Project #2. Discovery and mechanistic understanding of functionally-selectiveD2 dopamine receptor ligands (Richard Mailman, Project PI)A.
SPECIFIC AIMS The idea that some ligands can selectively activate only some of the signaling pathways mediated by asingle receptor isoform ('functional selectivity') is a novel concept that has markedly changed classic conceptsof receptor pharmacology. During the past few years, there has been overwhelming support for this conceptfrom more than a dozen receptor systems. We, and almost all of the other labs who have worked in this arena,have recognized that it has profound implications for drug discovery. As discussed in the overall introduction tothis application, the functional selectivity hypothesis predicts that functionally selective drugs (i.e., those thatdifferentially activate (or inhibition) signaling pathways linked to a single receptor) will be differentiated not onlyby their behavior in vitro, but that this will lead to different behavioral and physiological effects seen bothpreclinically and clinically. This project will focus on the dopamine D2L receptor, and utilize a battery ofsignaling assays which are independent (or largely independent) of each other. These assays allow thedifferential classification of functionally selective properties, and allow the rational selection of agents withnovel properties. These assays, as well as new assays in development, will allow for the discovery andrational classification of novel functionally selective dopamine agonists. Novel leads identified from in vitroscreening campaigns will be profiled computationally and physically for off-target actions and suitably selectivecompounds will subsequently be evaluated in a variety of behavioral assays that will offer clues predictive ofpotential therapeutic action.We recognize that the current state-of-the-art is not adequately advanced such that it is possible topredict from a novel pattern of signaling activity seen in vitro whether the candidate ligand might have clinicalsuperiority in discrete domains of psychopathology. In this regard, however, the examination of interestingcandidate compounds both in traditional behavioral models and in mice with specific genetic alterations willprovide a powerful way to select compounds that might become drug candidates. As an example, one inherenthypothesis is that functionally selective drugs can be discovered that cause significant facilitation ofdopaminergic functions that modulate working memory, while still effectively attenuating dopaminergic functionthat decreases positive symptoms of schizophrenia. The foundation of research from the past two decadesmakes us believe that we can confirm this optimistic hypothesis. We shall pursue this via the following aimsthat are heavily integrated with Projects 1 and 3, and will make use of both scientific cores.1.
AIM 1. CHARACTERIZE EXISTING AND DISCOVERY COMPOUNDS FOR FUNCTIONALLYSELECTIVE PROPERTIES IN NON-NEURONAL D2L DOPAMINE RECEPTOR SYSTEMSHypothesis: A battery of signaling assays that are independent or largely independent of each other willdifferentiate drugs with functionally selective properties, and allow selection of agents with novel properties.Corollary: The pattern of signaling effects will differentiate the ligands one from the other and be predictive ofpreclinical behavioral and clinical differences, although not necessary clinical superiority. The goal is todiscover novel functionally selective D2 signalers that can provide new lead molecules, and/or ligands that maybe useful research tools. The broad array of existing D2L functional assays are expressed in non-neuronal celllines (CHO and HEK). Although it would be ideal to know the exact mechanisms about how the D2L receptorsignals in situ in primate tissue, this knowledge does not currently exist (although it will be explored in Project1. On the other hand, the current battery can discriminate functionally selective D2 ligands that have uniquebehavioral properties in animals and humans, providing reasonable evidence of validity.We shall focus on relatively small subsets of rationally selected compounds from our Wyeth coinvestigators,from compounds from our recent research, from promising targets we have found in the publicdomain, and from selected structures available in commercial libraries predicted from our computationalapproaches. Reference compounds will be dopamine and two 'typical' D2L agonists (quinpirole and RNPA), aswell as known functionally selective compounds with different patterns of activity (e.g., aripiprazole and S-3-PPP). The compounds will be analyzed in four receptor-mediated functional assays (inhibition of adenylatecyclase, stimulation of MAPK kinase, arachidonic acid release, and stimulation of GTPase binding). Emax andED50 values will be determined, and functionally selective ligands for detailed study by this Project andProjects 2 and 3 will be those in which there is greater than a 30% difference intrinsic activity in one or morefunctional endpoints versus the others, or when the ED50 changes by more than ten-fold relative to thereference compounds.Finally, these assays are currently semi-automated, and provide adequate throughput for the focusedtypes of studies we are making now. In parallel, we shall make efforts to increase their throughput rate (withoutdecreasing either accuracy or precision), such that they can be applied to higher throughput analysis for theD2i_ and other receptors.2.
