ABSTRACT
Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. Results: RG7774 was found to be a potent (EC50: 2.8 nM and Ki: 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.
ABSTRACT
The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.
Subject(s)
Fluorescent Dyes , Receptor, Cannabinoid, CB1 , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Receptor, Cannabinoid, CB1/metabolism , Humans , Molecular Docking Simulation , HEK293 Cells , Ligands , Pyrroles/chemistry , Pyrroles/pharmacology , Pyrroles/chemical synthesis , Structure-Activity Relationship , Cyclic AMP/metabolismABSTRACT
We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB2R) selective inverse agonists (S)-1 and (R)-1, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem-dimethylheptyl side chain. Epimer (R)-1 exhibits high affinity for CB2R with Kd = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-mediated cAMP as well as G protein recruitment assays and do not trigger ß-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp2586.48 and thereby stabilize CB2R in its inactive state.
ABSTRACT
Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the ßγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 µM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.
ABSTRACT
Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.
Subject(s)
Fluorescent Dyes , Signal Transduction , Ligands , Protein Binding , Fluorescent Dyes/chemistry , Receptors, CannabinoidABSTRACT
The human neuropeptide neuromedin S (NMS) consists of 33 amino acids. The introduction of tritium atoms into NMS has not been described so far. This represents a gap for using [3H]NMS in radioreceptor binding assays or in tracking and monitoring their metabolic pathway. Two approaches for the incorporation of tritium into NMS were explored in this study: (1) halogenation at the His-18 residue followed by catalyzed iodine-127/tritium exchange and (2) conjugation of tritiated N-succinimidyl-[2,3-3H3]propionate ([3H]NSP) to at least one of the three available primary amines of amino acids Ile-1, Lys-15, and Lys-16 in the peptide sequence. Although iodination of histidine was achieved, subsequent iodine-127/deuterium exchange was unsuccessful. Derivatization at the three possible amino positions in the peptide using nonradioactive NSP resulted in a mixture of unconjugated NSM and 1- to 3-conjugations at different amino acids in the peptide sequence. Each labeling position in the mixture was assigned following detailed LC-MS/MS analysis. After separating the mixture, it was shown in an in vitro fluorometric imaging plate reader (FLIPR) and in a competitive binding assay that the propionyl-modified NMS derivatives were comparable to the unlabeled NMS, regardless of the degree of labeling and the labeling position(s). A molecular simulation with NMS in the binding pocket of the protein neuromedin U receptor 2 (NMUR2) confirmed that the possible labeling positions are located outside the binding region of NMUR2. Tritium labeling was achieved at the N-terminal Ile-1 using [3H]NSP in 7% yield with a radiochemical purity of >95% and a molar activity of 90 Ci/mmol. This approach provides access to tritiated NMS and enables new investigations to characterize NMS or corresponding NMS ligands.
ABSTRACT
Computational methods in medicinal chemistry facilitate drug discovery and design. In particular, machine learning methodologies have recently gained increasing attention. This chapter provides a structured overview of the current state of computational chemistry and its applications for the interrogation of the endocannabinoid system (ECS), highlighting methods in structure-based drug design, virtual screening, ligand-based quantitative structure-activity relationship (QSAR) modeling, and de novo molecular design. We emphasize emerging methods in machine learning and anticipate a forecast of future opportunities of computational medicinal chemistry for the ECS.
Subject(s)
Computational Chemistry , Endocannabinoids , Drug Design , Ligands , Machine Learning , Quantitative Structure-Activity RelationshipABSTRACT
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.
ABSTRACT
The Torsion Library is a collection of torsion motifs associated with angle distributions, derived from crystallographic databases. It is used in strain assessment, conformer generation, and geometry optimization. A hierarchical structure of expert curated SMARTS defines the chemical environments of rotatable bonds and associates these with preferred angles. SMARTS can be very complex and full of implications, which make them difficult to maintain manually. Recent developments in automatically comparing SMARTS patterns can be applied to the Torsion Library to ensure its correctness. We specifically discuss the implementation and the limits of such a procedure in the context of torsion motifs and show several examples of how the Torsion Library benefits from this. All automated changes are validated manually and then shown to have an effect on the angle distributions by correcting matching behavior. The corrected Torsion Library itself is available including both PDB as well as CSD histograms in the Supporting Information and can be used to evaluate rotatable bonds at https://torsions.zbh.uni-hamburg.de.
