Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.

Profiling of orthosteric and allosteric group-III metabotropic glutamate receptor ligands on various G protein-coupled receptors with Tag-lite® assays.

Group-III metabotropic glutamate (mGlu) receptors are important synaptic regulators and are potential druggable targets for Parkinson disease, autism and pain. Potential drugs include orthosteric agonists in the glutamate binding extracellular domain and positive allosteric modulators interacting with seven-pass transmembrane domains. Orthosteric agonists are rarely completely specific for an individual group-III mGlu subtype. Furthermore they often fail to pass the blood-brain barrier and they constitutively activate their target receptor. These properties limit the potential therapeutic use of orthosteric agonists. Allosteric modulators are more specific and maintain the biological activity of the targeted receptor. However, they bind in a hydrophobic pocket and this limits their bio-availability and increases possible off-target action. It is therefore important to characterize the action of potential drug targets with a multifaceted and deeply informative assay. Here we aimed at multifaceted deep profiling of the effect of seven different agonists, and seven positive allosteric modulators on 34 different G protein-coupled receptors by a Tag-lite® assay. Our results did not reveal off-target activity of mGlu orthosteric agonists. However, five allosteric modulators had either positive or negative effects on non-cognate G protein-coupled receptors. In conclusion, we demonstrate the power of the Tag-lite® assay for potential drug ligand profiling on G protein-coupled receptors and its potential to identify positive allosteric compounds.

From the Promiscuous Asenapine to Potent Fluorescent Ligands Acting at a Series of Aminergic G-Protein-Coupled Receptors.

Monoamine neurotransmitters such as serotonin, dopamine, histamine, and noradrenaline have important and varied physiological functions and similar chemical structures. Representing important pharmaceutical drug targets, the corresponding G-protein-coupled receptors (termed aminergic GPCRs) belong to the class of cell membrane receptors and share many levels of similarity as well. Given their pharmacological and structural closeness, one could hypothesize the possibility to derivatize a ubiquitous ligand to afford rapidly fluorescent probes for a large set of GPCRs to be used for instance in FRET-based binding assays. Here we report fluorescent derivatives of the nonselective agent asenapine which were designed, synthesized, and evaluated as ligands of 34 serotonin, dopamine, histamine, melatonin, acetylcholine, and adrenergic receptors. It appears that this strategy led rapidly to the discovery and development of nanomolar affinity fluorescent probes for 14 aminergic GPCRs. Selected probes were tested in competition binding assays with unlabeled competitors in order to demonstrate their suitability for drug discovery purposes.

G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome.

Stretches of guanines can associate in vitro through Hoogsteen hydrogen bonding to form four-stranded structures. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. This study demonstrates that oligonucleotides containing these G-clusters form highly stable G-quadruplexes of various structures in vitro, whose formation was controlled by an easy and reversible protocol using sodium hydroxide. Among these sequences, a G'2 hairpin dimer was the most stable structure adopted by the 5'-tail of the (+) downstream strand. Since the two (+) strands of the HIV-1 central DNA flap hold these G-clusters, and based on the properties of reverse branch migration in DNA flaps, constructions using HIV-1 sequences were assembled to mimic small DNA flaps where the G-clusters are neighbors. G-quartets were successfully probed in such flaps. They were induced by potassium and by a dibenzophenanthroline derivative already known to stabilize them. Such results suggest some function(s) for G-quartets associated with a DNA flap in the HIV-1 pre-integration steps, and argue for their transient formation during the processing of G-rich DNA flaps at the time of replication and/or repair.

Time-resolved FRET strategy to screen GPCR ligand library.

Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect. Such molecules are then expected to display fewer side effects. To reach such a goal, there is much interest in novel, efficient, simple, and direct screening assays that may help identify any drugs interacting with the target, these being then analyzed for their biased activity. Here, we present an efficient strategy to screen ligands on their binding properties. The method described is based on time-resolved FRET between a receptor and a ligand. This method has already been used to develop new assays called Tag-lite(®) binding assays for numerous G protein-coupled receptors, proving its broad application and its power.

Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer.

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.

Discovery of novel N-phenylphenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining high-throughput screening and synthesis.

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis -infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket.

A novel approach to indoloditerpenes by Nazarov photocyclization: synthesis and biological investigations of terpendole E analogues.

The application of the Nazarov photocyclization as a mild and efficient method for access to the basic core of novel indoloditerpenoid derivatives is reported. The detailed synthesis of these new analogues of terpendole E, as well as their evaluation as potential inhibitors of KSP, is described.

PDE5 inhibitors: An original access to novel potent arylated analogues of tadalafil.

A method to access totally new analogues of tadalafil was explored. The Buchwald reaction was adapted and used to replace the methyl group of tadalafil by various aryl groups. Inhibition potencies on PDE5 of these analogues were determined and proved to be comparable to the one of tadalafil. Using the same route, compounds with the same level of activity but improved water solubility were produced by introducing a pyridine or a pyrimidine ring. This original route also opens access to new unpatented compounds.

G-quadruplex recognition by quinacridines: a SAR, NMR, and biological study.

The synthesis of a novel group of quinacridine-based ligands (MMQs) is described along with an evaluation of their G-quadruplex binding properties. A set of biophysical assays was applied to characterize their interaction with DNA quadruplexes: FRET-melting experiments and equilibrium microdialysis were used to evaluate their quadruplex affinity and their ability to discriminate quadruplexes across a broad panel of DNA structures. All data collected support the proposed model of interaction of these compounds with G-quadruplexes, which is furthermore confirmed by a solution structure determined by 2D NMR experiments. Finally, the activity of the MMQ series against tumor cell growth is reported, and the data support the potential of quadruplex-interactive compounds for use in anticancer approaches.