Vadim Cherezov - US grants

DESCRIPTION (provided by applicant): Experimentally determining the 3-D structure of the G protein-coupled receptor (GPCR), CXCR4 and its complexes, the overall goal of the proposed study, is critical in developing a deeper understanding of its role in HIV viral entry and toxicity;availability of these data is expected to facilitate the development of new therapeutic strategies. Although CCR5 is the primary co-receptor responsible for HIV transmission, emergence of CXCR4-tropic (X4-tropic) viruses later in infection, correlates with a more rapid CD4 decline and a faster progression to AIDS. CXCR4 is also implicated in neuroaids, triggering neuronal cell death and possibly causing onset of dementia. Known conformational flexibility of GPCRs as well as results from preliminary studies have shown that stabilization of the CXCR4 receptor is a significant bottleneck in attempts to crystallize and solve its structures. The two PI's of this research proposal, one a chemist, and the other a crystallographer, propose to develop new chemical tools optimized for structural studies of the HIV co-receptor CXCR4, attempting crystallization and structural determination of the receptors and its complexes. Chemical tools will be designed and optimized for stabilizing, purifying, and detecting the receptor and for measuring its functional behavior. R21 studies will have two aims: (1) Develop tool compounds optimized for structural studies of CXCR4 and (2) Validate new ligands, detergents, and lipids by assessing their impact on protein sample production processes and by producing diffracting crystals of CXCR4. Results from R21 studies will be used to achieve the following in R33 phase of the work: (3) Determine the high-resolution X-ray structure of CXCR4, and (4) Assess the utility of new molecular tools and technologies in stabilizing and crystallizing CXCR4 in complex with gp120. Success of the R21 studies will be achieved by the production of diffracting CXCR4 crystals along with the production of new compound tools that significantly improves receptor sample behavior (e.g. improved stability over time, increased Tm). PUBLIC HEALTH RELEVANCE: Availability of a 3D structure of CXCR4 along with its complexes will lead to developing a deeper understanding of how the HIV virus enters the cell and will help in the design of new therapeutics. CXCR4 is also involved in important physiological process and have been implicated in a number of diseases such as cancer and availability of its structure will likewise have important biomedical implications.

DESCRIPTION (provided by applicant): Crystallization of membrane proteins in lipidic mesophases (in meso) is a promising technique, which recently yielded high-resolution structures of human ?2-adrenergic and adenosine A2A G protein-coupled receptors. A distinct feature of the in meso crystallization is that the protein of interest is embedded into a highly curved and folded in space lipid bilayer, forming a lipidic cubic phase. The structure of the cubic phase imposes spatial constrains on diffusion of large proteins or oligomeric protein aggregates. Our preliminary data indicate that one of the primary reasons for failure of the in meso crystallization trials is due to a fast nonspecific protein aggregation. There is no visual feedback to such event, the cubic phase remains clear, because the size of the non-diffusing stuck protein oligomeric aggregates is well below 100 nm. The aggregation behavior of a protein depends on the particular protein construct, host lipid and additives employed for crystallization. We propose to develop an automated system for carrying out Fluorescence Recovery after Photobleaching (FRAP) studies of membrane proteins in meso. This system will be used to develop a pre-screening assay which will use less than 10 ?L of fluorescently labeled protein solution to screen for diffusion of the protein in the lipidic cubic phase incubated with 96 solutions of carefully selected common precipitants. The results of this assay will help to select suitable for in meso crystallization protein constructs and to eliminate certain precipitants from subsequent crystallization trials. Such crystallization pre-screening approach will significantly increase chances of obtaining initial crystal hit leading to the determination of more high-resolution structures of challenging membrane proteins essential in designing new and improved highly specific drugs with lesser side effects. PUBLIC HEALTH RELEVENCE (provided by the applicant): This research project will develop new technologies that will accelerate the success rate of membrane protein crystallization. These proteins are involved in important cellular and physiological processes and therefore are important drug targets. Crystallization will facilitate solution of three-dimensional structures of the proteins providing necessary templates for the rational drug development process.

