The human genome encodes for over 600 kinases, which are important targets for drug discovery in many diseases, such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, which contributes to the difficulty of developing selective inhibitors. A lack of selectivity may contribute to high drug candidate attrition rates due to an unfavorable off-target profile. To prevent unforeseen side effects, as well as to accurately link inhibitor function to pharmacological effects and phenotypic readouts, efficient methods for determining target engagement across the cellular kinome are necessary. One way to address this problem is to profile the interaction landscape of kinase inhibitors in living cells using the broad-spectrum kinase probe XO44. Here, we describe a detailed protocol of the CellEKT workflow using XO44 to profile kinase inhibitors in a full dose-response manner across 200+ kinases. The protocol allows to determine the EC50 of up to three kinase inhibitors in a cell line of interest in 4 days.

The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular target engagement of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and non-covalent drugs across over 300 kinases, expressed as half maximum inhibitory concentration (IC50), which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, target engagement profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs.

Drug discovery is a costly and time-intensive process that is often limited by efficacy issues and unforeseen side effects. GPCR-targeting ligands, which account for one-third of marketed drugs, have been shown to exhibit biased signaling and preferential activation of one signaling pathway over another. While designing biased ligands is a recent advancement, their therapeutic benefits remain uncertain. However, the success of existing drugs raises the following question: do they inherently exhibit signaling bias that enhances efficacy or safety? This study examines the signaling profiles of short- and long-acting β2AR agonists (SABAs and LABAs), key treatments for asthma and COPD, using biosensors to measure G protein and β-arrestin coupling. Older SABAs, such as isoprenaline and isoetharine, show minor G protein bias, while newer agents, such as salbutamol, demonstrate significant G protein bias. Among LABAs, salmeterol shows greater G protein bias compared to that of the more balanced formoterol. This shift toward G protein bias over 50 years reflects efforts to improve asthma treatments. The increased bias results from reduced ligand-receptor residence times and weaker receptor-β-arrestin complex formation, contributing to the enhanced efficacy. Despite the potential advantages, a systematic evaluation of signaling bias remains underutilized in drug development. Early-stage, high-throughput tools to assess signaling profiles could improve candidate selection, reduce late-stage failures, and minimize side effects. We advocate for the routine integration of biosensors for quantifying signaling bias, optimizing compound selection, and enhancing therapeutic outcomes.

Studying biased signaling of G protein-coupled receptors (GPCRs) holds promise for the identification of ligands with a better therapeutic window. However, proper examination of biased signaling remains challenging by risking introduction of system or observation bias. Therefore, we developed a novel multiplex assay that simultaneously and kinetically detects cAMP production and ß-arrestin-2 recruitment in the same well. To investigate the applicability of the kinetic multiplex assay, we profiled seventeen clinically tested agonists for the cannabinoid CB2 receptor (CB2R), a promising GPCR for treating tissue injury and inflammation. Agonist-mediated CB2R activation and signaling was time sensitive, dependent on the agonist. Similar potency and efficacy parameters were obtained from semi-kinetic and kinetic analysis, while the latter provided additional signaling rate constants for signaling onset (k1) and decline (k2). Fast CB2R engagement (kon) of agonists resulted in increased affinity and potency. Slow dissociation of agonists extended the interaction between CB2R and ß-arrestin-2. Moreover, superagonists Tedalinab, Olorinab, PRS-211375 and ART-27.13 were characterized by fast k1 values. No significant biased signaling was observed for the investigated CB2R agonists. To validate the assay, the reported biased compound JWH145 was tested in a multiplex assay developed for CB1R and confirmed that our new method is able to detect bias. Altogether, this study accentuates the potential of multiplexing functional responses and performing kinetic analyses to provide an extensive preclinical profile for agonists that may better predict their in vivo pharmacological effects. Ultimately, providing full kinetic context for binding and signaling of GPCR agonists could advance drug discovery efforts.

Introduction:The kinetics of ligand binding to G protein-coupled receptors (GPCRs) is an important optimization parameter in drug discovery. Traditional radioligand assays are labor-intensive, preventing their application at the early stages of drug discovery. Fluorescence-based assays offer several advantages, including a possibility to develop a homogeneous format, continuous data collection, and higher throughput. This study sought to develop a fluorescence-based binding assay to investigate ligand-binding kinetics at human cannabinoid type 1 and 2 receptors (CB1R and CB2R). Methods:We synthesized D77, a novel tracer derived from the non-selective cannabinoid Δ8-THC. Using time-resolved Förster resonance energy transfer (TR-FRET), we developed an assay to study ligand-binding kinetics at physiological temperatures. For CB1R, we truncated the first 90 amino acids of its flexible N-terminal domain to reduce the FRET distance between the terbium cryptate (donor) and the fluorescent ligand (acceptor). The full-length CB2R construct was functional without modification due to its shorter N-terminus. The Motulsky-Mahan competition binding model was used to analyze the binding kinetics of the endocannabinoids and several other non-fluorescent ligands. Results:The D77 tracer showed nanomolar-range affinity for truncated CB1R (CB1R91-472) and full-length CB2R (CB2R1-360), displaying competitive binding with orthosteric ligands. D77 exhibited rapid dissociation kinetics from both CB1R and CB2R, which were similar to the fastest dissociating reference compounds. This was critical for accurately determining the on- and off-rates of the fastest dissociating compounds. Using D77, we measured the kinetic binding properties of various CB1R and CB2R agonists and antagonists at physiological temperature and sodium ion concentration. Discussion:The k on values for molecules binding to CB1R varied by three orders of magnitude, from the slowest (HU308) to the fastest (rimonabant). A strong correlation between k on and affinity was observed for compounds binding to CB1R, indicating that the association rate primarily determines their affinity for CB1R. Unlike CB1R, a stronger correlation was found between the dissociation rate constant k off and the affinity for CB2R, suggesting that both k on and k off dictate the overall affinity for CB2R. Exploring the kinetic parameters of cannabinoid drug candidates could help drug development programs targeting these receptors.