Subject(s)
Molecular Conformation , Databases, Factual , Gene LibraryABSTRACT
The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.
Subject(s)
Glycine Plasma Membrane Transport Proteins/antagonists & inhibitors , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine/metabolism , Binding Sites , Biological Transport/drug effects , Crystallography , Humans , Models, Molecular , Piperazines/chemistry , Piperazines/pharmacology , Protein Binding , Protein Conformation , Protein Stability , Single-Domain Antibodies , Sulfones/chemistry , Sulfones/pharmacology , SynchrotronsABSTRACT
Genetic, preclinical and clinical data link Parkinson's disease and Gaucher's disease and provide a rational entry point to disease modification therapy via enhancement of ß-Glucocerebrosidase (GCase) activity. We discovered a new class of pyrrolo[2,3-b]pyrazine activators effecting both Vmax and Km. They bind to human GCase and increase substrate metabolism in the lysosome in a cellular assay. We obtained the first crystal structure for an activator and identified a novel non-inhibitory binding mode at the interface of a dimer, rationalizing the observed structure-activity relationship (SAR). The compound binds GCase inducing formation of a dimeric state at both endoplasmic reticulum (ER) and lysosomal pHs, as confirmed by analytical ultracentrifugation. Importantly, the pyrrolo[2,3-b]pyrazines have central nervous system (CNS) drug-like properties. Our findings are important for future drug discovery efforts in the field of GCase activation and provide a deeper mechanistic understanding of the requirements for enzymatic activation, pointing to the relevance of dimerization.
Subject(s)
Enzyme Activators/metabolism , Glucosylceramidase/metabolism , Protein Multimerization/drug effects , Pyrazines/metabolism , Pyrroles/metabolism , Binding Sites , Crystallography, X-Ray , Enzyme Activators/chemistry , Glucosylceramidase/chemistry , Humans , Kinetics , Molecular Structure , Protein Binding , Pyrazines/chemistry , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Fluorescent Dyes/chemistry , Microglia/metabolism , Receptor, Cannabinoid, CB2/analysis , Animals , CHO Cells , Cricetulus , Disease Models, Animal , Flow Cytometry , Fluorescence Resonance Energy Transfer , Humans , Ligands , Mice , Molecular Docking Simulation , Molecular Probes/chemistry , Optical Imaging , Sensitivity and Specificity , Signal TransductionABSTRACT
The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptorâ 2 (CB2 R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood-likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB2 R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB2 R agonists with high selectivity over the closely related cannabinoid receptorâ 1 (CB1 R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB2 R to date.
Subject(s)
Amines/chemistry , Cannabinoids/chemistry , Dronabinol/analogs & derivatives , Receptor, Cannabinoid, CB2/metabolism , Binding Sites , Cannabinoids/metabolism , Dronabinol/chemistry , Dronabinol/metabolism , Humans , Kinetics , Ligands , Molecular Docking Simulation , Protein Binding , Protein Structure, Tertiary , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/chemistryABSTRACT
The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [11C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a Ki (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [18F]3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/µmol. Specific binding of [18F]3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405â¯833. Metabolite analysis revealed only intact [18F]3 in the rat brain. [18F]3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.
Subject(s)
Brain/metabolism , Fluorine Radioisotopes/metabolism , Neuroimaging/methods , Positron-Emission Tomography/methods , Pyridines/chemistry , Radiopharmaceuticals/metabolism , Receptor, Cannabinoid, CB2/metabolism , Animals , Brain/diagnostic imaging , Cyclic AMP/metabolism , Fluorine Radioisotopes/chemistry , Hepatocytes/metabolism , Humans , Ligands , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Protein Conformation , Radiochemistry , Radiopharmaceuticals/chemistry , Rats , Rats, Wistar , Receptor, Cannabinoid, CB2/chemistry , Structure-Activity RelationshipABSTRACT
(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.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Cannabinoid Receptor Agonists/administration & dosage , Endotoxins/adverse effects , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Uveitis/drug therapy , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cannabinoid Receptor Agonists/chemistry , Cannabinoid Receptor Agonists/pharmacology , Cell Adhesion/drug effects , Cells, Cultured , Disease Models, Animal , Leukocytes/drug effects , Leukocytes/metabolism , Male , Mice , Mice, Knockout , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Neutrophils/drug effects , Neutrophils/metabolism , Receptor, Cannabinoid, CB2/chemistry , Receptor, Cannabinoid, CB2/genetics , Signal Transduction , Uveitis/chemically induced , Uveitis/immunologyABSTRACT
The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.
