Solute carrier transporters (SLCs) are integral membrane proteins that play pivotal roles in maintaining cellular homeostasis by mediating the transport of a diverse range of substrates across cell membranes. With their involvement in fundamental physiological processes such as nutrient uptake, neurotransmitter signaling, and drug transport, SLCs have emerged as crucial players in health and disease. Dysregulation of SLC function has been implicated in a spectrum of disorders, including metabolic diseases, cancer, and neurological afflictions. Despite their significance, SLCs remain relatively understudied compared to other protein classes, resulting in a gap in understanding their molecular mechanisms of action and potential as therapeutic targets. This review aims to address this gap by providing a comprehensive overview of the diverse array of small-molecule probes utilized in the study of SLCs. Various types of functionalized probes, amongst which fluorescent probes, bivalent probes, covalent inhibitors, affinity-based probes, photoswitchable inhibitors and proteolysis targeting chimeras (PROTACs), have been designed to investigate transporter function, substrate specificity, and regulatory mechanisms. In this review, we describe the principles underlying the design and synthesis of these probes, highlights key examples of their application in elucidating transporter function and regulation, and discuss insights gained from such studies. Furthermore, we examine current challenges and future directions in the development and utilization of small-molecule probes for SLC transporter research. By shedding light on the intricate mechanisms involved in transporter function and regulation, this review not only enhances the understanding of SLCs but also highlights their potential as therapeutic targets in drug discovery and thereby may facilitate systematic implementation of these innovative research approaches and the refinement of existing methodologies.

Roughly one-third of all marketed drugs act by binding to one or more of the >800 human GPCRs, primarily through activation or inhibition via the orthosteric binding site. In addition, novel strategies to alter GPCR functioning are being developed, including allosteric, biased and covalently binding ligands. Molecular probes play an important role in verifying such drug molecules with new modes of action and providing information on all factors involved in GPCR signalling. Various types of molecular probes have been developed, ranging from small molecules to antibodies, each bearing its own advantages and disadvantages. In this mini-review, a closer look is taken at small molecular probes that functionalize GPCRs in a covalent manner, such as through the conjugation of reporter groups like fluorophores or biotin. Covalently bound reporter groups allow the investigation of GPCRs across an increasing range of biochemical assay types, yielding new insights into GPCR signalling pathways. Here, a broad range of recently developed ‘functionalized covalent probes’ is summarized. Furthermore, the use of these probes in biochemical assays and their applications in the field of GPCR research are discussed. Lastly, a view on possible future applications of these types of small molecular probes is provided.

Solute carrier (SLC) transporters form a protein superfamily that enables transmembrane transport of diverse substrates including nutrients, ions and drugs. There are about 450 different SLCs, residing in a variety of subcellular membranes. Loss-of-function of an unusually high proportion of SLC transporters is genetically associated with a plethora of human diseases, making SLCs a rapidly emerging but challenging drug target class. Knowledge of their protein environment may elucidate the molecular basis for their functional integration with metabolic and cellular pathways and help conceive pharmacological interventions based on modulating proteostatic regulation. We aimed at obtaining a global survey of the SLC-protein interaction landscape and mapped the protein-protein interactions of 396 SLCs by interaction proteomics. We employed a functional assessment based on RNA interference of interactors in combination with measurement of protein stability and localization. As an example, we detail the role of a SLC16A6 phospho-degron and the contributions of PDZ-domain proteins LIN7C and MPP1 to the trafficking of SLC43A2. Overall, our work offers a resource for SLC-protein interactions for the scientific community.

A robust endogenous clock is required for proper function of many physiological processes. The suprachiasmatic nucleus (SCN) constitutes our central circadian clock and allows us to adapt to daily changes in the environment. Aging can cause a decline in the amplitude of circadian rhythms in SCN and peripheral clocks, which contributes to increased risk of several chronic diseases. Strengthening clock function would therefore be an effective strategy to improve health. A high-throughput chemical screening has identified clock-enhancing molecule 3 (CEM3) as small molecule that increases circadian rhythm amplitude in cell lines and SCN explants. It is, however, currently not known whether CEM3 acts by enhancing the amplitude of individual single-cell oscillators or by enhancing synchrony among neurons. In view of CEM3's potential, it is of evident importance to clarify the mode of action of CEM3. Here, we investigated the effects of CEM3 on single-cell PERIOD2::LUCIFERASE rhythms in mouse SCN explants. CEM3 increased the amplitude in approximately 80%-90% of the individual cells in the SCN without disrupting the phase and/or period of their rhythms. Noticeably, CEM3's effect on amplitude is independent of the cell's initial amplitude. These findings make CEM3 a potential therapeutic candidate to restore compromised amplitude in circadian rhythms and will boost the development of other molecular approaches to improve health.