G protein-coupled receptors (GPCRs) are important therapeutic drug targets for a wide range of diseases. Upon activation, GPCRs can initiate several signaling pathways, each with unique therapeutic implications. Therefore, understanding how drugs selectively engage specific signaling pathways becomes paramount. However, achieving this selectivity remains highly challenging. To unravel the underlying multifaceted mechanisms, we integrate systematic mutagenesis of the CB2R, comprehensive profiling of Gαi2 and β-arrestin1 engagements and computer simulations to track the effects of mutations on receptor dynamics. Our research reveals multiple triggers within a complex allosteric communication network (ACN) that converge to preferential CB2R coupling by modulating evolutionarily conserved motifs. Utilizing network path analysis, we find that potent triggers are typically highly connected nodes and are located near regions of high information transmission within the ACN. Our insights highlight the complexity of GPCR signaling and provide a framework for the rational design of drug candidates tailored to evoke specific functional responses, ultimately enhancing the precision and efficacy of therapeutic interventions.

Allosteric modulation of cannabinoid receptor type 1 (CB1R) offers a promising alternative to conventional therapeutic approaches using orthosteric ligands (OLs). Currently, CB1R allosteric modulators (AMs) are characterized based on their ability to modulate binding or functional response of OLs, preventing isolation of individual contributions by allosteric and orthosteric ligands. Herein, we develop the first allosteric fluoroprobe and attendant FRET-based assay allowing for the direct profiling of CB1R AMs without coincubation with an OL. Our allosteric tracer enables differentiation of allosteric and orthosteric ligands as well as their pharmacological profiling at CB1R. The utility of this work is highlighted by addressing ambiguities surrounding the binding of cannabidiol (CBD). CBD was found to interact with both allosteric and orthosteric sites of CB1R with comparable affinity (pKi=5.34 and 5.67, respectively).

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

G protein-coupled receptors are important therapeutic drug targets for a wide range of diseases. Their ability to preferentially engage specific signaling pathways over others can be exploited to design drugs that target only disease-associated pathways leading to an improved safety profile. However, the underlying molecular mechanisms for preferential pathway engagement are complex and remain largely elusive. To elucidate the multifaceted actions at the receptor level that lead to preferential coupling, we employ a combination of techniques. Our approach integrates systematic mutagenesis of the CB2R and comprehensive profiling of Gαi2 and β-arrestin1 engagements with computer simulations to track mutant-induced impacts on receptor dynamics. Most importantly, our research discloses multiple triggers on a complex allosteric communication network (ACN) that converge to preferential CB2R coupling by modulating evolutionary conserved motifs (e.g., CWxP, NPxxY, sodium binding site). Potent triggers for a preferential Gαi2 response exhibit high levels of connectivity and are located in proximity to connections with high information transmission. Our insights highlight the complexity of GPCR signaling and can guide the rational design of drug candidates tailored to evoke specific functional responses that can enhance the precision and efficacy of therapeutic interventions.

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research.

Chemical probes allow us to identify, validate and confirm novel targets for therapeutic applications, enable the development of drug candidates, and open the way to new therapeutic strategies, vaccines and diagnostic tools.

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans-cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with 18F or 3H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with 18F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a 3H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.

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 βγ 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.

Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to β-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.

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.

Enzymes are essential, physiological catalysts involved in all processes of life, including metabolism, cellular signaling and motility, as well as cell growth and division. They are attractive drug targets because of the presence of defined substrate-binding pockets, which can be exploited as binding sites for pharmaceutical enzyme inhibitors. Understanding the reaction mechanisms of enzymes and the molecular mode of action of enzyme inhibitors is indispensable for the discovery and development of potent, efficacious, and safe novel drugs. The combination of classical concepts of enzymology with new experimental and data analysis methods opens new routes for drug discovery.

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 β-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.

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 context. 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 and to the detection of CB2R in human breast cancer cells.

We thank Alarcon and Borroto for commenting on our work (1, 2). Modulation of TCR signaling by AX-024 was shown previously by ZAP70 phosphorylation data (1, 3, 4). We deliberately used low concentrations of anti-CD3 to weakly stimulate primary human T cells but could not reproduce the published effect of AX-024 on ZAP70 phosphorylation, possibly due to technical differences. In line with this finding, we observed that AX-024 does not impede the CD3e/Nck1-SH3.1 interaction as measured by SPR and NMR (2). In Ref. 3, the response unit scale of the SPR data suggests very low protein-binding activity. In our opinion, the observed lag phase during association and the incomplete signal return to baseline upon dissociation preclude extraction of kinetic and thermodynamic parameters of CD3e peptide binding to Nck1-SH3.1. Our SPR and ligandobserved NMR studies did not reveal interaction of AX-024 with Nck1-SH3.1 (based on resonances, line widths, and chemical shifts). We noted that the average changes in H and N NMR chemical shifts at millimolar AX-024 concentrations are markedly smaller compared with the chemical shift perturbations observed with CD3e in Ref. 3. In addition, we could not recapitulate the binding mode of AX-024 to Nck1-SH3.1 proposed in Ref. 3. Together with the tendency for AX-024 to bind to other proteins (2), the mode of action of AX-024 remains to be established. Thus, publication of the announced structure-activity relationship (SAR)-enabling co-crystal structure of AX-024 bound to Nck will generate valuable insights.