(383) ELD607 Inhibits Excessive Proinflammatory Ca²⁺ Signaling in CF Neutrophils

Background: The CF lung is subject to chronic neutrophilic inflammation that is disproportionate to bacterial load and is poorly resolving. Exuberant inflammatory responses contribute significantly to the pathogenesis of CF lung disease by promoting mucus hypersecretion and tissue remodeling, as well as causing collateral damage to host tissues. Many anti-inflammatory drugs are unsuitable for the management of CF airway inflammation due to their immunosuppressive effects. Thus, there is an urgent and unmet need to develop anti-inflammatory therapeutics. Store-operated Ca²⁺ entry (SOCE) is mediated by the plasma membrane Ca²⁺ channel, Orai1. SOCE plays a central role in neutrophil activation and regulates degranulation, reactive oxygen species production and the release of neutrophil extracellular traps. Ca²⁺ signaling has been reported to be dysregulated in CF neutrophils, which may contribute to the hyper-inflammatory phenotype of the CF airway. We have developed a novel, fully-optimized, peptide-based Orai1 inhibitor called ELD607. We hypothesize that ELD607 can be used to reduce neutrophilic inflammation in CF lungs without immunosuppressive effects.

Methods: To investigate the effects of ELD607 on neutrophil Ca²⁺ signaling, neutrophils were isolated from CF and non-CF subject blood by immunomagnetic negative selection and treated for 3 hours with 30 mM ELD607, scrambled peptide control or vehicle. To measure Ca²⁺ signaling, neutrophils were loaded with the Ca²⁺ sensitive fluorescent dye, fluo-4 and fluorescence was measured in response to stimulation with thapsigargin (1 mM) using a Tecan plate reader.

Results: Neutrophils were collected from 4 non-CF subjects and 12 CF patients with a range of CFTR mutations including F508del, L467P, 4209TGTT >A, G551D and N1303K. 11/12 patients were undergoing Trikafta (elexacaftor/tezacaftor/ivacaftor) treatment at the time of collection. In non-CF neutrophils, thapsigargin induced a 39±5 % increase in cytosolic Ca²⁺ concentration over 10 minutes. CF neutrophils displayed a 53±6 % increase in cytosolic Ca²⁺ concentration. The rate of increase in cytosolic Ca²⁺ concentration and area under the curve (AUC) over 10 min were both significantly greater in CF neutrophils than non-CF controls (p< 0.01 and p=0.04 respectively), suggesting dysregulated Ca²⁺ signaling. ELD607, but not scrambled peptide exposure, reduced AUC by 30% (p< 0.01) in CF neutrophils. Non-CF neutrophils displayed a similar (29%) reduction in AUC that was not statistically significant, likely due to the small sample size.

Conclusions: Collectively, these data indicate that CF neutrophils display exaggerated Ca²⁺ signaling in response to thapsigargin that can be inhibited by ELD607. We predict ELD607 treatment may benefit CF patients by rebalancing Ca²⁺ signaling in CF neutrophils and reducing inflammation.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (grant number WRENNA21F0)

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