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.

Interest in affinity-based probes (AfBPs) as novel tools to interrogate G protein-coupled receptors (GPCRs) has gained traction in recent years. AfBPs represent an interesting and more versatile alternative to antibodies. In the present study, we report the development and validation of AfBPs that target the intracellular allosteric pocket of CCR2, a GPCR of interest for the development of therapies targeting autoimmune and inflammatory diseases and also cancer. Owing to the two-step labeling process of these CCR2 AfBPs through the incorporation of a click handle, we were successful in applying our most efficient probe in a variety of in vitro experiments and making use of multiple different detection techniques, such as SDS-PAGE and LC/MS-based proteomics. Collectively, this novel probe shows high selectivity, versatility, and applicability. Hence, this is a valuable alternative for CCR2-targeting antibodies and other traditional tool compounds and could aid in target validation and engagement in drug discovery.

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

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.

Bioactivity prediction is essential in computational drug discovery, particularly within virtual screening campaigns. Despite advancements in model architectures and features, the sparsity and quality of relevant training data remain a major bottleneck. Notably, genetic variance annotation, crucial for understanding variant-specific bioactivity, is often neglected. Key efforts to tackle these issues are conducted by public bioactivity databases such as ChEMBL, but these are not free of challenges. Here, a comprehensive analysis of the extent and distribution of bioactivity data tested on genetic variants across organisms, protein families, individual targets, and specific variants, for the first time characterises in detail the genetic variability landscape in the ChEMBL database and sheds light on the range and consequences of protein amino acid substitutions in bioactivity data distribution and modelling. Furthermore, an extensive set of analysis resources (Python package and notebooks) and a variant-annotated bioactivity dataset are made available to help replicate the analyses described here for any protein of interest and make informed decisions regarding the quality of data for modelling. Finally, the potential to extract variants and subsets of the chemical space with desirable inter-variant bioactivity profiles is demonstrated for data-rich proteins. This approach contributes to more reliable bioactivity modelling, aids noise reduction and informs decision-making in computational drug discovery.

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.

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.

CC chemokine receptor 2 and CCL2 are highly involved in cancer growth and metastasis, and immune escape. Raised sodium ion concentrations in solid tumours have also been correlated to metastasis and immune modulation. Sodium ions can modulate class A G protein-coupled receptors through the sodium ion binding site characterized by a highly conserved aspartic acid residue (D2.50), also present in CCR2. Hence, we further explored this binding site in CCR2 by radioligand binding studies and mutagenesis. Modulation of three distinctly binding radioligands by sodium ions and amiloride derivates was investigated. Sodium ions were observed to be relatively weak modulators of antagonist binding, but substantially increased 125I-CCL2 dissociation from CCR2. 6-Substituted Hexamethylene Amiloride (HMA) modulated all tested radioligands. Induced-fit docking of HMA in the presumed sodium ion binding site of CCR2 confirmed its binding site. Finally, investigation of (cancer-associated) mutations in the sodium ion binding site showed a markedly decreased expression compared to wild type. Only two mutants, G123A3.35 and G127K3.39, were able to be bound by [3H]INCB3344 and [3H]CCR2-RA-[R]. Thus, mutagenesis showed that the sodium ion binding site residues, which are distinct from other class A GPCRs and related to chemokine receptor evolution, are crucial for receptor integrity. Moreover, the tested mutations appeared to have no effect on modulation observed by HMA or a minor effect on sodium chloride modulation on the tested radioligands. All in all, these results invite further exploration of the CCR2 sodium ion binding site in (cancer) biology, and potentially as a third druggable binding site.

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.