DESCRIPTION (provided by applicant): This proposal is focused on the X-ray structure determination of a complex between the oxytocin receptor (OXTR), a member of the G Protein-Coupled Receptor (GPCR) family, and cholesterol, a ubiquitous lipid in mammalian cells known to modulate functions of a variety of membrane proteins. Significant efforts will be devoted to developments of technologies facilitating structure determination of membrane proteins through the use of the stabilizing effect of lipids. For a long time, the oxytocin system was linked with supporting two critical reproductive functions in mammals: uterine contraction during labor and milk ejection during lactation. Recently, a significant interest has emerged concerning the involvement of the OXTR in social and emotional behavior. The OXTR is activated by a cyclic nonapeptide hormone oxytocin. The OXTR has been demonstrated to have a remarkable sensitivity to the levels of cholesterol in the membrane. Depletion or addition of cholesterol reversibly transform the receptor between low (Kd>100 nM) and high (Kd~1 nM) affinity states. The main hypothesis of this proposal is that cholesterol modulates the activity of the OXTR by binding to a distinct allosteric site(s) thus inducing a change in the receptor conformation. The proposed structural and functional studies will take advantage of the Lipidic Cubic Phase (LCP) as a promising matrix for stabilizing and crystallizing difficult membrane proteins. The specific aims are: 1) Correlate the identity and amounts of natural lipids bound to GPCRs with successes of crystal nucleation and growth in LCP; 2) Establish the effect of cholesterol on modulating ligand binding affinities of the OXTR in LCP; 3) Obtain crystals of the OXTR in complex with cholesterol. For structural studies, we will follow the highly successful strategy of replacing the third intracellular loop of the receptor with T4 lysozyme combined with crystallization in LCP, which has recently yielded high resolution structures of human b2 adrenergic and adenosine A2A GPCRs.

DESCRIPTION (provided by applicant): This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipeline for the atomic-level structural characterization of membrane protein microcrystals grown in membrane-like environment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electron X-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals, effectively eliminate radiation damage issues through diffraction before destruction (i.e., diffraction data are collected prior to onset of any damage), simplify handling, as harvestig and freezing are not required, and significantly reduce the time from obtaining initial crystal hit to collecting full data sets. Our long term goal is the integration of this technology into our structural determination pipeline enabling the determination of a large number of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressing questions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Our goal will be achieved through the following specific aims. Aim 1: Develop protocols for the production of samples of GPCR-ligand complexes and for the generation, and characterization of large number of microcrystals that can be used for SFX studies. Aim 2: Develop protocols for SFX data collection, processing and structure solution of GPCR-ligand complexes. Aim 3: Integrate protocols developed in Aims 1 and 2 into the GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining the structure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largest family of membrane proteins in the human genome with approximately 800 members and are responsible for transmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses, including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; several GPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multiple effector pathways has profound therapeutic implications, which underscores the need to understand the receptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currently marketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limited by a lack of structural information. The structure determination of GPCRs is hampered by the difficulty of preparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high- resolution structure determination even when using state-of-the art microfocus beamlines at synchrotron sources.

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome with approximately 800 members. GPCR signaling through multiple effector pathways has profound therapeutic implications, making these receptors the targets of ~40% of currently marketed drugs. Modulation of GPCR activity by small molecule ligands is emerging as a new strategy for development of anticancer therapeutics. However, design of new drugs with higher efficacy and lower side effects, and detailed understanding of GPCR mechanism of action are hampered by the limited structural information. Structural studies of many GPCRs are in turn limited by a scarcity of high-affinity ligands that stabilize GPCRs and enable their crystallization. The project attempts to develop a new platform using the in situ click chemistry approach to discover novel ligands that bind GPCRs to serve as tool compounds for structural and functional studies or as starting point for drug discovery. Target-guided synthesis approaches including in situ click chemistry, by which reactive fragments are joined in the binding sites of a biological target, have shown great promise in drug discovery but have not been applied to GPCRs. The challenges lie in the GPCRs' membrane disposition, their high conformational dynamics, low stability and low expression levels. The proposed platform is being developed targeting all GPCRs, with initial focus primarily on those known to be cancer targets. In the two years of this study, we will focus on smoothened (SMO) receptor which is an orphan receptor and a key signal transducer in the Hedgehog (Hh) signaling pathway activated during development, and thus is a target of a number of antitumor drugs in clinical trials. Our goal will be attained by achieving the following two aims: (1) Establish an in situ click chemistry platform to generate high-affinity GPCR ligands, (2) Characterize the interaction between selected ligands from Aim 1 and SMO by crystallography. The study will be supported by a team that had developed the GPCR Structure Determination Pipeline as well as its underlying technologies which have led to the structure determination of 12 GPCR structures since 2007.

