Stefan Löber

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.

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.

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.

Fused azaindole derivatives: Molecular design, synthesis and in vitro pharmacology leading to the preferential dopamine D3 receptor agonist FAUC 725

Computational studies based on the similarity of molecular electrostatic potential maps initiated the synthesis of the tricyclic target compounds 1 (FAUC 725) and 2. Receptor binding studies at the dopamine receptor subtypes D1, D2(long), D2(short), D3 and D4 showed that the azaindole 1 revealed D3 affinity (K(i)=0.54 nM) comparable to the lead pramipexole and enhanced selectivity over D2 and D4. Mitogenesis experiments indicated substantial intrinsic activity for the D3 selective dipropylamine 1. Based on the structure of (S)-3-PPP, bioisosteric replacement and conformational restriction leading to the test compound 2 was not fruitful.

Synthesis, Radiofluorination, and In Vitro Evaluation of Pyrazolo[1,5-a]pyridine-Based Dopamine D4 Receptor Ligands: Discovery of an Inverse Agonist Radioligand for PET

A series of fluoro-substituted analogs structurally derived from the aminomethyl-substituted pyrazolo[1,5- a]pyridine lead compounds 9 (FAUC 113) and 10 (FAUC 213) were synthesized and evaluated as high-affinity D 4 receptor (D 4R) ligands ( 3a- 3h, K i = 1.3-28 nM). The para-fluoroethoxy-substituted derivatives 3f and 3h revealed an outstanding D 4 subtype selectivity of more than 3 orders of magnitude over both congeners D 2 and D 3 combined with inverse agonism at D 4R. The corresponding (18)F-labeled radioligands revealed high serum stability in vitro and log P values of 2-3. In vitro rat brain autoradiography showed specific binding of [ (18)F]3h in distinct brain regions, including the gyrus dentate of the hippocampus, that were inhibited by both eticlopride (65-80%) and the selective D 4R antagonist 10 (78-93%). The observed binding pattern was mainly consistent with the known D 4R distribution in the rat brain. Thus, [(18)F]3h (FAUC F41) represents a potential radioligand for studying the D 4R in vivo by positron emission tomography (PET).

Subtype selective tetracycline agonists and their application for a two-stage regulatory system

The selective control of gene expression by a drug-like effector is one of the current most promising strategies in molecular biology. Furthermore, it can provide a better understanding of gene function in development and disease, and offers many potential applications in biotechnology and gene therapy. The tetracycline-responsive gene regulation system is highly specific and efficient and is used widely for this purpose. Its main element is the homodimeric Tet repressor (TetR) that regulates resistance of Gram-negative bacteria against tetracycline-derived antibiotics. TetR tightly binds its effector—tetracycline, or the closely related derivatives chlorotetracycline, anhydrotetracycline, or doxycycline—as a Mg chelate to a regulatory core domain. This leads to an allosteric conformational change in TetR that results in its dissociation from the operator DNA sequence. Expansion of the biological space for TetRbased expression systems has led to novel eukaryotic transcriptional activators. Furthermore, random mutagenesis coupled with phenotypic screening has given rise to variants termed rtTA (reverse tetracycline-controlled transactivator), which possess reversed allosteric response and require the tetracycline agonists anhydrotetracycline or doxycycline for DNA binding. The application of this system is currently limited to an on/off switch for a single gene. However, many complex biological processes, such as organ development, depend on the coordinated and successive expression of several genes. Such coordinated expression patterns can currently not be accomplished with “chemical regulation”. Here we present the development of such an expression system by combining a chemical approach that diversifies the inducer with a biological approach that adjusts the regulatory protein to a newly synthesized compound. We put forward the generation of a sequential rtTA-based regulatory setup that allows the consecutive expression of two transgenes by the addition of two different tetracycline agonists. This work complements our recent results on a Tet-based expression system that has high specificity for the nonantibiotic inducer, 4-(dedimethylamino)anhydrotetracycline. The success of our plan depended on the discovery of subtype-selective tetracycline agonists capable of differentiating between two reverse transactivators. To avoid crosstalk between the expression systems, the two variants used contained mutations that ensured selective effector recognition and exclusive DNA-binding specificity, but prevented heterodimerization. The highly sensitive variants rtTA-1 and rtTA-D (for an overview of transregulator nomenclature and source, see Table S1 in the Supporting Information) were chosen because they not only harbor mutations in the DNA-reading head (S12G and E19G in rtTA-1, and E15A, L17G, and L25V in rtTA-D), but rtTA-1 also contains amino-acid substitutions (A56P, D148E, H179R) in the effector-binding core domain. A conceptual homology model, which we derived from the X-ray crystal structure of TetR–[chlorotetracycline–Mg] , shows that the bcarbon atoms of the mutated amino acids are located within a 10–13 G distance from C4, C9, and C10 of the bound chlorotetracycline (Figure 1A). This indicates that chemical modifica-

