Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins.

Derivatives of the rhodamine-based dye 5-TAMRA (5-carboxy-tetramethylrhodamine) and the indocarbocyanine-type Cy3B (cyclized derivative of the cyanine dye Cy3), both representing important fluorophores frequently used for the labeling of biomolecules (proteins, nucleic acids) and bioactive compounds, such as receptor ligands, were photophysically investigated in aqueous solution, i.e., in neat phosphate-buffered saline (PBS) and in PBS supplemented with 1 wt % bovine serum albumin (BSA). The dyes exhibit comparable absorption (λabs,max: 550-569 nm) and emission wavelengths (λem,max: 580-582 nm), and similar S1 lifetimes (2.27-2.75 ns), and their excited state deactivation proceeds mainly via the lowest excited singlet state (triplet quantum yield ca. 1%). However, the probes show marked differences with respect to their fluorescence quantum yield and photostability. While 5-TAMRA shows a lower quantum yield (37-39%) than the Cy3B derivative (ca. 57%), its photostability is considerably higher compared to Cy3B. Generally, the impact of the protein on the photophysics is low. However, on prolonged illumination, both fluorescent dyes undergo a photocatalytic reaction with tryptophan residues of BSA mediated by sensitized singlet oxygen resulting in a tryptophan photoproduct with an absorption maximum around 330 nm. The overall results of this work will assist in choosing the right dye for the labeling of bioactive compounds, and the study demonstrates that experiments performed with 5-TAMRA or Cy3B-labeled compounds in a biological environment may be influenced by photochemical modification of experimentally relevant proteins at aromatic amino acid residues.

A Metabolomic Analysis of Sensitivity and Specificity of 23 Previously Proposed Biomarkers for Renal Transporter-Mediated Drug-Drug Interactions.

Endogenous biomarkers are discussed as tools for detection of drug-drug interactions (DDIs) mediated by renal transport proteins, such as organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATE1 and MATE2-K) and organic anion transporters (OAT1 and OAT3). Whereas sensitivity of some endogenous biomarkers against at least one clinical transporter inhibitor has frequently been shown, intra-study comparisons of the extent of effects of inhibitors on different biomarkers are frequently lacking. Moreover, in vivo specificity of such discussed biomarkers has frequently not been studied. We therefore investigated changes of 10 previously described putative biomarkers for inhibition of OCT2/MATEs, as well as 15 previously described putative biomarkers for OATs in human plasma and urine samples of healthy volunteers in response to treatment with 4 inhibitors of transport proteins [verapamil (P-glycoprotein), rifampin (organic anion transporting polypeptides), cimetidine (OCT2/MATEs), and probenecid (OATs)]. Two of the putative biomarkers had been suggested for both OCT2/MATEs and OATs. All substances were unequivocally identified in an untargeted metabolomics assay. The OCT2/MATE biomarkers choline and trimethylamine N-oxide were both sensitive and specific (median log2-fold changes -1.18 in estimated renal elimination and -0.85 in urinary excretion, respectively). For renal OATs, indoleacetyl glutamine and indoleacetic acid (median log2-fold changes -3.77 and -2.85 in estimated renal elimination, respectively) were the candidates for sensitive and specific biomarkers with the most extensive change, followed by taurine, indolelactic acid, and hypoxanthine. This comprehensive study adds further knowledge on sensitivity and specificity of 23 previously described biomarkers of renal OCT2/MATE- and OAT-mediated DDIs.

Exploring Structural Determinants of Bias among D4 Subtype-Selective Dopamine Receptor Agonists.

The high affinity dopamine D4 receptor ligand APH199 and derivatives thereof exhibit bias toward the Gi signaling pathway over ß-arrestin recruitment compared to quinpirole. Based on APH199, two novel groups of D4 subtype selective ligands were designed and evaluated, in which the original benzyl phenylsemicarbazide substructure was replaced by either a biphenylmethyl urea or a biphenyl urea moiety. Functional assays revealed a range of different bias profiles among the newly synthesized compounds, namely, with regard to efficacy, potency, and GRK2 dependency, in which bias factors range from 1 to over 300 and activation from 15% to over 98% compared to quinpirole. These observations demonstrate that within bias, an even more precise tuning toward a particular profile is possible, which─in a general sense─could become an important aspect in future drug development. Docking studies enabled further insight into the role of the ECL2 and the EPB in the emergence of bias, thereby taking advantage of the diversity of functionally selective D4 agonists now available.

Cyanine- and Rhodamine-Derived Alkynes for the Selective Targeting of Cancerous Mitochondria through Radical Thiol-Yne Coupling in Live Cells.

Despite their long history and their synthetic potential underlined by various recent advances, radical thiol-yne coupling reactions have so far only rarely been exploited for the functionalization of biomolecules, and no examples yet exist for their application in live cells - although natural thiols show widespread occurrence therein. By taking advantage of the particular cellular conditions of mitochondria in cancer cells, we have demonstrated that radical thiol-yne coupling represents a powerful reaction principle for the selective targeting of these organelles. Within our studies, fluorescently labeled reactive alkyne probes were investigated, for which the fluorescent moiety was chosen to enable both mitochondria accumulation as well as highly sensitive detection. After preliminary studies under cell-free conditions, the most promising alkyne-dye conjugates were evaluated in various cellular experiments comprising analysis by flow cytometry and microscopy. All in all, these results pave the way for improved future therapeutic strategies relying on live-cell compatibility and selectivity among cellular compartments.

