Vsevolod Katritch - US grants

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.

? DESCRIPTION (provided by applicant): Opioid analgesics are the most efficient tool in pain management; however, high tolerance and addiction liabilities also make them the most abused class of medication, resulting in millions of cases of drug overdose and more than 15,000 deaths in the US each year. Specific modulation of d-opioid receptor (DOR) represents one of the most promising strategies to control tolerance and addiction to opioid drugs; however, only a few chemotypes of DOR-selective compounds have been explored. A new unique opportunity to expand the repertoire of DOR compounds by discovery of novel chemotypes with desirable functional profiles is enabled by structural characterization of opioid receptors, including new high resolution structures of DOR/naltrindole complex at 1.8 Å, and DOR with bifunctional peptide DIPP-NH2. The structures reveal atomic details of ligand binding and provide a reliable 3D framework for rational discovery of new ligands targeting orthosteric or allosteric pockets of DOR. The project will primarily focus on two therapeutically beneficial profiles: (a) bifunctional MOR agonists/DOR antagonists known to reduce tolerance and side effects of opioid therapy, and (b) allosteric ligands that modulate DOR activity only in the presence of endogenous opioid. In the exploratory R21 phase of the project we will perform large scale structure-based virtual ligand screening of more than 6 million available compounds to select small sets of candidate hits, and then experimentally test and identify new bifunctional and allosteric ligands. The most promising chemical scaffolds, and validated structural models will be employed then in an iterative lead optimization at the R33 phase. Both initial screening and lead optimization phases will employ state-of-the-art structure-based approaches which have proven to be highly effective in recent applications to GPCRs. Success of the program will result in new insights into drug action, lead to new DOR ligands with desired properties, and establish a solid platform for rational structure-based discovery of safer and less addictive opioid 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.

PROJECT SUMMARY The central focus of this Multi-PI R01 research proposal is the structure-function characterization of the human cannabinoid receptor 2 (CB2), a key protein component of the endocannabinoid system. We aim to develop a fundamental understanding of the structural basis of CB2 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program. The ECS is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes. There have been important implications that targeting CB2 may be useful as a means for treating inflammation, pain, neurological disorders and addiction. As with other G protein-coupled receptors (GPCRs), CB2 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to refine therapeutic approaches, to improve beneficial properties, and reduce side effect liability. The study will provide the structural basis for the design and development of pharmacologically distinct CB2-selective compounds as useful biological probes and/or leads for the future development of therapeutics. To enhance our effort in obtaining high quality crystal structures, we shall use carefully designed ligands with high affinities and selectivities for CB2, and which are also capable of tight attachment at or near the receptor?s binding domain(s) coupled with their abilities to form crystallizable ligand-receptor complexes. The study has three specific aims: (1) Design and synthesize novel irreversible ligands representing key classes of CB2 selective compounds with distinct functional profiles. (2) Extensive characterization of the newly synthesized ligands in order to identify compounds with pharmacologically diverse profiles, including the partial agonists, inverse agonists, neutral antagonists and allosteric modulators. The crystallization candidates and their chemical derivatives will also be characterized for their reversible binding nature using functional assays. (3) Develop a clear understanding of CB2 ligand binding sites by determining the 3-D structures of the several receptor-ligand complexes. Towards these goals, several crystal structures will be solved to better understand molecular recognition, signaling, and to assist in the design of novel compounds that could then serve as prototypes for later generation leads and drug candidates.

? DESCRIPTION (provided by applicant): Structure-function characterization of a key protein component of the endocannabinoid system, the human cannabinoid receptor 1 (CB1), is the central focus of this research proposal. It aims to develop a fundamental understanding of the structural basis of CB1 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program based on experimentally determined 3-dimensional structures. The endocannabinoid system is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes, including nociception, inflammation, sleep, and drug addiction. As with other G protein coupled receptors, CB1 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to discover new medications with improved pharmacological profiles, enhanced therapeutic properties and reduced side effects. The study will provide the structural basis for the design and development of functionally distinct CB1 selective compounds as useful pharmacological tools and/or leads for the future development of therapeutics. Several crystal structures will be solved to better understand molecular recognition, signaling, and to assist in the design of novel compounds that could then serve as prototypes for later generation leads and drug candidates. The study has three specific aims: (1) Design and synthesize covalent ligands representing key classes of cannabinergic ligands that have been shown to have distinct functional profiles, (2) Develop a better understanding of the CB1 orthosteric binding site by solving the 3D structure of several receptor-ligand complexes, and (3) Develop a better understanding of the CB1 active state by solving the structure of the CB1 signaling complex.