Di- and trisubstituted pyrazolo[1,5-a]pyridine derivatives: Synthesis, dopamine receptor binding and ligand efficacy

Based on the lead molecule FAUC 113, a series of di- and trisubstituted pyrazolo[1,5-a]pyridine derivatives was synthesized and investigated for their dopamine receptor binding profile. The carbonitrile 11a (FAUC 327) showed excellent pharmacological properties combining high D4 affinity (K(i)=1.5 nM) and selectivity with significant intrinsic activity (31%) in low nanomolar concentrations (EC50=1.5 nM).

18F‐Labeled FAUC 346 and BP 897 Derivatives as Subtype‐Selective Potential PET Radioligands for the Dopamine D3 Receptor

Disturbances of neutrotransmission at the dopamine D3 receptor are related to several neuropsychiatric diseases and in particular to drug addiction. Herein, we report the computer‐assisted prediction of D3 selectivities of new fluoroalkoxy‐substituted receptor ligands by means of 3D‐QSAR analysis. As close analogues of the D3‐selective lead compound FAUC 346 and BP 879, the 19F‐substituted test compounds 4 a–d were synthesized and evaluated. In vitro investigation of their binding characteristics in transfected Chinese Hamster Ovary (CHO) cells led to excellent Ki values between 0.12 and 0.69 nM at the dopamine D3 subtype. The benzothiophene‐substituted carboxamide 4 a (Ki=0.12 nM) displayed 133 and 283‐fold selectivity over the structurally related D2Long and D4 subtypes, respectively. Mitogenesis assays showed the behavior of partial agonists. Based on these data, we synthesized the [18F]fluoroethoxy‐substituted radioligands [18F]4 a–d. The N‐[4‐[4‐(2‐hydroxyphenyl)piperazin‐1‐yl]butyl]‐2‐carboxamides 3 a–d were prepared and labeled with 2‐[18F]fluoroethyltosylate in a two‐step procedure. Optimization of the 18F‐labeling conditions led to radiochemical yields between 24 and 65 %.

Bivalent molecular probes for dopamine D2-like receptors

Merging two arylamidoalkyl substituted phenylpiperazines as prototypical recognition elements for dopamine D(2)-like receptors by oligoethylene glycol linkers led to a series of bivalent ligands. These dimers were investigated in comparison to their monomeric analogues for their dopamine D(2long), D(2short), D(3) and D(4) receptor binding. Radioligand binding experiments revealed strong bivalent effects for some para-substituted benzamide derivatives. For the D(3) subtype, the target compounds 32, 34 and 36 showed an up to 70-fold increase of affinity and a substantial enhancement of subtype selectivity when compared to the monovalent analogue 24. Analysis of the binding curves displayed Hill slopes very close to one indicating that the bivalent ligands displace 1equiv of radioligand. Obviously, the two pharmacophores occupy an orthosteric and an allosteric binding site rather than adopting a receptor-bridging binding mode.