AIM 2 : DETERMINATION OF THE STRUCTURAL BASIS FOR ACTIVATION AND FUNCTIONALSELECTIVITY OF D2L RECEPTORS.Our hypothesis that functional selectivity results from the ligand-unique sets of induced conformations(rather than selection of discrete active states) makes it important to understand some of the structuraldeterminants that are involved. Thus, we hypothesize that functional selectivity can result either fromconformational perturbation of the D2|_ receptor done by ligands 'sterically' (close to the residues that binddopamine) and/or 'allosterically' (involving both the steric sites and aspects of the receptor not normallyengaged by dopamine).
This aim will elucidate some of the subtle structural interactions that differentiate hownon-selective and functionally selective ligands affect the D2i_ receptor, and how this results in selectiveactivation of specific effector pathways. Computationally-predicted mutations of the D2|_ receptor will be made,and ligand analysis conducted using rigid or semi-rigid compounds that minimize possible clocking poses, aswell as functionally selective ligands emerging from Aim 1.3.
AIM 3. DEVELOP NEURONALLY-RELEVANT CELL SPECIFIC ASSAY SYSTEMSProject #1 seeks to develop physiologically relevant ex vivo models for screening for functionallyselective drugs. In parallel, we wish to find immortal cell lines that functionally mirror dopamine neurons. Priorwork with the MN9D cell line has shown that these cells provide excellent predictability on which functionallyselective drugs produce novel behavioral characteristics in vivo. Unfortunately, we have found this like to beunstable, and seek a line that can replace it. We have identified two excellent candidates for our purposes(N27 and CAD), and we shall first characterize these lines for appropriate phenotype, and for their stabilitywhen molecularly-manipulated.
| Che, Tao; Majumdar, Susruta; Zaidi, Saheem A et al. (2018) Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor. Cell 172:55-67.e15 |
| Wang, Sheng; Che, Tao; Levit, Anat et al. (2018) Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone. Nature 555:269-273 |
| Rose, Samuel J; Pack, Thomas F; Peterson, Sean M et al. (2018) Engineered D2R Variants Reveal the Balanced and Biased Contributions of G-Protein and ?-Arrestin to Dopamine-Dependent Functions. Neuropsychopharmacology 43:1164-1173 |
| McCorvy, John D; Butler, Kyle V; Kelly, Brendan et al. (2018) Structure-inspired design of ?-arrestin-biased ligands for aminergic GPCRs. Nat Chem Biol 14:126-134 |
| McCorvy, John D; Wacker, Daniel; Wang, Sheng et al. (2018) Structural determinants of 5-HT2B receptor activation and biased agonism. Nat Struct Mol Biol 25:787-796 |
| Peng, Yao; McCorvy, John D; Harpsøe, Kasper et al. (2018) 5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology. Cell 172:719-730.e14 |
| Wang, Sheng; Wacker, Daniel; Levit, Anat et al. (2017) D4 dopamine receptor high-resolution structures enable the discovery of selective agonists. Science 358:381-386 |
| Wacker, Daniel; Wang, Sheng; McCorvy, John D et al. (2017) Crystal Structure of an LSD-Bound Human Serotonin Receptor. Cell 168:377-389.e12 |
| Wacker, Daniel; Stevens, Raymond C; Roth, Bryan L (2017) How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170:414-427 |
| Arnsten, Amy F T; Girgis, Ragy R; Gray, David L et al. (2017) Novel Dopamine Therapeutics for Cognitive Deficits in Schizophrenia. Biol Psychiatry 81:67-77 |
Showing the most recent 10 out of 136 publications
