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In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells. [Display omitted] • Small molecules LDC7559 and NA-11 inhibit the phagocytic oxidative burst • NA-11 dampens flux through the pentose phosphate pathway to limit cellular NADPH • Cryo-EM confirms agonism of PFKL through binding at the AMP/ADP allosteric pocket • PFKL is a regulatory node for NOX2-dependent NETosis and tissue damage The small molecule LDC7559 and its more potent analog, NA-11, suppress excessive NOX2-dependent oxidative burst and NETosis, as well as subsequent tissue damage and inflammation, without compromising basal ROS production. They selectively activate the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) to suppress glycolytic flux through the pentose phosphate pathway that leads to NOX2-dependent outputs. [ABSTRACT FROM AUTHOR]