Connecting Ruthenium Photocatalysis to 1,2-Dioxetane-Mediated Chemiluminescence: a Versatile Combination for Optical Detection and Read-Out.

Optical storage and photon quantification systems based on sensitive photoreactions have numerous applications. Herein, we report a highly efficient photocatalytic reaction, in which ruthenium photoredox catalysis is combined with a 1,2-dioxetane from which chemiluminescence can be triggered. In this system, blue light irradiation as optical input enables a defined inverse correlation with base-triggered, blue light emission as optical output. Comparison of readout by 1 H NMR and chemiluminescence, relative to previous optical input, underlines the reliability and usefulness of the ruthenium-dioxetane system for optical storage, sensing and ruthenium detection.

Tuning the Polarity of Antibiotic-Cy5 Conjugates Enables Highly Selective Labeling of Binding Sites.

Multidrug-resistant bacteria pose a major threat to global health, even as newly introduced antibiotics continue to lose their therapeutic value. Against this background, deeper insights into bacterial interaction with antibiotic drugs are urgently required, whereas fluorescently labeled drug conjugates can serve as highly valuable tools. Herein, the preparation and biological evaluation of 13 new fluorescent antibiotic-Cy5 dye conjugates is described, in which the tuning of the polarity of the Cy5 dye proved to be a key element to achieve highly favorable properties for various fields of application.

Selectfluor-Mediated Chlorination and Fluorination of Arenes: Late-Stage Functionalization of APIs and Its Biological Effects.

By using active pharmaceutical ingredients (APIs) previously recovered from expired drugs, it is shown that Selectfluor can act as a reagent for operationally simple late-stage fluorination and chlorination of electron-rich arenes under mild reaction conditions. As shown in mechanistic experiments, aromatic fluorination thereby competes with chlorine-for-fluorine exchange on Selectfluor and subsequent aromatic chlorination, whereat the chloride ions may either be provided by the hydrochloride salt of the respective API or by triethylammonium chloride. Biological testing of the fluorinated or chlorinated APIs at adrenergic, dopaminergic, muscarinergic, opioid or serotoninergic receptors demonstrated that improved binding affinities can be achieved via this straightforward strategy.

Behavioural effects of APH199, a selective dopamine D4 receptor agonist, in animal models.

RATIONALE: The dopamine D4 receptors (DRD4) play a key role in numerous brain functions and are involved in the pathogenesis of various psychiatric disorders. DRD4 ligands have been shown to moderate anxiety, reward and depression-like behaviours, and cognitive impairments. Despite a series of promising but ambiguous findings, the therapeutic advantages of DRD4 stimulation remain elusive. OBJECTIVES: The investigation focused on the behavioural effects of the recently developed DRD4 agonist, APH199, to evaluate its impact on anxiety, anhedonia, behavioural despair, establishment and retrieval of alcohol reinforcement, and amphetamine (AMPH)-induced symptoms. METHODS: Male C57BL/6 J mice and Sprague-Dawley rats were examined in five independent experiments. We assessed APH199 (0.1-5 mg/kg, i.p.) effects on a broad range of behavioural parameters in the open field (OF) test, conditioned place preference test (CPP), elevated plus maze (EPM), light-dark box (LDB), novelty suppressed feeding (NSF), forced swim test (FST), sucrose preference test (SPT), AMPH-induced hyperlocomotion test (AIH), and prepulse inhibition (PPI) of the acoustic startle response in AMPH-sensitized rats. RESULTS: APH199 caused mild and sporadic anxiolytic and antidepressant effects in EPM and FST, but no remarkable impact on behaviour in other tests in mice. However, we found a significant increase in AMPH-induced hyperactivity, suggesting an exaggeration of the psychotic-like responses in the AMPH-sensitized rats. CONCLUSIONS: Our data challenged the hypothesis of the therapeutic benefits of DRD4 agonists, pointing out a possible aggravation of psychosis. We suggest a need for further preclinical studies to ensure the safety of antipsychotics with DRD4 stimulating properties.

Visible-Light-Mediated Radical Arylations Using a Fluorescein-Derived Diazonium Salt: Reactions Proceeding via an Intramolecular Forth and Back Electron Transfer.

Functionalizations of arenes and alkenes via additive-free radical reactions using highly photosensitive, fluorescein-derived diazonium salts are described. The particular properties of the diazonium salts enable unique Meerwein-type carbohydroxylations of non-activated alkenes, which can be rationalized by a reaction mechanism involving forth and back electron transfer from and to the xanthene subunit of the fluorescein moiety.

Synthesis, Characterization, and Application of Muscarinergic M3 Receptor Ligands Linked to Fluorescent Dyes.

