Harald Hübner

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.

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

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.

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.

1,1'-Disubstituted ferrocenes as molecular hinges in mono- and bivalent dopamine receptor ligands.

On the basis of previous work on dopaminergic partial agonists of type 1 and 2, disubstituted ferrocenes are presented as valuable arene bioisosteres. Because substituents at the distal cyclopentadienyl ring are able to adjust the relative disposition that is required for ligand binding, disubstituted ferrocenes can act as molecular hinges. Taking advantage of click chemistry, the regioselective construction and functionalization of the target molecules is reported. Thus triazole derived appendages were used for the fine-tuning of biological activity and for the attachment of linker units generating bivalent GPCR ligands. Receptor binding was evaluated by radioligand displacement experiments, revealing superaffinity with sub- to single-digit nanomolar K(i) values for particular test compounds. As a neutral antagonist at the dopamine receptors D3 and D4 and a potent partial agonist at the D2 subtype (intrinsic activity = 57%, EC(50) = 2.5 nM), the bifunctional ferrocene 10b revealed a novel and unique activity profile.

Bivalent Dopamine D2 Receptor Ligands: Synthesis and Binding Properties

Dopamine D(2) receptor homodimers might be of particular importance in the pathophysiology of schizophrenia and, thus, serve as promising target proteins for the discovery of atypical antipsychotics. A highly attractive approach to investigate and control GPCR dimerization may be provided by the exploration and characterization of bivalent ligands, which can act as molecular probes simultaneously binding two adjacent binding sites of a dimer. The synthesis of bivalent dopamine D(2) receptor ligands of type 1 is presented, incorporating the privileged structure of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) and triazolyl-linked spacer elements. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for bivalent ligands with particular spacer lengths and a comparative analysis with respective monovalent control ligands and unsymmetrically substituted analogues indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites.

Highly Potent 5-Aminotetrahydropyrazolopyridines: Enantioselective Dopamine D3 Receptor Binding, Functional Selectivity, and Analysis of Receptor−Ligand Interactions

Heterocyclic dopamine surrogates of types 5 and 7 were synthesized and investigated for their dopaminergic properties. The enantiomerically pure biphenylcarboxamide (S)-5a displayed an outstanding K(i) of 27 pM at the agonist-labeled D(3) receptor and significant selectivity over the D(2) subtype. Measurement of [(35)S]GTPγS incorporation in the presence of a coexpressed PTX-insensitive G(α0-1) subunit indicated highly efficient G-protein coupling. Comparison of ligand efficacy data from cAMP accumulation and [(3)H]thymidine incorporation experiments revealed that ligand biased signaling is exerted by the test compound (S)-5a. Starting from the D(3) crystal structure, a combination of homology modeling and site directed mutagenesis gave valuable insights into the binding mode and the intermolecular origins of stereospecific receptor recognition. According to these data, the superior affinity of the eutomer 5a is caused by the favorable binding energy that results from interaction between the ligands central ammonium unit and the aspartate residue in position 3.32 of the receptor.

PIP3EA and PD-168077, two selective dopamine D4 receptor agonists, induce penile erection in male rats: site and mechanism of action in the brain

PIP3EA (2‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl‐methyl]imidazo[1,2‐a]pyridine) and PD‐168077 (N‐[4‐2‐cyanophenylpiperazin‐1‐ylmethyl]‐3‐methylbenzamide maleate), two selective dopamine D4 agonists, administered systemically, intracerebroventricularly or into the paraventricular nucleus of the hypothalamus induce penile erection in male Sprague–Dawley rats. A U‐inverted dose–response curve was found with either compound when given subcutaneously (1–100 µg/kg) or intracerebroventricularly (0.1–20 µg/rat), but not into the paraventricular nucleus (10–200 ng/rat). The pro‐erectile effect of PIP3EA and of PD‐168077 occurs concomitantly with an increased nitric oxide (NO) production in the paraventricular nucleus, as measured by the increased concentration of nitrites and nitrates found in the dialysate obtained from the paraventricular nucleus by intracerebral microdialysis. These effects of PIP3EA and PD‐168077 were reduced by L‐745,870 (3‐[4‐(4‐chlorophenyl)piperazin‐1‐ylmethyl]‐1H‐pyrrolo[2,3‐b]pyridine trihydrochloride), a selective dopamine D4 receptors antagonist, by ω‐conotoxin, a blocker of voltage‐dependent Ca2+ channels of the N‐type, by S‐methyl‐thiocitrulline, a neuronal nitric oxide synthase inhibitor, and by d(CH2)5Tyr(Me)2‐Orn8‐vasotocin, an oxytocin receptor antagonist, given into the lateral ventricles, but not into the paraventricular nucleus. Comparison of the dose–response curves of PIP3EA and PD‐168077 revealed that PIP3EA is as potent as PD‐168077 when given into the paraventricular nucleus, but more potent when given systemically. However, both compounds are less efficacious (e.g. induce a lower number of penile erection episodes) than apomorphine, a classical mixed dopamine receptor agonist, irrespective of the route of administration. These results confirm previous findings showing that central D4 receptors mediate penile erection and show that dopamine D4 receptor agonists act in the paraventricular nucleus to facilitate penile erection by increasing central oxytocinergic neurotransmission.

Functionally Selective Dopamine D2, D3 Receptor Partial Agonists

Dopamine D2 receptor-promoted activation of Gα(o) over Gα(i) may increase synaptic plasticity and thereby might improve negative symptoms of schizophrenia. Heterocyclic dopamine surrogates comprising a pyrazolo[1,5-a]pyridine moiety were synthesized and investigated for their binding properties when low- to subnanomolar K(i) values were determined for D(2L), D(2S), and D3 receptors. Measurement of [(35)S]GTPγS incorporation at D(2S) coexpressed with G-protein subunits indicated significant bias for promotion of Gα(o1) over Gα(i2) coupling for several test compounds. Functionally selective D(2S) activation was most striking for the carbaldoxime 8b (Gα(o1), pEC50 = 8.87, E(max) = 65%; Gα(i2), pEC50 = 6.63, E(max) = 27%). In contrast, the investigated 1,4-disubstituted aromatic piperazines (1,4-DAPs) behaved as antagonists for β-arrestin-2 recruitment, implying significant ligand bias for G-protein activation over β-arrestin-2 recruitment at D(2S) receptors. Ligand efficacy and selectivity between D(2S) and D3 activation were strongly influenced by regiochemistry and the nature of functional groups attached to the pyrazolo[1,5-a]pyridine moiety.