Neutrophils are a necessary player in host-defense against pathogenic insult, yet, can play a harmful role promoting inflammation and tissue damage in a number of acute and chronic diseases.  Understanding mediators of neutrophil oxidative burst, phagocytic activity, degranulation and NETosis may lead to opportunities for disease-modifying therapies.  Stemming from a phenotypic screen, as small-molecule inhibitor of NETosis (LDC7559) was recently discovered and hypothesized to act through inhibition of GSDMD (Zchlinsky <i>Sci. Immun. </i><b>2018</b>).   We improved activity of this tool compound and then used proteome-wide approaches (PAL, TSA) to identify PFKL as the primary target of this series of molecules.  Biochemical and biophysical mechanistic characterization concluded these compounds act as allosteric activators of PFKL.  Metabolomic analysis indicates activation of PFKL promotes a metabolic shunt toward glycolysis, thus depleting the neutrophils of necessary NADPH required for phagocytic oxidative burst.  The first cryo-EM structure of optimized tool compound, NA-11, was enabled and well-explains SAR observed in the optimization process.  These molecules represent the first non-natural, and most potent, activators of PFKL activity.  PFKL represents an interesting target in diseases exacerbated by neutrophil over-activation.

From phenotype to molecular mechanism of action – small molecule activators of PFKL inhibit neutrophil oxidative burst and NETosis

ACS Spring 2022

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