Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation

Abstract

Edited by Dennis VoelkerNeutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the upregulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

Therapeutic targeting of neutrophil exocytosis

“…In particular, JFC1 interacts with the small GTPase Rab8a in addition to Rab27a . Tyr‐122 in Rab27a, a residue involved in the interaction of this GTPase with its effector and recognized by neutrophil exocytosis inhibitors (Nexinhibs), is not conserved in Rab8a. Thus, small‐molecule inhibitors of the Rab27a‐Slp1/JFC1 interaction are not expected to affect Rab8a‐Slp1/JFC1 interaction.…”

“…We next use a terbium‐conjugated anti‐Myc antibody, which specifically binds to the tag moiety in Myc‐JFC1. FRET signal is triggered by the close proximity between the donor (terbium) and acceptor (EGFP) in response to the specific binding of Myc‐JFC1 to EGFP‐Rab27a . To increase the likelihood of identifying specific inhibitors with physiological significance, the reactions are performed with lysates obtained by nondenaturing methods, and the integrity of the organelles is maintained throughout the analysis.…”

“…Using this approach and high‐throughput screening analysis, counterscreens and orthogonal validation, we identified Nexinhibs, small‐molecule inhibitors of the JFC1‐Rab27a interaction which constitute the first neutrophil‐specific exocytosis inhibitors (Fig. ).…”

“…). A binding pocket for Nexinhib20 with the highest druggability score was identified within a Rab27a domain associated with JFC1 family effector binding . This pocket contains a Rab27a domain associated with the binding to the synaptotagmin‐like homology domains of Slp/JFC1 family members.…”

“…Structural modeling identified Tyr‐122 in Rab27a as a NEI20‐interacting residue, an important observation because this tyrosine mediates Rab‐effector specificity . Initial structure‐activity relationship analysis of Nexinhibs identified potentially important groups in 3 compounds, Nexinhibs 4, 20, and 20 analog . Nexinhibs inhibit azurophilic granule exocytosis without affecting phagocytosis, NET production, or cell viability .…”