Structure-Activity Relationship Studies and Optimization of 4-Hydroxypyridones as GPR84 Agonists.

GPR84 is a putative medium-chain fatty acid receptor that is implicated in regulation of inflammation and fibrogenesis. Studies have indicated that GPR84 agonists may have therapeutic potential in diseases such as Alzheimer's disease, atherosclerosis, and cancer, but there is a lack of quality tool compounds to explore this potential. The fatty acid analogue LY237 (4a) is the most potent GPR84 agonist disclosed to date but has unfavorable physicochemical properties. We here present a SAR study of 4a. Several highly potent agonists were identified with EC50 down to 28 pM, and with SAR generally in excellent agreement with structure-based modeling. Proper incorporation of rings and polar groups resulted in the identification of TUG-2099 (4s) and TUG-2208 (42a), both highly potent GPR84 agonists with lowered lipophilicity and good to excellent solubility, in vitro permeability, and microsomal stability, which will be valuable tools for exploring the pharmacology and therapeutic prospects of GPR84.

Discovery of Potent Tetrazole Free Fatty Acid Receptor 2 Antagonists.

The free fatty acid receptor 2 (FFA2), also known as GPR43, mediates effects of short-chain fatty acids and has attracted interest as a potential target for treatment of various metabolic and inflammatory diseases. Herein, we report the results from bioisosteric replacement of the carboxylic acid group of the established FFA2 antagonist CATPB and SAR investigations around these compounds, leading to the discovery of the first high-potency FFA2 antagonists, with the preferred compound TUG-2304 (16l) featuring IC50 values of 3-4 nM in both cAMP and GTPγS assays, favorable physicochemical and pharmacokinetic properties, and the ability to completely inhibit propionate-induced neutrophil migration and respiratory burst.

Identification of galectin-1 and other cellular targets of alpha,beta-unsaturated carbonyl compounds, including dimethylfumarate, by use of click-chemistry probes.

α,ß-Unsaturated carbonyls are a common motif in environmental toxins (e.g. acrolein) as well as therapeutic drugs, including dimethylfumarate (DMFU) and monomethylfumarate (MMFU), which are used to treat multiple sclerosis and psoriasis. These compounds form adducts with protein Cys residues as well as other nucleophiles. The specific targets ('adductome') that give rise to their therapeutic or toxic activities are poorly understood. This is due, at least in part, to the absence of antigens or chromophores/fluorophores in these compounds. We have recently reported click-chemistry probes of DMFU and MMFU (Redox Biol., 2022, 52, 102299) that allow adducted proteins to be visualized and enriched for further characterization. In the current study, we hypothesized that adducted proteins could be 'clicked' to agarose beads and thereby isolated for LC-MS analysis of DMFU/MMFU targets in primary human coronary artery smooth muscle cells. We show that the probes react with thiols with similar rate constants to the parent drugs, and give rise to comparable patterns of gene induction, confirming similar biological actions. LC-MS proteomic analysis identified ∼2970 cellular targets of DMFU, ∼1440 for MMFU, and ∼140 for the control (succinate-probe) treated samples. The most extensively modified proteins were galectin-1, annexin-A2, voltage dependent anion channel-2 and vimentin. Other previously postulated DMFU targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cofilin, p65 (RELA) and Keap1 were also identified as adducted species, though at lower levels with the exception of GAPDH. These data demonstrate the utility of the click-chemistry approach to the identification of cellular protein targets of both exogenous and endogenous compounds.

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls.

Humans are commonly exposed to α,ß-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the 'adductome') are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,ß-unsaturated carbonyls. Here we report a 'proof of concept' study in which we synthesize novel α,ß-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,ß-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows 'click-chemistry' to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,ß-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites.

The Two Formyl Peptide Receptors Differently Regulate GPR84-Mediated Neutrophil NADPH Oxidase Activity.

Neutrophils express the two formyl peptide receptors (FPR1 and FPR2) and the medium-chain fatty acid receptor GPR84. The FPRs are known to define a hierarchy among neutrophil G protein-coupled receptors (GPCRs), that is, the activated FPRs can either suppress or amplify GPCR responses. In this study, we investigated the position of GPR84 in the FPR-defined hierarchy regarding the activation of neutrophil nicotine adenine dinucleotide phosphate (NADPH) oxidase, an enzyme system designed to generate reactive oxygen species (ROS), which are important regulators in cell signaling and immune regulation. When resting neutrophils were activated by GPR84 agonists, a modest ROS release was induced. However, vast amounts of ROS were induced by these GPR84 agonists in FPR2-desensitized neutrophils, and the response was inhibited not only by a GPR84-specific antagonist but also by an FPR2-specific antagonist. This suggests that the amplified GPR84 agonist response is achieved through a reactivation of desensitized FPR2s. In addition, the GPR84-mediated FPR2 reactivation was independent of ß-arrestin recruitment and sensitive to a protein phosphatase inhibitor. In contrast to FPR2-desensitized cells, FPR1 desensitization primarily resulted in a suppressed GPR84 agonist-induced ROS response, indicating a receptor hierarchical desensitization of GPR84 by FPR1-generated signals. In summary, our data show that the two FPRs in human neutrophils control the NADPH oxidase activity with concomitant ROS production by communicating with GPR84 through different mechanisms. While FPR1 desensitizes GPR84 and by that suppresses the release of ROS induced by GPR84 agonists, amplified ROS release is achieved by GPR84 agonists through reactivation of the desensitized FPR2.