Production of large quantities of hiighly purified receptors is an underlying requirement for all the projects in ttie Program Project -Structure-Function of Opioid Receptors for Drug Discovery. The production of these samples will be carried out using well established protocols that have now been optimized for use with GPCRs as part of GPCR Network's GPCR Structure Determination Pipeline. These production protocols now include the use of newly developed GPCR fusion partner toolchest for stabilization and crystallization that has increased the quantity and quality of structures that we are able to determine. Most notably, with the use of a new fusion partner, apo-cytochrome b562 (RIL) mutant, BRIL, we were able to determine the structure of the A2A adenosine receptor- ZM241385 to 1.8 A resolution, the structure of NOP, and more recently 2 agonist bound serotonin receptors in an activated state. Structural studies will demand the use of large volumes of highly purified protein for crystallization studies while others doing functional studies will need much less. Studies that led to the structure solution of the K-opioid receptor and the nociception/oprhanin FQ peptide receptor required a large number of constructs and an average of 25 mgs of highly purified protein or about 100 liters of biomass. For receptor-ligand complex structural studies we estimate that 5-10 mgs will be required per structure.

We propose to develop a deeper understanding of the structural basis for the behavior/function of the opioid receptor subfamily of G protein-coupled receptors (GPCR). Structures of all three opioid receptors: ? -opioid receptor (MOR), ? -opioid receptor (KOR), ?-opioid receptor (DOR), and the opioid-like nociceptin/orphanin FQ opioid-like receptor (NOP), have recently been solved. This breakthrough created a unique opportunity to pursue a comprehensive understanding of ligand recognition and selectivity, as well as structural mechanisms of opioid receptor activation and biased signaling. Attaining this goal will require solving a large number of structures of ligand-receptor complexes similar to what is routinely done in structure based drug design targeting soluble proteins. This is now possible since our GPCR Structure Determination Pipeline (GSDP) and our newly developed GPCR Protein Fusion Toolchest has enabled large-scale structure determination of ligand-receptor complexes. The GSDP has been used by us in the successful structure determination of 14 different human GPCRs and their complexes, including two new receptors in activated states with bound agonist. In this proposed study, we will conduct similar efforts toward the crystallization and structural determination of ligand-receptor complexes of all of the receptors in the opioid family using agonists, antagonists, inverse agonists, and allosteric modulators. Criteria for opioid receptor and signaling pathway selectivity will be approached by attempting structure determination of several complexes of functional near-wild type constructs and rationally designed mutants. Within three specific aims we will solve several ligand-receptor structures on (1) KOR, (2) NOP, and (3) MOR and DOR.