Through the linkage of two muscarinergic M3 receptor ligands to fluorescent tetramethylrhodamine- and cyanine-5-type dyes, two novel tool compounds, OFH5503 and OFH611, have been developed. Based on the suitable binding properties and kinetics related to the M3 subtype, both ligand-dye conjugates were found to be useful tools to determine binding affinities via flow cytometric measurements. In addition, confocal microscopy underlined the comparably low unspecific binding and the applicability for studying M3 receptor expression in cells. Along with the proven usefulness regarding studies on the M3 subtype, the conjugates OFH5503 and OFH611 could, due to their high affinity to the M1 receptor, evolve as even more versatile tools in the field of research on muscarinergic receptors.

Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

pH-Dependent Conformational Switching of Amide Bonds─from Full trans to Full cis and Vice Versa.

Strategies enabling the pH-dependent conformational switching of amide bonds from trans to cis, and vice versa, are yet limited in the sense that, in a suitable pH range, one rotamer may be stabilized to a large extent while the complementary pH range only leads to a mixture of isomers. By exploiting the effects of steric demand and the interaction of the amide carbonyl with a positive charge, we herein present the first examples for reversible pH-dependent switching from full trans to full cis.

Cleavage of Organosolv Lignin to Phenols Using Nitrogen Monoxide and Hydrazine.

From the variety of methods known for the depolymerization of organosolv lignin, a broad range of diversely substituted aromatic compounds are available today. In the present work, a novel two-step reaction sequence is reported, which is focused on the formation of phenols. While the first step of the depolymerization strategy comprises the 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-catalyzed oxidation of organosolv lignin with nitrogen monoxide so that two waste materials are combined, cleavage to the phenolic target compounds is achieved in the second step employing hydrazine and potassium hydroxide under Wolff-Kishner-type conditions. Besides the fact that the novel strategy proceeds via an untypical form of oxidized organosolv lignin, the two-step sequence is further able to provide phenols as cleavage products, which bear no substituent at the 4-position.

Synthetic Route to Phenyl Diazenes and Pyridazinium Salts from Phenylazosulfonates.

The synthesis of pyridazinium salts was achieved from readily available phenylazosulfonates in a single reaction step. The reaction proceeds via the formation of short-lived phenyldiazenes, which-owing to the strongly acidic conditions-are partially protonated. The phenyldiazenes then undergo a rapid cycloaddition to furans to give pyridazinium salts via elimination of water. The fact that the pyridazinium synthesis shows a low sensitivity toward oxygen, although phenyldiazenes occur as intermediates, can be explained by the very fast cycloaddition step and the partial protonation of the phenyldiazene.

2-Fluoro-5-nitrophenyldiazonium: A Novel Sanger-Type Reagent for the Versatile Functionalization of Alcohols.

As a novel Sanger-type reagent, 2-fluoro-5-nitrophenyldiazonium tetrafluoroborate enabled the versatile functionalization of primary and secondary aliphatic alcohols. Based on a mild nucleophilic aromatic substitution of the fluorine atom under unprecedented, base-free conditions, the diazonium unit on the aromatic core of the resulting aryl-alkyl ether could be employed for such diverse transformations as radical C-H activation and cyclization, as well as palladium catalyzed cross-coupling reactions.

Visible Light Promoted, Catalyst-Free Radical Carbohydroxylation and Carboetherification under Mild Biomimetic Conditions.

Metal and catalyst-free carbohydroxylations and carboetherifications at room temperature have been achieved by a combination of beneficial factors including high aryl diazonium concentration and visible light irradiation. The acceleration of the reaction by visible light irradiation is particularly remarkable against the background that neither the aryldiazonium salt nor the alkene show absorptions in the respective range of wavelength. These observations point to weak charge transfer interactions between diazonium salt and alkene, which are nevertheless able to considerably influence the reaction course. As highly promising perspective, many more aryldiazonium-based radical arylations might benefit from simple light irradiation without requiring a photocatalyst or particular additive.

Shifted Selectivity in Protonation Enables the Mild Deuteration of Arenes Through Catalytic Amounts of Bronsted Acids in Deuterated Methanol.

Taking advantage of the "differentiating effect" of the solvent methanol, deuterations of electron-rich aromatic systems can be carried out under mild acid catalysis and thus under far milder conditions than known so far. The exceptional functional group tolerance observed under the optimized conditions, which even includes highly acid-labile groups, results from a hitherto unexploited shifted selectivity in protonation, and enabled simple and straightforward access to complex deuterium-labeled compounds.

N-Alkylaminoferrocene-Based Prodrugs Targeting Mitochondria of Cancer Cells.

Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2-• that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.

Oxidative Formation of Disulfide Bonds by a Chemiluminescent 1,2-Dioxetane under Mild Conditions.

The oxidation of alkyl thiols to disulfides has been achieved under mild conditions using a chemiluminescent 1,2-dioxetane as a stoichiometric oxidant. Besides the mild and biocompatible reaction conditions, this approach offers the possibility to monitor the presence of thiols through oxidation and chemiluminescence of the remaining dioxetane.