
• An analytical review of published data, scientific and technical developments and patents in the field of structural and functional studies of endothelin G protein-coupled receptors has been prepared, and detailed plans and strategies for new experiments have been developed • An ICM software package from MOLSOFT ( has been selected for designing receptor constructs and computer modeling of protein-ligand interactions. • Three young researchers-project participants received practical training in the Bridge Institute at the University of Southern California in Los Angeles, USA. Purified samples of stabilized engineered endothelin receptor constructs for crystallization have been prepared and characterized: • Twenty genetically engineered constructs for crystallization of endothelin receptors have been cloned. • Engineered endothelin receptor constructs have been expressed in insect cells using the baculovirus-based system. • Receptor expression, solubilization and purification have been optimized. Purified receptor constructs have been characterized for their homogeneity and thermal stability. Effects of different ligands on the receptors' stability have been evaluated. • The mobility of fluorescently-labeled receptors in the crystallization matrix - lipidic cubic phase has been analyzed using the fluorescence recovery after photobleaching method (LCP-FRAP), and the most promising constructs, ligands and other conditions have been selected for subsequent crystallization trials.



1. Р. Асташкин, Н.Вартанян, А.Гусач, А.Лугинина, М.Эргашева, М.Шевцов, А.Мишин, В.Борщевский, В.Черезов Pre-crystallization assays of an engineered human endothelin receptor type B. The FEBS journal, Volume 283, S1, p.292 (year - 2016).

Annotation of the results obtained in 2017
During 2017 year in the frame of this project we conducted comprehensive studies of GPCR receptors associated with the pathogenesis of inflammatory and autoimmune diseases: A. Conducting functional tests for comparison of the obtained recombinant receptors with native ones. A1. Use of microscale thermophoresis and surface plasmon resonance methodologies to determine the binding constant of obtained recombinant receptor constructs with the native receptor ligand. A2 Measurement of protein expression and fluorescently labeled receptor localization using flow cytometry techniques A3. Carrying out of cellular functional tests using FRET-based second messenger assays.. B. Crystallization of target receptors and study of crystals: B1. Crystallization tests, search for optimal conditions for crystallization. B2. Characterization of the diffraction quality of crystals, determination of the maximum resolution of the diffraction pattern for the resulting crystals. B3. X-ray analysis of the obtained crystals on microfocus beamlines at the third generation European Synchrotron Radiation Facility (ESRF, Grenoble, France) and PETRA III (Hamburg, Germany). Based on the results of the work, 4 publications were prepared.



1. Попов П.А., Грудинин С. Eurecon: Equidistant Uniform Rigid-body Ensemble Constructor Journal of Molecular Graphics and Modelling, - (year - 2018).

2. Peng Y., McCorvy J.D., Harpsoe K.,Lansu K., Yuan S.,Popov P., ......... Bjorn-Yoshimoto W. E., Ding K., Wacker D., Han G. W.,Cheng J., Katritch V., Jensen A. A., Hanson M. A., Zhao S., Gloriam D.E., Roth B. L., Stevens R. C., Liu Z.-J. The Structural Basis of G Protein-Coupled Receptor Polypharmacology Cell, - (year - 2017).

3. E. Lyapina, N. Safronova, A. Burdakova, A. Gusach, A. Luginina, M. Semelina, R. Astashkin, M. Shevtsov, A. Mishin, V. Cherezov Crystallization trials of human endothelin receptor B The FEBS Journal, Volume 284 Supplement 1 September 2017 (year - 2017).

4. I. Melnikov, V. Polovinkin, K. Kovalev, I. Gushchin, M. Shevtsov, V. Shevchenko, A. Mishin, A. Alekseev, F. Rodriguez-Valera, V. Borshchevskiy, V. Cherezov, G.A. Leonard, V. Gordeliy, A. Popov Fast iodide-SAD phasing for high-throughput membrane protein structure determination. Science Advances, Sci Adv 3: e1602952; (year - 2017).