Complex pharmacology of opioid drugs cannot be fully understood without deciphering molecular and structural details of drug-receptor interactions and functional mechanisms. This knowledge is critical for development of new generation of effective and side-effect free therapies for pain control, mood disorders and other indications. A new unique opportunity for such structure-based analysis is provided by recently solved crystal structures of all four subtypes of opioid receptors and continuing efforts for crystallographic characterization of their complexes with different ligands. These structures create a reliable 3-dimensional framework for computational analysis and modeling of ligand-receptor interactions and their functional and pharmacological consequences. The overall goal of this project is to decipher structural basis of opioid drugs action at atomic level and apply this knowledge to rationally design new tool compounds and candidate leads by using state ofthe art computational technologies for molecular modeling, ligand docking and virtual ligand screening. The specific Aims are (i) Development of 3D structural models of opioid receptors complexes with all known drugs and drug candidates, as well as systematic mapping of the ligand-receptor interactions for determinants of selectivity to a specific opioid subtype or specific function (agonism, antagonism, biased signaling); (ii) Virtual ligand screening of drug-like compounds against all opioid receptor subtypes in different functional states for discovery of new opioid ligand chemotypes; (iii) Computer-assisted structure- based design of lead compounds, including allosteric and bitopic ligands, for development new tool compounds for opioid receptors with desired functional and pharmacologic profile. Each Aim involves thorough experimental validation of the proposed hypotheses, as well as synthesis and biochemical/biophysical testing of the candidate ligands for binding and signaling properties by the collaborative Projects and the Chemistry Core. Receptor complexes for new compounds with most interesting structural and functional features will be tried for crystallization. Success of the program will not only result in new insights into drug action and lead to new opioid ligands with desired properties as tool compounds, but it will establish a platform for rational structure-based discovery of safer opioid therapies. RELEVANCE (See instructions): Development of new generation opioid medications with reduced risks of addiction, tolerance and other unwanted side effects is of paramount importance for pain and anxiety relief therapies. This project will combine molecular modeling with in vitro and in vivo experimental testing to decipher basic mechanisms of opioid drug action on their receptors and suggest molecular ways to separate their pain and anxiety relief effects from side effects. New knowledge will be applied to design of chemical compounds and peptides with specific functional features for pain research and pave the path for new candidate therapies.

Project Summary: This proposal outlines the development of a fundamentally new optogenetic technology capable of flexibly manipulating the activity of thousands of neurons contributing to the dynamic activity of distributed neural circuits with single neuron resolution. No method that currently exists even remotely meets the need of flexible, selective control of thousands of neurons distributed across large volumes of the brain. Filling this methodological gap is a central research objective of the BRAIN Initiative, because doing so will transform our ability to investigate how the nervous system encodes, processes, utilizes, stores, and retrieves information. The overall objective for this application is to acquire critical structural knowledge of photoactive states of a red-shifted channelrhodopsin and use these to engineer a photoselectable channel prototype that demonstrates the potential of our approach for future development in behaving animals. This would allow opsin-expressing neurons to be flexibly selected, activated, and deselected with light. By leveraging new structural knowledge, we anticipate that we can develop a fundamentally new approach to optogenetics that takes us beyond genetically targeted control and into an era of functionally targeted, flexible control of any neural ensemble. The aims of our research are to obtain the first atomic structures of red-shifted channelrhodopsin mutants in three channel states, engineer a three-state ReaChR mutant with high open conductance and optimized action spectra, and demonstrate reversible photoselective control of neurons in vivo with PReaChR prototypes. We anticipate that completion of these aims will yield the following expected outcomes. First, it will produce new knowledge of the underlying structural transformations between channelrhodopsin photostates that will enable efficient computational design of photoselectable optogenetic tools. Second, it will produce the first examples of photoselective channelrhodopsins useful for neural excitation. Third, it will assess the utility of these new opsins for flexible control of distributed sets of neurons. Collectively, these will provide a roadmap to extending the transformative new trait of photoselectabilty to a wide range of existing optogenetic tools for excitation, inhibition and modulation of neural activity. Further research in this direction should ultimately enable flexible control of spatially complex distributions of neurons in head-fixed and freely moving animals during behavior, a key to furthering our understanding of the intricate neural dynamics that underlie our thoughts, feeling, and actions and how circuit dynamics are disrupted by neurological disorders.

DESCRIPTION (provided by applicant): This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipeline for the atomic-level structural characterization of membrane protein microcrystals grown in membrane-like environment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electron X-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals, effectively eliminate radiation damage issues through diffraction before destruction (i.e., diffraction data are collected prior to onset of any damage), simplify handling, as harvestig and freezing are not required, and significantly reduce the time from obtaining initial crystal hit to collecting full data sets. Our long term goal is the integration of this technology into our structural determination pipeline enabling the determination of a large number of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressing questions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Our goal will be achieved through the following specific aims. Aim 1: Develop protocols for the production of samples of GPCR-ligand complexes and for the generation, and characterization of large number of microcrystals that can be used for SFX studies. Aim 2: Develop protocols for SFX data collection, processing and structure solution of GPCR-ligand complexes. Aim 3: Integrate protocols developed in Aims 1 and 2 into the GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining the structure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largest family of membrane proteins in the human genome with approximately 800 members and are responsible for transmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses, including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; several GPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multiple effector pathways has profound therapeutic implications, which underscores the need to understand the receptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currently marketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limited by a lack of structural information. The structure determination of GPCRs is hampered by the difficulty of preparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high- resolution structure determination even when using state-of-the art microfocus beamlines at synchrotron sources.

