Functional selectivity explains the capacity of a ligand to preferentially orientate the coupling of a GPCR with a subset of G proteins or other responses as well, such as β-arrestin recruitment. Structural studies of individual ternary complexes and the creation of functionally selective molecular probes will provide a mechanistic basis by identifying pathway specific receptor conformations. This will help to develop functionally selective drugs displaying increased clinical efficacy with lower unwanted side effects.
The D2 dopamine receptor, which is of particular relevance for the treatment of neuropsychiatric diseases, exemplifies a number of aspects of functional selectivity at different signaling readouts. The atypical antipsychotic properties of aripiprazole were associated to functional selectivity at the D2 receptor. We have synthesized various aripiprazole derivatives including the azaindole derivative FAUC238, which turned out to behave as a partial agonist in Gi activation and as a full agonist in the stimulation of Go. Additionally, FAUC238 did not show any D2 promoted internalization indicating functional selectivity between G protein coupling and β-arrestin recruitment. Very recently, we contributed to the discovery of PZM21 a G protein-biased agonist at the m-opioid receptor. Interestingly, PZM21 was very efficacious in analgesia but was devoid of both respiratory depression and morphine-like reinforcing activity in mice (A. Manglik et. al, Nature 2016).
To better understand and to rationally control the phenomenon of functional selectivity, we aim to synthesize novel molecular probes and lead compounds. Because docking studies displayed spatial proximity with amino acid residues of the binding pocket that are expected to be crucial for receptor activation, our initial synthetic efforts will focus on bioisosteric replacement and conformational restriction of the ligands’ bioactive conformation.
In-vitro profiles of the target compounds will be studied by radioligand binding, GTPγS assays and β-arrestin recruitment studies. Additionally, diagnostic mutants will be designed and characterized to discover receptor - ligand contacts that are responsible for functional selectivity. An extension of the programme towards the discovery of functionally selective ligands for other receptors is envisioned.
Research stay abroad
The qualification programme will facilitate that the graduate(s) conduct internalization and in vivo studies using diagnostic knock out animals in Jennifer Whistler's laboratory (UCSF).
Histamine is a neurotransmitter and local mediator and exerts its effects through four histamine receptor subtypes (H1R, H2R, H3R, H4R). After discovery of the H3R and the H4R, the purported H2R selectivity of numerous compounds turned out to be compromised. Moreover, besides the selectivity for the H4R vs. the closely related H3R, the activity ratios at H2R vs. H4R proved to be a critical issue, especially when different species were considered.
The H4R is assumed to play a proinflammatory role in various diseases. The analysis of the (patho)physiological role of the H4R and its validation as a drug target in translational animal models are seriously hampered by substantial species-dependent differences regarding potencies, receptor selectivity and even by opposite activities of available ligands. Generally, the responses to H2R or H4R ligand treatment strongly depend on species, test model and readout, and there is strong evidence of functional selectivity in both classes of compounds.
The project is aiming at optimized selective H2R and H4R agonists to explore ligand-receptor interactions more closely, to gain a deeper insight into molecular determinants of subtype- and species-selectivity of ligands and to provide validated pharmacological tools, including fluorescent and radiolabeled compounds, for in vitro and in vivo studies. The medicinal chemistry part comprises bioisosteric concepts, preparation of conformationally constrained compounds and stereoisomers to identify optimal 3D structures, development of bivalent and hybrid agonists and the synthesis of radio- and fluorescence-labelled ligands. Supported by molecular modelling, the key amino acids involved in species- and HxR subtype-selective interactions will be identified by radioligand binding and functional assays (GTPase-, GTPγS binding-, gene reporter- and fluorescence/bioluminescence-based assays; arrestin assays) using recombinantly expressed wildtype and mutant human and orthologous (mouse, rat, dog, guinea pig) HxR subtypes.
The project is divided into two parts:
B.1 will focus on H2R selectivity, functional selectivity and the exploration of the binding mode of bivalent ligands with emphasis on the target side, but also include the synthesis of special tools (e.g. radiolabeled bivalent ligands, irreversibly binding or fluorescent agonists).
B.2 will focus on H4R agonists, functional selectivity and molecular determinants of ortholog selective interactions. Ligands for both, human and murine H4R, will be considered, taking into account the differences in potency and efficacy and HxR subtype-selectivity of currently available compounds.
