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 β-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.