Abstract G protein-coupled receptors (GPCRs) constitute the largest membrane protein superfamily in the human genome, with over 800 unique sequences. GPCR-mediated signaling pathways play a key role in all physiological systems as well as many pathophysiological conditions, and therefore represent important drug targets. GPCRs have a seven-transmembrane-helix (7TM) topology and contain multiple binding sites for orthosteric ligands and allosteric modulators. Upon recognition of their native ligands receptors transmit signals across the cell membrane to intracellular partner proteins, such as G proteins or ?-arrestins. Developing a detailed understanding of functional mechanisms of GPCRs and facilitating design of novel drugs with high selectivity and potency require access to high-resolution three-dimensional structures, determination of which, however, remains a challenging task. We propose here a comprehensive research program which combines technology development with integrated structure-function studies focused on the GPCR superfamily. The proposed research directions are designed to accelerate high-resolution structure determination of membrane proteins, improve our understanding of the GPCR superfamily and answer specific questions on ligand specificity and selectivity, as well as molecular mechanisms of action using several specific receptors as targets. Our approach integrates structural information on new receptors and complexes with data obtained from biophysical, biochemical and functional experiments through computer-based analysis and modeling. The long-term goal of our laboratory is to develop a deeper understanding of the molecular mechanisms of action of GPCRs using the tools of structural biology, and to use the achieved insights to accelerate the design and development of novel and efficacious therapeutics.

Project Summary Neuropathic chronic pain affects ~20 million of Americans and bears more than US$500 Billion burden on the US economy. Moreover, the widespread use of addictive opioid painkillers for chronic pain is the major root of the opioid abuse epidemics threatening the whole society. As only one in four patients with neuropathic pain experiences pain relief with the current treatment options, discovery of new approaches to treating neuropathic pain is an unmet medical need with a major impact on society. Targeting pain in peripheral neurons is a key paradigm for effective and safe analgesia, which can naturally bypass the CNS-mediated side effects and addiction. One of the most promising and advanced peripheral targets, angiotensin AT2 receptor is involved in regulations of neuronal membrane excitability and neurite outgrowth of peripheral sensory neurons. Inhibition of AT2R in PNS has shown effect in preclinical models of neuropathic pain, as well as in phase II clinical trials, where the EMA401 drug demonstrated analgesia in patients with post-herpetic neuralgia. However, EMA401, which had modest potency and suboptimal drug-like properties, has been terminated in May 2019 due to off- target hepatotoxicity at high therapeutic doses. Lack of suitable AT2R candidates in pipeline calls for discovery and development of new highly potent and safe AT2R antagonists for neuropathic pain. We have established a structure-based drug discovery platform and used it to identify new lead chemotypes for AT2R antagonists. Our current lead has affinity (Ki =56 nM) on par with the previous clinical candidate, but much higher ligand efficiency, better drug-like properties and initial SAR suggesting high optimization potential. Moreover, recent breakthroughs in understanding the neuroimmune functional role of AT2R in neuropathic pain helped us to establish a set of cell-based functional assays, lack of which hampered previous development efforts. Using these platforms, we plan to establish comprehensive SAR for our main and backup lead series, and develop a screening funnel in the 1-year UG3 phase of the project. In close collaboration with the BPN steering committee, consultants and contractors, this screening funnel will then be employed in UH3 phase to optimize the lead potency, selectivity, ADMET and PK properties relevant for AT2R lead development, including peripheral restriction that precludes CNS side effects. This would allow selection of clinical development candidate for pre- development, IND-enabling studies and preparation of IND targeting post-herpetic neuralgia.