Daan van der Es

Direct-to-biology (D2B) has emerged as a powerful accelerant in early drug discovery, bypassing compound purification to compress the design-make-test-analyse (DMTA) cycle. However, the chemistry underpinning D2B libraries is limited to just a few sequential transformations using assay-compatible reagents and solvents. By contrast, solid-phase synthesis (SPS) empowers a far broader range of reactions and is highly amenable to multistep synthesis, yet its potential as a source for D2B libraries remains largely underexplored. In this study, we develop AutoSPS-D2B, a fully automated solid-phase synthesis workflow that converts multistep, resin-bound chemistry into crude screening libraries that can be assayed directly, expanding reaction scope well beyond the reach of solution-phase D2B. We applied AutoSPS-D2B to explore several distinct chemotypes as allosteric ligands for the immuno-oncology target CCR2. Good agreement was observed in the relative binding affinity trends between crude and pure screening compounds, enabling streamlined ranking of analogues and prioritisation of hits for further investigation. When applied to the advancement of a previously underexplored CCR2 hit, AutoSPS-D2B enabled an analogue space of more than 1,700 building block combinations to be rapidly sampled, leading to optimised compounds with up to six-fold binding affinity improvements in just two DMTA cycles. This work establishes automated SPS as a versatile and efficient engine for D2B-driven hit-to-lead discovery.

The intestinal epithelium is exposed to diverse combinations of microbiota-derived compounds; however, the mechanisms by which the host integrates these signals remain poorly defined. Studying two highly abundant microbial metabolites, we identified the purine metabolite hypoxanthine as an effector metabolite that directly drives signaling and the short-chain fatty acid butyrate as a regulatory metabolite that conditions host responsiveness. Specifically, hypoxanthine activates the adenosine A1 receptor–TRPC4 axis in enterochromaffin (EC) cells, triggering calcium influx and serotonin release resulting in accelerated gastrointestinal transit locally and increased platelet activation systemically. In contrast, butyrate epigenetically upregulates specific G protein–coupled receptors and ion channels to enhance response to hypoxanthine and the neurotransmitters norepinephrine and dopamine. These findings define a cooperative signaling framework and highlight the role of EC cells as a distinct epithelial signaling hub that senses and integrates microbial metabolite signals to drive physiological responses. Our findings provide a mechanistic foundation for therapeutic strategies that leverage combinatorial microbial signaling.

A novel series of N ,2-cyclopentylamine-based dopamine reuptake inhibitors was developed from earlier dual inhibitors. MAT selectivity was assessed in vitro and rationalized in silico . In vivo efficacy was shown in fruit fly locomotor assays.

Targeted protein degradation (TPD) is one of the most prominent and rapidly advancing modalities in drug discovery. However, only few degraders have been reported for the membrane-bound G protein-coupled receptors (GPCRs). Therefore, using CC chemokine receptor 2 (CCR2) as a GPCR model, we synthesized potential CCR2 molecular degraders. The putative proteolysis-targeting chimeras (PROTACs) employed an allosteric intracellular CCR2 ligand tethered to commonly used E3 ligase ligands. Among these compounds, LUF7996 (8) demonstrated engagement of both CCR2 and the E3 ligase cereblon and displayed sustained and concentration-dependent degradation of CCR2 over 24 h. Mechanistic studies revealed the reliance of LUF7996 on the lysosomal pathway to induce CCR2 degradation. Finally, LUF7996 (8) efficiently inhibited monocyte migration in a transwell assay. Collectively, the developed assessment workflow led to identification of the first CCR2 molecular degraders and has the potential to expand the repertoire of degraders targeting the pharmacologically rich GPCRs.

CC chemokine receptor (CCR) 2 and 5 are G protein-coupled receptors that play a crucial role in immunohomeostasis. Accordingly, overactivation of their signaling pathways is involved in various immunopathologies and cancer. Extensive research focusing on discovering CCR2 and CCR5 orthosteric antagonists, ultimately resulted in some clinical success, but the area of intracellular allosteric modulators is still underexplored and the move from orthosteric to allosteric modulation could be an interesting paradigm shift. To this end, we document the development of novel CCR2 and CCR5 intracellular allosteric antagonists through a virtual screen on a small combinatorial library derived from existing CCR2, CCR5, and CCR4 ligands. Using a molecular docking approach, the created library was screened in its entirety utilizing a refined AlphaFold model of CCR5 based on the crystal structure of its close homologue, CCR2. The screening resulted in the identification of several virtual hits, out of which one was developed further by in-house synthesis. In total, 18 analogues were prepared and experimentally evaluated for their binding affinity for CCR2 and functional inhibition on CCR5. This expeditious and simple workflow beginning from docking to compound evaluation identified 3 hits for CCR2 (K_i = 1.3-6 μM) and 1 hit (IC₅₀ = 10.8 μM) for CCR5. The obtained structure-activity relationships were also further rationalized using structural information available for both CCR5 and CCR2 providing valuable insights for future development of intracellular allosteric ligands.

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.

Adenosine receptors (ARs: A₁AR, A_2AAR, A_2BAR, and A₃AR) are crucial therapeutic targets; however, developing selective, efficacious drugs for them remains a significant challenge. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the human A₃AR in three distinct functional states: bound to the endogenous agonist adenosine, the clinically relevant agonist Piclidenoson, and the covalent antagonist LUF7602. These structures, complemented by mutagenesis and pharmacological studies, reveal an A₃AR activation mechanism that involves an extensive hydrogen bond network from the extracellular surface down to the orthosteric binding site. In addition, we identify a cryptic pocket that accommodates the N⁶-iodobenzyl group of Piclidenoson through a ligand-dependent conformational change of M174^5.35. Our comprehensive structural and functional characterisation of A₃AR advances our understanding of adenosine receptor pharmacology and establishes a foundation for developing more selective therapeutics for various disorders, including inflammatory diseases, cancer, and glaucoma.

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.

Optimization of previously identified N ,2-substituted cycloalkylamine scaffold for norepinephrine transport inhibition.

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.