Exploring novel dilazep derivatives as hENT1 inhibitors and potentially covalent molecular tools.

The human equilibrative nucleoside transporter 1 (SLC29A1, hENT1) is a solute carrier that modulates the passive transport of nucleosides and nucleobases, such as adenosine. This nucleoside regulates various physiological processes, such as vasodilation and -constriction, neurotransmission and immune defense. Marketed drugs such as dilazep and dipyridamole have proven useful in cardiovascular afflictions, but the application of hENT1 inhibitors can be beneficial in a number of other diseases. In this study, 39 derivatives of dilazep's close analogue ST7092 were designed, synthesized and subsequently assessed using [3H]NBTI displacement assays and molecular docking. Different substitution patterns of the trimethoxy benzoates of ST7092 reduced interactions within the binding pocket, resulting in diminished hENT1 affinity. Conversely, [3H]NBTI displacement by potentially covalent compounds 14b, 14c, and 14d resulted in high affinities (Ki values between 1.1 and 17.5 nM) for the transporter, primarily by the ability of accommodating the inhibitors in various ways in the binding pocket. However, any indication of covalent binding with amino acid residue C439 remained absent, conceivably as a result of decreased nucleophilic residue reactivity. In conclusion, this research introduces novel dilazep derivatives that are active as hENT1 inhibitors, along with the first high affinity dilazep derivatives equipped with an electrophilic warhead. These findings will aid the rational and structure-based development of novel hENT1 inhibitors and pharmacological tools to study hENT1's function, binding mechanisms, and its relevance in (patho)physiological conditions.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

Label-free detection of prostaglandin transporter (SLCO2A1) function and inhibition: insights by wound healing and TRACT assays.

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies.

Computational Characterization of Membrane Proteins as Anticancer Targets: Current Challenges and Opportunities.

Cancer remains a leading cause of mortality worldwide and calls for novel therapeutic targets. Membrane proteins are key players in various cancer types but present unique challenges compared to soluble proteins. The advent of computational drug discovery tools offers a promising approach to address these challenges, allowing for the prioritization of "wet-lab" experiments. In this review, we explore the applications of computational approaches in membrane protein oncological characterization, particularly focusing on three prominent membrane protein families: receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and solute carrier proteins (SLCs). We chose these families due to their varying levels of understanding and research data availability, which leads to distinct challenges and opportunities for computational analysis. We discuss the utilization of multi-omics data, machine learning, and structure-based methods to investigate aberrant protein functionalities associated with cancer progression within each family. Moreover, we highlight the importance of considering the broader cellular context and, in particular, cross-talk between proteins. Despite existing challenges, computational tools hold promise in dissecting membrane protein dysregulation in cancer. With advancing computational capabilities and data resources, these tools are poised to play a pivotal role in identifying and prioritizing membrane proteins as personalized anticancer targets.

Pharmacological characterization of allosteric modulators: A case for chemokine receptors.

Chemokine receptors are relevant targets for a multitude of immunological diseases, but drug attrition for these receptors is remarkably high. While many drug discovery programs have been pursued, most prospective drugs failed in the follow-up studies due to clinical inefficacy, and hence there is a clear need for alternative approaches. Allosteric modulators of receptor function represent an excellent opportunity for novel drugs, as they modulate receptor activation in a controlled manner and display increased selectivity, and their pharmacological profile can be insurmountable. Here, we discuss allosteric ligands and their pharmacological characterization for modulation of chemokine receptors. Ligands are included if (1) they show clear signs of allosteric modulation in vitro and (2) display evidence of binding in a topologically distinct manner compared to endogenous chemokines. We discuss how allosteric ligands affect binding of orthosteric (endogenous) ligands in terms of affinity as well as binding kinetics in radioligand binding assays. Moreover, their effects on signaling events in functional assays and how their binding site can be elucidated are specified. We substantiate this with examples of published allosteric ligands targeting chemokine receptors and hypothetical graphs of pharmacological behavior. This review should serve as an effective starting point for setting up assays for characterizing allosteric ligands to develop safer and more efficacious drugs for chemokine receptors and, ultimately, other G protein-coupled receptors.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB2 Receptor.

