Pharmacological characterisation of small molecule C5aR1 inhibitors in human cells reveals biased activities for signalling and function.

The complement fragment C5a is a core effector of complement activation. C5a, acting through its major receptor C5aR1, exerts powerful pro-inflammatory and immunomodulatory functions. Dysregulation of the C5a-C5aR1 axis has been implicated in numerous immune disorders, and the therapeutic inhibition of this axis is therefore imperative for the treatment of these diseases. A myriad of small-molecule C5aR1 inhibitors have been developed and independently characterised over the past two decades, however the pharmacological properties of these compounds has been difficult to directly compare due to the wide discrepancies in the model, read-out, ligand dose and instrumentation implemented across individual studies. Here, we performed a systematic characterisation of the most commonly reported and clinically advanced small-molecule C5aR1 inhibitors (peptidic: PMX53, PMX205 and JPE1375; non-peptide: W545011, NDT9513727, DF2593A and CCX168). Through signalling assays measuring C5aR1-mediated cAMP and ERK1/2 signalling, and ß-arrestin 2 recruitment, this study highlighted the signalling-pathway dependence of the rank order of potencies of the C5aR1 inhibitors. Functional experiments performed in primary human macrophages demonstrated the high insurmountable antagonistic potencies for the peptidic inhibitors as compared to the non-peptide compounds. Finally, wash-out studies provided novel insights into the duration of inhibition of the C5aR1 inhibitors, and confirmed the long-lasting antagonistic properties of PMX53 and CCX168. Overall, this study revealed the potent and prolonged antagonistic activities of selected peptidic C5aR1 inhibitors and the unique pharmacological profile of CCX168, which thus represent ideal candidates to fulfil diverse C5aR1 research and clinical therapeutic needs.

Synthesis and evaluation of NLRP3-inhibitory sulfonylurea [11C]MCC950 in healthy animals.

The diaryl sulfonylurea MCC950/CRID3 is a potent NLRP3 inhibitor (IC50 = 8 nM) and, in animal models, MCC950 protects against numerous NLRP3-related neurodegenerative disorders. To evaluate the brain uptake and investigate target engagement of MCC950, we synthesised [11C-urea]MCC950 via carrier added [11C]CO2 fixation chemistry (activity yield = 237 MBq; radiochemical purity >99%; molar activity = 7 GBq/µmol; radiochemical yield (decay-corrected from [11C]CO2) = 1.1%; synthesis time from end-of-bombardment = 31 min; radiochemically stable for >1 h). Despite preclinical efficacy in neurodegeneration studies, preclinical positron emission tomography (PET) imaging studies in mouse, rat and rhesus monkey revealed poor brain uptake of low molar activity [11C]MCC950 and rapid washout. In silico prediction tools suggest efflux transporter liabilities for MCC950 at microdoses, and this information should be taken into account when developing next generation NLRP3 inhibitors and/or PET radiotracers.

MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

Characterisation of small molecule ligands 4CMTB and 2CTAP as modulators of human FFA2 receptor signalling.

Short chain fatty acids (SCFAs) are protective against inflammatory diseases. Free fatty acid receptor 2 (FFA2), is a target of SCFAs however, their selectivity for FFA2 over other FFA receptors is limited. This study aimed to functionally characterise 2-(4-chlorophenyl)-3-methyl-N-(thiazole-2-yl)butanamide (4CMTB) and 4-((4-(2-chlorophenyl)thiazole-2-yl)amino)-4oxo-3-phenylbutanoic acid (2CTAP), and their enantiomers, in modulating FFA2 activity. The racemic mixture (R/S) and its constituents (R-) and (S-) 4CMTB or 2CTAP were used to stimulate human (h)FFA2 in the absence or presence of acetate. Calcium ions (Ca2+), phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) and cyclic adenosine monophosphate (cAMP) were measured. R/S-4CMTB is a functionally selective ago-allosteric ligand that enhances Ca2+ response to acetate. Both R/S-4CMTB and S-4CMTB are more potent activators of pERK1/2 and inhibitors of forskolin-induced cAMP than acetate. S-4CMTB increased neutrophil infiltration in intestinal ischemia reperfusion injury (IRI). 2CTAP inhibited constitutive Ca2+ levels, antagonised acetate-induced pERK1/2 and prevented damage following IRI. This study characterises enantiomers of functionally selective ligands for FFA2 in cells stably expressing hFFA2. It highlights the novel roles of selective FFA2 enantiomers 4CMTB and 2CTAP on Ca2+, pERK1/2 and cAMP and their roles as allosteric modulators which, may assist in efforts to design novel therapeutic agents for FFA2-driven inflammatory diseases.

Synthesis of deuterium-labelled analogues of NLRP3 inflammasome inhibitor MCC950.

This study describes the syntheses of di, tetra and hexa deuterated analogues of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor MCC950. In di and tetra deuterated analogues, deuteriums were incorporated into the 1,2,3,5,6,7-hexahydro-s-indacene moiety, whereas in the hexa deuterated MCC950 deuteriums were incorporated into the 2-(furan-3-yl)propan-2-ol moiety. The di deuterated MCC950 analogue was synthesised from 4-amino-3,5,6,7-tetrahydro-s-indacen-1(2H)-one 5. Tetra deuterated analogues were synthesised in 10 chemical steps starting with 5-bromo-2,3-dihydro-1H-inden-1-one 9, whereas the hexa deuterated analogue was synthesised in four chemical steps starting with ethyl-3-furoate 24. All of the compounds exhibited similar activity to MCC950 (IC50 = 8 nM). These deuterated analogues are useful as internal standards in LC-MS analyses of biological samples from in vivo studies.