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We developed and characterized Nexinhib20, a small-molecule inhibitor that targets azurophilic granule exocytosis in neutrophils by blocking the interaction between the small GTPase Rab27a and its effector JFC1. Its therapeutic potential has been demonstrated in several preclinical models of inflammatory disease. Here, using neutrophils from Jfc1-KO mice, we show that JFC1 regulates the mobilization of a small subpopulation of CD11b+ granules. Nexinhib20 inhibits the mobilization of \u03b22-integrins from a subset of CD11b+ granules to the plasma membrane in human and mouse neutrophils. The putative impact of Nexinhib20 on integrin activation is caused by decreased avidity, secondary to its effect on \u03b22-integrin mobilization. CD11b mobilization and integrin activation were unaffected by pharmacological inhibition or activation of Rac1. Using quantitative 3D enhanced resolution microscopy, we show that neutrophil activation induces the recruitment of JFC1 to CD11b+ granules. Nexinhib20 decreased the localization of JFC1 at CD11b+ granules without affecting the association of Rac1 with CD11b. Nexinhib20 inhibits JFC1 recruitment but not endogenous Rac1 activation in living cells. Using orthogonal analyses of Rac1 activity consisting of a sensitive, time-resolved fluorescence energy transfer, Rac1-PAK1-binding assay, and endogenous Rac1-GTP examination, we show that Nexinhib20 does not interfere with Rac1 activation. Instead, we confirm its molecular mode of action as the inhibition of the Rab27a-JFC1 binding. Thus, Nexinhib20 limits \u03b22-integrin mobilization to the cell surface, decreasing avidity and affecting active integrin availability in a JFC1-dependent but Rac1-independent manner.<\/jats:p>","DOI":"10.1093\/jleuko\/qiaf012","type":"journal-article","created":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T19:27:55Z","timestamp":1738265275000},"source":"Crossref","is-referenced-by-count":5,"title":["Nexinhib20 inhibits JFC1-mediated mobilization of a subset of CD11b\/CD18+ vesicles decreasing integrin avidity, but does not inhibit Rac1"],"prefix":"10.1093","volume":"117","author":[{"given":"Kasra","family":"Askari","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, CA 92037, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer L","family":"Johnson","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular 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