Objectives Activation of neutrophils and release of neutrophil extracellular traps (NETs), proteases and reactive oxygen species (ROS) is pathogenic in immune-mediated inflammatory diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), driving inflammation and damaging host tissues. The aim of this research was to identify small molecule inhibitors of NET production using a highly-curated panel of narrow-spectrum small molecule kinase inhibitors termed the Kinase Chemogenomic Set (KCGS).

Methods Neutrophils were isolated from healthy controls (HC) and people with RA or SLE. Over 220 small molecules were screened for their ability to inhibit NET production and decrease ROS production without impairing neutrophil apoptosis or killing of S. aureus bacteria.

Results Fifty compounds, nominally targeting 29 individual protein kinase targets, were found to inhibit NOX2-dependent (PMA-stimulated) and NOX2-independent (A23187-stimulated) NET production (p<0.05, n=5 HC, RA and SLE). Of these, seven compounds did not significantly impair ROS production or apoptosis. The deconvoluted targets of these small molecules inhibit kinases that operate in three cellular pathways: autophagy, cell cycle checkpoint and epidermal growth factor (EGF) tyrosine kinase signalling. Of these, only inhibitors of ULK1, JNK and ROCK1/2, broadly implicated in the regulation of autophagy, did not significantly impair bacterial killing (n=5 HC, p>0.05). Autophagy inhibitors were also able to inhibit immune-complex driven NET production (p<0.05, n=5 HC, RA and SLE).

Conclusion We propose that autophagy signalling pathways represent novel and exciting targets for the development of small molecule therapeutics to block unwanted neutrophil activation and NET release in immune-mediated inflammatory disease.

The authors have declared no competing interest.

This research was funded by a Connect Immune Research and The Lorna & Yuti Chernajovsky Biomedical Research Foundation Grant (Grant No. 22925).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was approved by the University of Liverpool Central University Research Ethics Committee C (Ref: 10956), and NRES Committee North West (Greater Manchester West, UK) (Ref: 11/NW/0206). All participants gave written, informed consent in accordance with the declaration of Helsinki.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data are available from the corresponding author upon reasonable request.