Personalized oncology has revolutionized cancer treatment by targeting specific genetic aberrations in tumors. However, the identification of suitable targets for anti-cancer therapies remains a challenge. In this study, we introduce a knowledge graph approach to prioritize cancer mutations with clinical, functional, and structural significance as potential therapeutic targets. Focusing on the human kinome, we integrate protein-protein interaction and patient-centric mutation networks to construct a comprehensive network enriched with transcriptomic, structural, and drug response data, together covering five layers of information. Moreover, we make the constructed knowledge graph publicly available, along with a plethora of scripts to facilitate further annotation and expansion of the network. Interactive visualization resources are also provided, ensuring accessibility for researchers regardless of computational expertise and enabling detailed analysis by cancer type and individual layers of information. This comprehensive resource has the potential to identify relevant mutations for targeted therapeutic interventions, thereby advancing personalized oncology and improving patient outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Gold complexes have emerged as promising anticancer metallodrugs due to their efficient thioredoxin reductase (TrxR) inhibition, which disturbs the redox balance of cancer cells.However, in this model, the role of the ligand(s) coordinated to gold is often overlooked.In this work, we present a series of tetrapyridyl Au(III) complexes that exhibit thiol-induced release of a Au(I) ion and a tetrapyridyl ligand.The formation of a free Au(I) center is responsible for the expected TrxR inhibition.Additionally, the released ligand, which was visible in cells due to its intense blue fluorescence, showed excellent binding properties to the hERG potassium channel.Moreover, these ligands ended up in the lysosomes, resulting in significant lysosome damage.Altogether, the Au(III) complexes presented in this work showed broad-spectrum anticancer properties, both in hypoxic 2D monolayers and 3D tumor spheroids.We suggest that the interaction of the released Au(I) center and the tetrapyridyl ligand with two different protein targets may combine into prodrugs that overcome hypoxia-induced drug deactivation.

The norepinephrine transporter (NET), encoded by the SLC6A2 gene, is one of three key monoamine neurotransmitter transporters. Inhibition of NET-mediated reuptake of norepinephrine by monoamine reuptake inhibitors has been the main therapeutic strategy to treat disorders such as depression, ADHD and Parkinson's disease. Nevertheless, lack of efficacy as well as risk of adverse effects are still common for these treatments underscoring the necessity to improve drug discovery efforts for this target. In this study, we developed new inhibitors based on 4-((2-(3,4-dichlorophenyl)cyclopentyl)amino)butan-1-ol (8), a potent NET inhibitor, which emerged from earlier virtual screening efforts using a predictive proteochemometric model. Hence, we optimized the N,2-substituted cycloalkylamine scaffold in three regions to design twenty new derivatives. To establish structure-activity relationships for these NET inhibitors, all novel compounds were tested utilizing an impedance-based 'transporter activity through receptor activation' assay. Moreover, all stereoisomers of the most potent compound (27) were synthesized and evaluated for their inhibitory potencies. Initial screening indicated that modifications in the cyclopentylamine moiety and phenyl substitutions decreased NET inhibition compared to 8, emphasizing the importance of the five-membered ring, secondary amine and dichloro-substitution pattern in NET binding. Substituting the original butylalcohol at the R-2 position with a rigid cyclohexanol yielded lead compound 27, with potency similar to reference inhibitor nisoxetine. Pharmacological characterization of all eight stereoisomers of 27 revealed varying inhibitory potencies, favoring a trans-orientation of the N,2-substituted cyclopentyl moiety. Molecular docking highlighted key interactions and the impact of a hydrophilic region in the binding pocket. This study presents a novel set of moderate to highly potent NET inhibitors, elucidating the influence of molecular orientation in the NET binding pocket and offering valuable insights into drug discovery efforts for monoamine transport-related treatments.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at . G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Due to the large drug-like chemical space available to search for feasible drug-like molecules, rational drug design often starts from specific scaffolds to which side chains/substituents are added or modified. With the rapid growth of the application of deep learning in drug discovery, a variety of effective approaches have been developed for de novo drug design. In previous work, we proposed a method named DrugEx, which can be applied in polypharmacology based on multi-objective deep reinforcement learning. However, the previous version is trained under fixed objectives similar to other known methods and does not allow users to input any prior information (i.e. a desired scaffold). In order to improve the general applicability, we updated DrugEx to design drug molecules based on scaffolds which consist of multiple fragments provided by users. In this work, the Transformer model was employed to generate molecular structures. The Transformer is a multi-head self-attention deep learning model containing an encoder to receive scaffolds as input and a decoder to generate molecules as output. In order to deal with the graph representation of molecules we proposed a novel positional encoding for each atom and bond based on an adjacency matrix to extend the architecture of the Transformer. Each molecule was generated by growing and connecting procedures for the fragments in the given scaffold that were unified into one model. Moreover, we trained this generator under a reinforcement learning framework to increase the number of desired ligands. As a proof of concept, our proposed method was applied to design ligands for the adenosine A2A receptor (A2AAR) and compared with SMILES-based methods. The results demonstrated the effectiveness of our method in that 100% of the generated molecules are valid and most of them had a high predicted affinity value towards A2AAR with given scaffolds.

The authors regret that the abstract stated "One of the mutant receptors, namely S101P 2.63 with a mutation near the orthosteric binding site, did not express on the cell surface".However, the correct statement should be "One of the mutant receptors, namely P258L 6.50 with a mutation near the allosteric binding site, did not express on the cell surface".