Subject(s)
Ligands , Receptors, CCR7/metabolism , Allosteric Regulation , Binding Sites , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Neuraminidase/genetics , Neuraminidase/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, CCR2/chemistry , Receptors, CCR2/metabolism , Receptors, CCR7/antagonists & inhibitors , Receptors, CCR7/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purificationABSTRACT
BACKGROUND: Several factors contribute to the development failure of novel pharmaceuticals, one of the most important being adverse effects in pre-clinical and clinical studies. Early identification of off-target compound activity can reduce safety-related attrition in development. In vitro profiling of drug candidates against a broad range of targets is an important part of the compound selection process. Many compounds are synthesized during early drug discovery, making it necessary to assess poly-pharmacology at a limited number of targets. This paper describes how a rational, statistical-ranking approach was used to generate a cost-effective, optimized panel of assays that allows selectivity focused structure-activity relationships to be explored for many molecules. This panel of 50 targets has been used to routinely screen Roche small molecules generated across a diverse range of therapeutic targets. Target hit rates from the Bioprint® database and internal Roche compounds are discussed. We further describe an example of how this panel was used within an anti-infective project to reduce in vivo testing. METHOD: To select the optimized panel of targets, IC50 values of compounds in the BioPrint® database were used to identify assay "hits" i.e. IC50â¯≤â¯1⯵M in 123 different in vitro pharmacological assays. If groups of compounds hit the same targets, the target with the higher hit rate was selected, while others were considered redundant. Using a step-wise analysis, an assay panel was identified to maximize diversity and minimize redundancy. Over a five-year period, this panel of 50 off-targets was used to screen ≈1200 compounds synthesized for Roche drug discovery programs. Compounds were initially tested at 10⯵M and hit rates generated are reported. Within one project, the number of hits was used to refine the choice of compounds being assessed in vivo. RESULTS: 95% of compounds from the BioPrint® panel were identified within the top 47-ranked assays. Based on this analytical approach and the addition of three targets with established safety concerns, a Roche panel was created for external screening. hERG is screened internally and not included in this analysis. Screening at 10⯵M in the Roche panel identified that adenosine A3 and 5HT2B receptors had the highest hit rates (~30%), with 50% of the targets having a hit rate of ≤4%. An anti-infective program identified that a high number of hits in the Roche panel was associated with mortality in 19 mouse tolerability studies. To reduce the severity and number of such studies, future compound selections integrated the panel hit score into the selection process for in vivo studies. It was identified that compounds which hit less targets in the panel and had free plasma exposures of ~2⯵M were generally better tolerated. DISCUSSION: This paper describes how an optimized panel of 50 assays was selected on the basis of hit similarity at 123 targets. This reduced panel, provides a cost-effective screening panel for assessing compound promiscuity, whilst also including many safety-relevant targets. Frequent use of the panel in early drug discovery has provided promiscuity and safety-relevant information to inform pre-clinical drug development at Roche.
ABSTRACT
Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.
Subject(s)
Morpholines/chemistry , Photoaffinity Labels/chemistry , Pyridines/chemistry , Receptor, Cannabinoid, CB2/biosynthesis , Receptor, Cannabinoid, CB2/metabolism , Alkynes/chemistry , HL-60 Cells , Humans , Ligands , Molecular Structure , Morpholines/chemical synthesis , Photoaffinity Labels/chemical synthesis , Pyridines/chemical synthesisABSTRACT
Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidß (Aß) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aß levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aß. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/metabolism , Peptide Fragments/administration & dosage , Administration, Intravenous , Amyloid Precursor Protein Secretases/chemistry , Animals , Aspartic Acid Endopeptidases/chemistry , Blood-Brain Barrier/metabolism , Catalytic Domain/drug effects , Dose-Response Relationship, Drug , Humans , Mice , Peptide Fragments/pharmacology , Receptors, Transferrin/metabolismABSTRACT
We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.