Peter Gmeiner

Structure and function of an irreversible agonist-β 2 adrenoceptor complex

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR–T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

Activation and allosteric modulation of a muscarinic acetylcholine receptor

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

Structural insights into µ-opioid receptor activation

Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Structure-based discovery of opioid analgesics with reduced side effects

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids—which include fatal respiratory depression—are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21—a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography

In the field of molecular imaging, positron emission tomography (PET) has emerged as an imaging modality with excellent sensitivity for in vivo studies. PET labeling is challenging since short-lived positron-emitting isotopes such as F and C are used as labeling agents. The optimization and efficient application of rapid and reliable labeling strategies are prerequisites for obtaining access to new radiopharmaceuticals for both research and clinical trials. Bioactive peptides that specifically address molecular targets in vivo represent an important class of PET tracers to facilitate predictive imaging and PET-guided therapy. Diverse strategies for the synthesis of peptide-based radiopharmaceuticals using F-labeled prosthetic groups have been elaborated, including chemoselective oxime conjugation and the use of F-labeled maleimide derivatives as cysteinereactive reagents. Following the concept of click chemistry introduced by Sharpless et al., the Huisgen [3+2] azide– alkyne cycloaddition has been adapted to F-radiosynthetic methods in order to take advantage of its selectivity, reliability, and speed under aqueous mild Cupromoted reaction conditions. The versatility of peptide imaging agents is frequently hampered by their instability in vivo because of rapid degradation by endogenous peptidases. As an example, the synthesis of radiolabeled peptide-based imaging agents for the neurotensin receptor-1 (NTR-1), which is overexpressed in a number of human cancers, requires modifications to improve the metabolic stability. Synthetic approaches to RGD tracers targeting avb3 integrin, which plays a key role in angiogenesis, capitalize on the pioneering studies by Kessler et al. , who successfully developed cyclic pentameric RGD peptides that selectively recognize integrin avb3. [9] Various radiolabeled cyclic RGD peptides have been described. Among these, [F]galactoRGD has been extensively evaluated in clinical studies. Since glycosylation of peptides is known to frequently improve the biokinetic and in vivo clearance properties, [F]galacto-RGD and further radiopeptides have been approached. 13] However, the multistep radiosynthesis of [F]galacto-RGD is time-consuming and laborious. In proposals to overcome this drawback, F-labeling by 2-deoxy-2[F]fluoroglucose (FDG) has been discussed. 15] The major disadvantages of the F-peptide-labeling strategies currently used are 1) harsh reaction conditions, 2) laborious multiple-step syntheses with a limited decayuncorrected radiochemical yield (RCY), which would complicate the automation for large-scale production, and 3) lipophilic derivatization, which impair the biokinetic properties of the tracer. Based on our previous work on click chemistry in drug discovery and the synthesis of b-mannosyl azides, we herein present an efficient strategy toward F-labeling with concomitant glycosylation for the synthesis of F-glycopeptides as imaging agents for PET. We combined this strategy with the development of a metabolically stable glycopeptoid analogue of NT(8–13), which is the highly potent C-terminal hexapeptide of the natural agonist neurotensin (NT). As a proof of concept, two F-glycopeptides derived from NT(8– 13) and c(RGDfPra), respectively, were applied to biodistribution studies and mPET for imaging NTR and avb3-integrin expression in vivo using xenograft nude mice models. In detail, 2-deoxy-2-fluoroglucosyl azide (3) could be obtained starting from tetraacetylated 2-deoxy-2-fluoroglucose. The glucosyl azide 3 was applied for the Cu-catalyzed azide–alkyne coupling with a series of alkyne-functionalized peptides to evaluate the influence of the appended glycosyl residue on receptor recognition. Commercially available propargylglycine (Pra) was introduced by solid-phase-supported synthesis at position X into the sequence of the bioactive peptide c(RGDfX) and at the N terminus of NT(8– 13) and metabolically stabilized derivatives thereof. Considering our studies on the influence of peptide backbone modifications and ligand conformation on affinity changes for a series of NT(8–13) analogues, metabolic stabilization was envisioned by alteration of three amino acids in the sequence [*] Dr. S. Maschauer, Dr. C. Hocke, Prof. T. Kuwert, Dr. O. Prante Nuklearmedizinische Klinik, Labor f r Molekulare Bildgebung Friedrich-Alexander-Universit t Erlangen-N rnberg Schwabachanlage 6, 91054 Erlangen (Germany) Fax: (+ 49)9131-853-4325 E-mail: olaf.prante@uk-erlangen.de

Tricyclic antidepressants, quinacrine and a novel, synthetic chimera thereof clear prions by destabilizing detergent-resistant membrane compartments

Prion diseases are invariably fatal, neurodegenerative diseases transmitted by an infectious agent, PrPSc, a pathogenic, conformational isoform of the normal prion protein (PrPC). Heterocyclic compounds such as acridine derivatives like quinacrine abolish prion infectivity in a cell culture model of prion disease. Here, we report that these compounds execute their antiprion activity by redistributing cholesterol from the plasma membrane to intracellular compartments, thereby destabilizing membrane domains. Our findings are supported by the fact that structurally unrelated compounds with known cholesterol‐redistributing effects – U18666A, amiodarone, and progesterone – also possessed high antiprion potency. We show that tricyclic antidepressants (e.g. desipramine), another class of heterocyclic compounds, displayed structure‐dependent antiprion effects and enhanced the antiprion effects of quinacrine, allowing lower doses of both drugs to be used in combination. Treatment of ScN2a cells with quinacrine or desipramine induced different ultrastructural and morphological changes in endosomal compartments. We synthesized a novel drug from quinacrine and desipramine, termed quinpramine, that led to a fivefold increase in antiprion activity compared to quinacrine with an EC50 of 85 nm. Furthermore, simvastatin, an inhibitor of cholesterol biosynthesis, acted synergistically with both heterocyclic compounds to clear PrPSc. Our data suggest that a cocktail of drugs targeting the lipid metabolism that controls PrP conversion may be the most efficient in treating Creutzfeldt‐Jakob disease.

Dopamine D2, D3, and D4 Selective Phenylpiperazines as Molecular Probes To Explore the Origins of Subtype Specific Receptor Binding

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

GPCR crystal structures: Medicinal chemistry in the pocket

Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy.

Azaindole derivatives with high affinity for the dopamine D4 receptor: Synthesis, ligand binding studies and comparison of molecular electrostatic potential maps

Piperazinylmethyl substituted pyrazolo[1,5-a]pyridines and related heterocycles were synthesized and found to recognize selectively the dopamine D4 receptor. For the most potent derivative 10d a Ki value of 2.0 nM was observed. SAR studies including the comparison of molecular isopotential surfaces were performed.

Class A G-Protein-Coupled Receptor (GPCR) Dimers and Bivalent Ligands

G-protein-coupled receptors (GPCRs) represent the largest family of membrane proteins involved in cellular signal transduction and are activated by various different ligand types including photons, peptides, proteins, but also small molecules like biogenic amines. Therefore, GPCRs are involved in diverse physiological processes and provide valuable drug targets for numerous diseases. Emerging body of evidence suggests that GPCRs exist as monomers or cross-react forming dimers and higher-ordered oligomers. In this Perspective we will review current biochemical and biophysical techniques to visualize GPCR dimerization, functional consequences of homo- and heterodimers, and approaches of medicinal chemists to target these receptor complexes with homo- and heterobivalent ligands.