Ontwerp, synthese en biologische evaluatie van nieuwe intracellulaire modulatoren die CCR5-signaaltransductie afzwakken.

The C-C chemokine receptor type 5 (CCR5) is a G protein-coupled receptor (GPCR) involved in various diseases, including HIV, cancer, immune diseases and acute graft-versus-host disease. These diseases present significant challenges in medical research and therapy. Developing agents that can modulate CCR5's activity in these conditions represents a promising area of research, potentially offering new therapeutic avenues. The central aim is to design and synthesize allosteric intracellular antagonists that can modulate CCR5 signaling and influence downstream signaling events resulting from CCR5 activation. We hypothesize that a well-designed small molecule targeting the druggable intracellular allosteric binding site (IABS) can selectively attenuate CCR5 signaling, thereby reducing the pathological effects associated with its overactivation. The goal is to develop robust CCR5 antagonists that can significantly advance research in chemokine receptor biology and potentially lead to novel therapeutic approaches. In preliminary screening work, a promising hit structure was identified, serving as a starting point for further optimization. The hit candidate IV-1, a disubstituted [1,2,5]oxadiazolo- [3,4-b]pyrazine, emerged from a pharmacophore-based in silico screen of commercially available libraries. IV-1 was the most potent of the identified hits, with an IC50 of 4.40 µM for the inhibition of CCR5-induced Ca2+ signaling. Additionally, it exhibited a high structural similarity to known chemokine receptor antagonists and featured three distinct regions that could be explored, providing ample opportunities for structural investigation. The optimization process focused on enhancing the potency, selectivity, and solubility of these compounds. Compound potency was assessed using a CCR5-mediated Ca2+ mobilization assay to ensure that the antagonists identified effectively inhibit CCR5 activity at low concentrations. High potency is crucial for creating reliable antagonists that can accurately modulate CCR5 activity in experimental settings. Target selectivity was determined by comparing the activity of the antagonists on CCR5 versus CCR2, the closest evolutionary receptor to CCR5. Ensuring that the antagonists are selective for CCR5 reduces off-target effects and provides precise tools for studying CCR5-related pathways and functions. Kinetic aqueous solubility was evaluated using mass spectrometry methods to ensure that the antagonists are sufficiently soluble in biological environments. Adequate solubility is necessary for the proper application of these compounds in various experimental conditions, as poor solubility can limit the effectiveness of an antagonist, regardless of its potency and selectivity. A total of 90 final compounds were synthesized and biologically evaluated. Two compounds of high interest emerged: IV-3ad and V-2a. Compound IV-3ad demonstrated high potency (IC50 of 1.09 µM) and selectivity (28-fold preference for CCR5 over CCR2) but had poor solubility. In contrast, compound V-2a showed high solubility (> 250 µM) and retained activity (IC50 of 1.80 µM) but had lower selectivity (9-fold preference for CCR5 over CCR2). While both compounds require further optimization to fully meet all criteria, they represent promising starting points for the development of selective, soluble, and potent CCR5 antagonists. The hypothesized intracellular binding mode of the disubstituted [1,2,5]oxadiazolo- [3,4-b]pyrazine series was supported by molecular dynamics simulations indicating that these compounds were highly likely to occupy the IABS. Following these in silico studies, the occupation of the IABS, which partially overlaps with the G protein binding site, was confirmed through an assay demonstrating direct competition between a Gαq11-mimetic and compound IV-3f. The results showed that compound IV-3f could compete with the Gαq11-mimetic for binding pocket occupation, with a dissociation constant (Kd) of 4.55 µM. A selection of compounds was subjected to more rigorous biological evaluation to assess their effects on the proliferation and wound healing behavior of two selected cancer cell lines: the engineered CCR5-overexpressing glioblastoma-derived cell line U87.CD4.CCR5 and the metastatic breast cancer cell line MDA-MB-231. Utilizing automated live-cell imaging and image analysis, the impact of these compounds on cell motility and proliferation was systematically assessed, providing a comprehensive understanding of how these compounds influence cellular behavior. The ultimate goal was to identify promising candidates that can modulate cancer cell proliferation and migration, providing insights into their potential therapeutic applications. In conclusion, this research presents the development of a novel class of potent and selective intracellular CCR5 antagonists, laying the foundation for further therapeutic development and contributing to the broader field of chemokine receptor research. The binding mode of these compounds was validated through molecular dynamics simulations and competitive binding assays, and their biological activity was confirmed in proliferation and migration studies in selected cancer cell lines. Future work will focus on further structural investigations to optimize the potency, solubility, and selectivity of the lead compounds. Comprehensive selectivity studies should be conducted across a full chemokine receptor panel to identify potential off-target activities. Further biological validation and assessment of the pharmacokinetic properties of these compounds are also crucial. Finally, the development of molecular tools from these novel CCR5 will advance the understanding of CCR5 biology and facilitate new therapeutic strategies.