Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS.

Astrocytes are involved in non-cell-autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF-κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF-κB activation. While NF-κB activation in astrocytes induced a Wnt-dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF-κB-dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB2R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage-dependent microglia modulation may be an effective therapeutic strategy in ALS.

Novel Triazolopyrimidine-Derived Cannabinoid Receptor 2 Agonists as Potential Treatment for Inflammatory Kidney Diseases.

The cannabinoid receptor 2 (CB2) system is described to modulate various pathological conditions, including inflammation and fibrosis. A series of new heterocyclic small-molecule CB2 receptor agonists were identified from a high-throughput screen. Lead optimization gave access to novel, highly potent, and selective (over CB1) triazolopyrimidine derivatives. A preliminary structure-activity relationship was established, and physicochemical properties in this compound class were significantly improved toward better solubility, lipophilicity, and microsomal stability. An optimized triazolopyrimidine derivative, (3S)-1-[5-tert-butyl-3-[(1-cyclopropyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol (39), was tested in a kidney ischemia-reperfusion model, in which it showed efficacy at a dose of 10 mg kg(-1) (p.o.). A significant depletion of the three measured kidney markers indicated a protective role of CB2 receptor activation toward inflammatory kidney damage. Compound 39 was also protective in a model of renal fibrosis. Oral treatment with 39 at 3 mg kg(-1) per day significantly decreased the amount of fibrosis by ∼ 40% which was induced by unilateral ureter obstruction.

Challenges and Rewards in Medicinal Chemistry Targeting Cardiovascular and Metabolic Diseases.

Medicinal chemistry has been transformed by major technological and conceptual innovations over the last three decades: structural biology and bioinformatics, structure and property based molecular design, the concepts of multidimensional optimization (MDO), in silico and experimental high-throughput molecular property analysis. The novel technologies advanced gradually and in synergy with biology and Roche has been at the forefront. Applications in drug discovery programs towards new medicines in cardiovascular and metabolic diseases are highlighted to show impact and advancement: the early discovery of endothelin antagonists for endothelial dysfunction (Bosentan), 11-beta hydroxysteroid dehydrogenase (11ß-HSD1) inhibitors for dysregulated cellular glucocorticoid tonus (type 2 diabetes and metabolic syndrome) and non-covalent hormone sensitive lipase (HSL) inhibitors to study the scope of direct inhibition of lipolysis in the conceptual frame of lipotoxicity and type 2 diabetes.

Challenges and Rewards in Medicinal Chemistry Targeting Cardiovascular and Metabolic Diseases.

Medicinal chemistry has been transformed by major technological and conceptual innovations over the last three decades: structural biology and bioinformatics, structure and property based molecular design, the concepts of multidimensional optimization (MDO), in silico and experimental high-throughput molecular property analysis. The novel technologies advanced gradually and in synergy with biology and Roche has been at the forefront. Applications in drug discovery programs towards new medicines in cardiovascular and metabolic diseases are highlighted to show impact and advancement: the early discovery of endothelin antagonists for endothelial dysfunction (Bosentan), 11-beta hydroxysteroid dehydrogenase (11ß-HSD1) inhibitors for dysregulated cellular glucocorticoid tonus (type 2 diabetes and metabolic syndrome) and non-covalent hormone sensitive lipase (HSL) inhibitors to study the scope of direct inhibition of lipolysis in the conceptual frame of lipotoxicity and type 2 diabetes.

6-Alkoxy-5-aryl-3-pyridinecarboxamides, a new series of bioavailable cannabinoid receptor type 1 (CB1) antagonists including peripherally selective compounds.