Research stay abroad
The graduate(s) will have the opportunity to work, e. g., in the laboratory of Takeaki Ozawa (University of Tokyo, Japan) to learn and apply the technique of engineering self-associating complementary proteins to establish cell-based (e.g. arrestin) assays.
Numerous ligands of the four histamine receptor subtypes are reported in literature. Over the last 10 years, a large number of different H4R ligands, like benzimidazoles, aminopyrimidines and quinazolines have been developed. Due to distinct differences in amino acid sequence between hH1R and hH4R, these H4R ligands are usually devoid of affinity to H1R. However, very few compounds are known to possess affinity to both, H1R and H4R, for example, variousdibenzo[b,f][1,4]oxazepine and quinazoline derivatives. Besides, bivalent approaches have often been applied to modify the affinity and selectivity of GPCR ligands. The H4R is discussed to be in-volved in inflammatory processes. Therefore, the H4R is regarded as a drug target for the treatment of immune diseases, like asthma, allergy and autoimmune disorders. Furthermore, a synergistic effect between H1R and H4R is described. Thus, it is a worthwhile goal, to develop dual H1/H4receptor ligands and to identify key amino acids for H1R and H4R selectivity, respectively.
Previously, aiming at dual H1/H4 receptor ligands, two different strategies have been explored: Firstly, H1 and H4pharmacophores were connected by spacers of different chemical structures and lengths. Secondly, clozapine-like moieties, supposed to possess both, H1 and H4 receptor affinity, were used as core structure.
This project is concerned with the synthesis, pharmacological characterisation and the elaboration of the structure-activity relationships of dual histamine H1/H4 receptor ligands. Mutant receptors will be generated to identify the molecular determinants of hH1R and hH4R selectivity, and molecular modelling studies will be performed to explain the pharmacological data and to suggest new dual H1/H4R ligands:
To increase the understanding of the pharmacological selectivity profile of the H1R and H4R, mutagenesis studies of hot-spot amino acids in the orthosteric binding pocket of the H1R and the H4R should be performed. After generation of these mutants , in-house H1R and/or H4R ligands should be characterized pharmacologically at these receptor mutants by radioligand competition binding and GTP[gS] binding assays.
Research stay abroad
The graduate(s) will have the opportunity to work in the laboratory of Irving W. Wainer (Gerontology Research Center, Baltimore) to transfer ‘the immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening’ to the H1R/H4R system.
The family of muscarinic acetylcholine receptors (mAChR or MR) comprises five receptor subtypes (M1 - M5), which control numerous physiological functions and were found to be involved, e.g., in neurological disorders. As the orthosteric binding pocket of MRs is highly conserved, the development of subtype-selective MR ligands has been very challenging. Highly selective MR agonists and antagonists are needed as pharmacological tools and potential therapeutic agents.
The project is aiming at the synthesis of homo- and heterodimeric MR ligands by connecting known MR binding moieties either directly or through linkers of different size and rigidity. This approach should alter the MR selectivity profiles, as already demonstrated for other GPCRs. The MR subtype selectivity of the new bivalent ligands will be investigated using radioligand competition binding and functional assays (e.g. Ca2+-assay). The design of the structures and the exploration of binding modes will be supported by computational chemistry based on reported crystal structures of MRs. The project also includes the preparation and pharmacological characterization of bivalent radioligands as special molecular tools.
Research stay abroad
The graduates will have the opportunity to work with Roger Read (UNSW, Sydney; organic synthesis) or with Arthur Christopoulos (Monash University, Melbourne, Australia; pharmacology of MR ligands, allosteric modulation).
Dysfunctions in the chemokine receptor CXCR3 signalling are associated with numerous pathologies including autoimmune diseases, cancer, vascular disease and transplant rejection. The efforts so far failed to produce therapeutics, which would specifically modulate the activity of CXCR3 and contribute to the symptom relieve or cure.
With the intention to develop a novel medicinal chemistry strategy, we focus our work on the development of molecular tools for the investigations of allosterism at CXCR3. The structure-activity-relationship studies on allosteric modulators needs to differentiate chemical modifications that influence compound affinity from those affecting the cooperatively exhibited towards the orthosteric ligand, as the two properties are not correlated. The existence of ‘molecular switches’, which are responsible for the switching between positive and negative efficacy of allosteric modulators, represents a further challenge. We will focus on the development of novel negative allosteric modulators that will selectively inhibit CXCR3, and the identification of crucial molecular determinants involved in the allosteric modulation of CXCR3. The main task of this subproject is thus de novo synthesis of allosteric modulators targeting CXCR3 and their precise functional categorization combined with the site directed mutagenesis to determine crucial amino acid residues involved in the ligand-receptor interactions.
