Selective activation of PFKL suppresses the phagocytic oxidative burst

Neri Amara; Madison P Cooper; Maria A Voronkova; Bradley A Webb; Eric M Lynch; Justin M Kollman; Taylur Ma; Kebing Yu; Zijuan Lai; Dewakar Sangaraju; Nobuhiko Kayagaki; Kim Newton; Matthew Bogyo; Steven T Staben; Vishva M Dixit

doi:10.1016/j.cell.2021.07.004

Selective activation of PFKL suppresses the phagocytic oxidative burst

Cell. 2021 Aug 19;184(17):4480-4494.e15. doi: 10.1016/j.cell.2021.07.004. Epub 2021 Jul 27.

Authors

Affiliations

¹ Physiological Chemistry Department, Genentech, South San Francisco, CA 94080, USA.
² Biochemistry Department, West Virginia University, Morgantown, WV 26506, USA.
³ Biochemistry Department, University of Washington, Seattle, WA 98195, USA.
⁴ Microchemistry, Proteomics, and Lipidomics Department, Genentech, South San Francisco, CA 94080, USA.
⁵ Drug Metabolism and Pharmacokinetics Department, Genentech, South San Francisco, CA 94080, USA.
⁶ Pathology Department, Stanford University, Stanford, CA 94305, USA.
⁷ Discovery Chemistry Department, Genentech, South San Francisco, CA 94080, USA.
⁸ Physiological Chemistry Department, Genentech, South San Francisco, CA 94080, USA. Electronic address: dixit@gene.com.

Abstract

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.

Keywords: LDC7559; NA-11; NADPH; NETosis; NOX2; PFKL; ROS; neutrophils.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Diphosphate / metabolism
Adenosine Monophosphate / metabolism
Allosteric Regulation / drug effects
Enzyme Activation / drug effects
Epithelial Cells / drug effects
Epithelial Cells / metabolism
Glycolysis / drug effects
Humans
Intracellular Signaling Peptides and Proteins / metabolism
Kinetics
Microbial Viability / drug effects
Models, Molecular
NADPH Oxidases / metabolism
Neutrophils / drug effects
Neutrophils / metabolism
Phagocytosis* / drug effects
Phosphate-Binding Proteins / metabolism
Phosphofructokinase-1, Liver Type / antagonists & inhibitors
Phosphofructokinase-1, Liver Type / metabolism*
Phosphofructokinase-1, Liver Type / ultrastructure
Protein Kinase Inhibitors / chemistry
Protein Kinase Inhibitors / pharmacology
Recombinant Proteins / isolation & purification
Respiratory Burst* / drug effects
Tetradecanoylphorbol Acetate / pharmacology

Substances

GSDMD protein, human
Intracellular Signaling Peptides and Proteins
Phosphate-Binding Proteins
Protein Kinase Inhibitors
Recombinant Proteins
Adenosine Monophosphate
Adenosine Diphosphate
NADPH Oxidases
Phosphofructokinase-1, Liver Type
Tetradecanoylphorbol Acetate

Abstract

Publication types

MeSH terms

Substances

Grants and funding