We identified 6-alkoxy-5-aryl-3-pyridinecarboxamides as potent CB1 receptor antagonists with high selectivity over CB2 receptors. The series was optimized to reduce lipophilicity compared to rimonabant to achieve peripherally active molecules with minimal central effects. Several compounds that showed high plasma exposures in rats were evaluated in vivo to probe the contribution of central vs peripheral CB1 agonism to metabolic improvement. Both rimonabant and 14g, a potent brain penetrant CB1 receptor antagonist, significantly reduced the rate of body weight gain. However, 14h, a molecule with markedly reduced brain exposure, had no significant effect on body weight. PK studies confirmed similarly high exposure of both 14h and 14g in the periphery but 10-fold lower exposure in the brain for 14h. On the basis of these data, which are consistent with reported effects in tissue-specific CB1 receptor KO mice, we conclude that the metabolic benefits of CB1 receptor antagonists are primarily centrally mediated as originally believed.

Conformationally restricted pyrrolidines by intramolecular [2+2] photocycloaddition reactions.

Intramolecular [2+2] photocycloaddition reactions of diversely substituted N-Boc protected 4-(allylaminomethyl)-2(5H)-furanones resulted in rigid products (53-75%) with three spatially defined positions for further functionalisation.

Highly potent and selective cannabinoid receptor 2 agonists: initial hit optimization of an adamantyl hit series identified from high-through-put screening.

A series of highly potent & selective adamantane derived CB2 agonists was identified in a high-throughput screen. A SAR was established and physicochemical properties were significantly improved. This was accompanied by potency of the compounds on the Q63R variant and varying ß-arrestin data which will support the insight into their relevance for the in vivo situation.

Rational design of novel pyrrolidine derivatives as orally active neurokinin-3 receptor antagonists.

The rational design of a novel series of pyrrolidine derivatives as neurokinin-3 receptor antagonists is reported starting from a selective neurokinin-1 receptor antagonist. Typical representatives in this series showed in vivo efficacy after oral administration in a NK3 mediated functional assay. This series of NK3 antagonists shows promise to deliver a novel antipsychotic.

Identification of a critical residue in the transmembrane domain 2 of tachykinin neurokinin 3 receptor affecting the dissociation kinetics and antagonism mode of osanetant (SR 142801) and piperidine-based structures.

In this study, we show that compound 3 (osanetant) binds with a pseudoirreversible, apparent noncompetitive mode of antagonism at the guinea pig NK(3), while it behaves competitively at the human NK(3). This difference is caused by a slower dissociation rate of compound 3 at the guinea pig NK(3) compared to human NK(3). The only amino acid difference between the human and guinea pig NK(3) in the binding site (Thr139(2.58) in human, corresponding to Ala114(2.58) in guinea pig) has been shown to be responsible for the different behavior. Compound 1 (talnetant), however, behaves competitively at both receptors. Using these data, 3D homology modeling, and site-directed mutagenesis, a model has been developed to predict the mode of antagonism of NK(3) antagonists based on their binding mode. This model was successfully used to predict the mode of antagonism of compounds of another chemical series including piperidine-based structures at human and guinea pig NK(3).

Selective naphthalene H(3) receptor inverse agonists with reduced potential to induce phospholipidosis and their quinoline analogs.

We reported earlier the refinement of our initial five-point pharmacophore model for the Histamine 3 receptor (H(3)R), with a new acceptor feature important for binding and selectivity against the other histamine receptor subtypes 1, 2 and 4. This approach was validated with a new series of H(3)R inverse agonists: the naphthalene series. In this Letter, we describe our efforts to overcome the phospholipidosis flag identified with our initial lead compound (1a). During the optimization process, we monitored the potency of our molecules toward the H(3) receptor, their selectivity against H(1)R, H(2)R and H(4)R, as well as some key molecular properties that may influence phospholipidosis. Encouraged by the promising profile of the naphthalene series, we used our deeper understanding of the H(3)R pharmacophore model to lead us towards the quinoline series. This series is perceived to have intrinsic advantages with respect to its amphiphilic vector.

5-hydroxyindole-2-carboxylic acid amides: novel histamine-3 receptor inverse agonists for the treatment of obesity.