Research stay abroad
A research visit of the Christopoulos group at the Monash University (Melbourne, Australia) will enable further theoretical and experimental dissection of allosteric modulation of CXCR3.
Project A6 has made great progress in characterizing the binding sites and mechanisms of action of the vasopressin receptor with its endogenous ligand  and investigating the cooperativity between ligand binding and the presence of an intracellular binding partner such as a G-protein, b-arrestin or the protein nanobodies often used to obtain crystals of activated receptors. Using modern metadynamics simulations, [2-4] we have been able to predict both ligand binding free energies and the effect (agonist, antagonist, partial agonist, or reverse agonist) of small molecules for a variety of receptors using both X-ray structures and homology models for the receptors
The project will now be extended to investigate the generality of the multi-site mechanism first found for the vasopressin receptors and to provide an in-depth mechanism for both unbiased and biased activation of different signaling pathways. Large grants (a total of 85 million cpu hours) of computer time on the SuperMUC machine at the Leibniz Rechenzentrum in Munich has been obtained for the project.
Research stay abroad
Research stays abroad are planned University College London to work with Prof. Francesco Gervasio, who has pioneered the use of metadynamics in predicting ligand-binding thermodynamics and kinetics.
The structurally related 36 amino acid (aa) peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) bind to GPCRs of the NPY receptor family and are involved in the regulation of numerous physiological processes such as blood pressure, food intake, pain sensitivity, anxiety/anxiolysis and hormone release. Among the functionally expressed NPY receptor subtypes in humans (Y1, Y2, Y4 and Y5 receptor) the Y4 receptor (Y4R) is unique due to preferential binding of PP. In contrast to the Y1, Y2 and Y5 receptor, there is still a lack of potent and selective Y4R agonists and antagonists, compounds, which are needed, e.g., as pharmacological tools for detailed investigations on the physiological role of the Y4R.
The aim of the project is the development of peptidic and non-peptidic selective Y4R ligands (agonists and antagonists), driven by structure-activity/selectivity relationship studies and bioisosteric approaches. The new receptor ligands will be pharmacologically characterized by receptor binding studies (radiochemical and fluorescent) and functional assays (e.g. Ca2+, reporter (luciferase), cAMP). Computational chemistry will be used for the exploration of ligand-receptor interactions and the design of new structures. The preparation, characterization and application of radio- and fluorescence labeled Y4R ligands is part of the project, too.
The project is subdivided into a peptidomimetic approach (project G.1) and a non-peptidic ligand approach (project G.2). G.1 includes the synthesis of unnatural cyclic β- and γ-amino acids and their use as building blocks for the preparation of truncated, conformationally constrained C-terminal NPY analogues. G.2 is focused on the development of non-peptidic selective Y4R antagonists by structural variation of dimeric argininamide-type Y1R antagonists, which recently turned out to be Y4R antagonists as well.
GPCRs differ in their sequence to a variable extent. The smallest sequence differences are single amino acid exchanges due to the variability of the human genome. A higher number of exchanges is characteristic for orthologous receptors from different organisms or paralogous receptor subtypes. Previous research has shown that even genetic variations of single amino acids may be sufficient to cause an altered efficacy of drugs targeting GPCRs. Despite extensive research in the past few years, another aspect, which remains hardly understood, is the selectivity of some ligands for orthologous receptors and receptor subtypes.
An identification of the residues responsible for altered selectivity requires an accurate determination of the ligand binding mode for subsequent energetic analyses. For this purpose, the project will use metadynamics simulations in addition to conventional MD (cMD) simulations. This strategy is currently applied to the histamine H1 and H2 receptor to determine the ligand binding mode and to assess the effect of mutations on the GPCR-ligand interaction. In addition, we plan to develop a more general metadynamics-based protocol for the determination of GPCR-ligand binding modes, which relies on a clustering of metadynamics simulations followed by a refinement using cMD.
Research stay abroad
Research stays abroad are planned at the University of Kansas to work with Prof. Yinglong Miao, who has pioneered the development of Gaussian-accelerated molecular dynamics to study conformational changes in GPCRs.