The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.

Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors.

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches.

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Dual allosteric and orthosteric pharmacology of synthetic analog cannabidiol-dimethylheptyl, but not cannabidiol, on the cannabinoid CB2 receptor.

Cannabinoid CB2 receptor (CB2R) is a class A G protein-coupled receptor (GPCR) involved in a broad spectrum of physiological processes and pathological conditions. For that reason, targeting CB2R might provide therapeutic opportunities in neurodegenerative disorders, neuropathic pain, inflammatory diseases, and cancer. The main components from Cannabis sativa, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been therapeutically exploited and synthetically-derived analogs have been generated. One example is cannabidiol-dimethylheptyl (CBD-DMH), which exhibits anti-inflammatory effects. Nevertheless, its pharmacological mechanism of action is not yet fully understood and is hypothesized for multiple targets, including CB2R. The aim of this study was to further investigate the molecular pharmacology of CBD-DMH on CB2R while CBD was taken along as control. These compounds were screened in equilibrium and kinetic radioligand binding studies and various functional assays, including G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. In dissociation studies, CBD-DMH allosterically modulated the radioligand binding. Furthermore, CBD-DMH negatively modulated the G protein activation of reference agonists CP55,940, AEA and 2-AG, but not the agonist-induced ß-arrestin-2 recruitment. Nevertheless, CBD-DMH also displayed competitive binding to CB2R and partial agonism on G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. CBD did not exhibit such allosteric behavior and only very weakly bound CB2R without activation. This study shows a dual binding mode of CBD-DMH, but not CBD, to CB2R with the suggestion of two different binding sites. Altogether, it encourages further research into this dual mechanism which might provide a new class of molecules targeting CB2R.

The 17th EFMC Short Course on Medicinal Chemistry on Small Molecule Protein Degraders.

The 17th EFMC Short Course on Medicinal Chemistry took place April 23-26, 2023 in Oegstgeest, near Leiden in the Netherlands. It covered for the first time the exciting topic of Targeted Protein Degradation (full title: Small Molecule Protein Degraders: A New Opportunity for Drug Design and Development). The course was oversubscribed, with 35 attendees and 6 instructors mainly from Europe but also from the US and South Africa, and representing both industry and academia. This report summarizes the successful event, key lectures given and topics discussed.

3DDPDs: describing protein dynamics for proteochemometric bioactivity prediction. A case for (mutant) G protein-coupled receptors.

Proteochemometric (PCM) modelling is a powerful computational drug discovery tool used in bioactivity prediction of potential drug candidates relying on both chemical and protein information. In PCM features are computed to describe small molecules and proteins, which directly impact the quality of the predictive models. State-of-the-art protein descriptors, however, are calculated from the protein sequence and neglect the dynamic nature of proteins. This dynamic nature can be computationally simulated with molecular dynamics (MD). Here, novel 3D dynamic protein descriptors (3DDPDs) were designed to be applied in bioactivity prediction tasks with PCM models. As a test case, publicly available G protein-coupled receptor (GPCR) MD data from GPCRmd was used. GPCRs are membrane-bound proteins, which are activated by hormones and neurotransmitters, and constitute an important target family for drug discovery. GPCRs exist in different conformational states that allow the transmission of diverse signals and that can be modified by ligand interactions, among other factors. To translate the MD-encoded protein dynamics two types of 3DDPDs were considered: one-hot encoded residue-specific (rs) and embedding-like protein-specific (ps) 3DDPDs. The descriptors were developed by calculating distributions of trajectory coordinates and partial charges, applying dimensionality reduction, and subsequently condensing them into vectors per residue or protein, respectively. 3DDPDs were benchmarked on several PCM tasks against state-of-the-art non-dynamic protein descriptors. Our rs- and ps3DDPDs outperformed non-dynamic descriptors in regression tasks using a temporal split and showed comparable performance with a random split and in all classification tasks. Combinations of non-dynamic descriptors with 3DDPDs did not result in increased performance. Finally, the power of 3DDPDs to capture dynamic fluctuations in mutant GPCRs was explored. The results presented here show the potential of including protein dynamic information on machine learning tasks, specifically bioactivity prediction, and open opportunities for applications in drug discovery, including oncology.

Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression.

The adenosine A3 receptor (A3AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A3AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A3AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A3AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A3AR in pathologies.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Microwave-Assisted Synthesis, Structure, and Preliminary Biological Evaluation of Novel 6-Methoxy-5,6-dihydro-5-azapurines.

We herein disclose the microwave-assisted synthesis of previously unreported 6-methoxy-5,6-dihydro-5-azapurines, whose purine-like scaffold is promising for drug discovery. The method is simple, fast, and relies on easily accessible reagents such as trimethyl orthoformate, acetic acid, and aminotriazole-derived N,N'-disubstituted formamidines. The preliminary biological evaluation revealed that selected representatives of synthesized 6-methoxy-5,6-dihydro-5-azapurines dose-dependently reduce the viability of HepG2 and A549 cancer cells having little to no influence on five tested purinergic receptors.

Proteochemometric Modeling Identifies Chemically Diverse Norepinephrine Transporter Inhibitors.

Solute carriers (SLCs) are relatively underexplored compared to other prominent protein families such as kinases and G protein-coupled receptors. However, proteins from the SLC family play an essential role in various diseases. One such SLC is the high-affinity norepinephrine transporter (NET/SLC6A2). In contrast to most other SLCs, the NET has been relatively well studied. However, the chemical space of known ligands has a low chemical diversity, making it challenging to identify chemically novel ligands. Here, a computational screening pipeline was developed to find new NET inhibitors. The approach increases the chemical space to model for NETs using the chemical space of related proteins that were selected utilizing similarity networks. Prior proteochemometric models added data from related proteins, but here we use a data-driven approach to select the optimal proteins to add to the modeled data set. After optimizing the data set, the proteochemometric model was optimized using stepwise feature selection. The final model was created using a two-step approach combining several proteochemometric machine learning models through stacking. This model was applied to the extensive virtual compound database of Enamine, from which the top predicted 22,000 of the 600 million virtual compounds were clustered to end up with 46 chemically diverse candidates. A subselection of 32 candidates was synthesized and subsequently tested using an impedance-based assay. There were five hit compounds identified (hit rate 16%) with sub-micromolar inhibitory potencies toward NET, which are promising for follow-up experimental research. This study demonstrates a data-driven approach to diversify known chemical space to identify novel ligands and is to our knowledge the first to select this set based on the sequence similarity of related targets.

Structural basis of selective cannabinoid CB2 receptor activation.

Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

Cellular Assay to Study ß-Arrestin Recruitment by the Cannabinoid Receptors 1 and 2.

Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of ß-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit ß-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a ß-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here.

A Chemical Biological Approach to Study G Protein-Coupled Receptors: Labeling the Adenosine A1 Receptor Using an Electrophilic Covalent Probe.

G protein-coupled receptors (GPCRs) have been known for decades as attractive drug targets. This has led to the development and approval of many ligands targeting GPCRs. Although ligand binding effects have been studied thoroughly for many GPCRs, there are multiple aspects of GPCR signaling that remain poorly understood. The reasons for this are the difficulties that are encountered upon studying GPCRs, for example, a poor solubility and low expression levels. In this work, we have managed to overcome some of these issues by developing an affinity-based probe for a prototypic GPCR, the adenosine A1 receptor (A1AR). Here, we show the design, synthesis, and biological evaluation of this probe in various biochemical assays, such as SDS-PAGE, confocal microscopy, and chemical proteomics.