Obesity is a major risk factor in the development of conditions such as hypertension, hyperglycemia, dyslipidemia, coronary artery disease, and cancer. Several pieces of evidence across different species, including primates, underscore the implication of the histamine 3 receptor (H(3)R) in the regulation of food intake and body weight and the potential therapeutic effect of H(3)R inverse agonists. A pharmacophore model, based on public information and validated by previous investigations, was used to design several potential scaffolds. Out of these scaffolds, the 5-hydroxyindole-2-carboxylic acid amide appeared to be of great potential as a novel series of H(3)R inverse agonist. Extensive structure-activity relationships revealed the interconnectivity of microsomal clearance and hERG (human ether-a-go-go-related gene) affinity with lipophilicity, artificial membrane permeation, and basicity. This effort led to the identification of compounds reversing the (R)-alpha-methylhistamine-induced water intake increase in Wistar rats and, further, reducing food intake in diet-induced obese Sprague-Dawley rats. Of these, the biochemical, pharmacokinetic, and pharmacodynamic characteristics of (4,4-difluoropiperidin-1-yl)[1-isopropyl-5-(1-isopropylpiperidin-4-yloxy)-1H-indol-2-yl]methanone 36 are detailed.

Discovery of benzoylpiperazines as a novel class of potent and selective GlyT1 inhibitors.

Screening of the Roche compound library led to the identification of the benzoylpiperazine 7 as a structurally novel GlyT1 inhibitor. The SAR which was developed in this series resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo efficacy after oral administration.

Refinement of histamine H3 ligands pharmacophore model leads to a new class of potent and selective naphthalene inverse agonists.

The refinement of our original five point pharmacophore model for the H(3) receptor with the addition of a new acceptor feature is presented. The importance of this new acceptor feature for the binding and the selectivity against H(1), H(2) and H(4) has been validated using a newly synthesized naphthalene series. With the SAR deduced from several hundred naphthalene derivatives in various sub-classes the specific role of each pharmacophoric feature, by varying the geometry, size and charge of the molecules, was elucidated. This led to the discovery of a highly potent and selective new compounds series.

Benzodioxoles: novel cannabinoid-1 receptor inverse agonists for the treatment of obesity.

The application of the evolutionary fragment-based de novo design tool TOPology Assigning System (TOPAS), starting from a known CB1R (CB-1 receptor) ligand, followed by further refinement principles, including pharmacophore compliance, chemical tractability, and drug likeness, allowed the identification of benzodioxoles as a novel CB1R inverse agonist series. Extensive multidimensional optimization was rewarded by the identification of promising lead compounds, showing in vivo activity. These compounds reversed the CP-55940-induced hypothermia in Naval Medical Research Institute (NMRI) mice and reduced body-weight gain, as well as fat mass, in diet-induced obese Sprague-Dawley rats. Herein, we disclose the tools and strategies that were employed for rapid hit identification, synthesis and generation of structure-activity relationships, ultimately leading to the identification of (+)-[( R)-2-(2,4-dichloride-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone ( R)-14g . Biochemical, pharmacokinetic, and pharmacodynamic characteristics of ( R)-14g are discussed.

Isomeric thiazole derivatives as ligands for the neuropeptide Y5 receptor.

Sets of isomeric thiazole derivatives 1 and 2 have been synthesised in a parallel iterative solution-phase synthesis approach guided by the SAR analysis derived from biological results and computer-aided design and analysis. This synergistic and streamlined working procedure led to highly active isomeric NPY5 receptor ligands. However, a 10-fold difference at least in their respective binding affinities was consistently found for all isomeric pairs 1 and 2. The analysis of conformational differences due to heteroatom interactions in 1 and 2 revealed a favourable C=O...S interaction in 1, whereas thiazoles 2 showed a repulsive C=O...N interaction.

Novel and potent NPY5 receptor antagonists derived from virtual screening and iterative parallel chemistry design.

In the quest for NPY5 receptor antagonists a virtual screening approach yielded a novel and potent hit class from a limited compound selection. The tight and seamless integration between virtual screening and rapid parallel chemistry within the framework of the Roche Lead Generation unit led in only two rounds of iterative chemistry optimisation to a much broader understanding of the factors which influence the potency of the thiazole hit class.