Rational design of small molecule inhibitors targeting the Rac GTPase-p67(phox) signaling axis in inflammation.
ABSTRACT The NADPH oxidase enzyme complex, NOX2, is responsible for reactive oxygen species production in neutrophils and has been recognized as a key mediator of inflammation. Here, we have performed rational design and in silico screen to identify a small molecule inhibitor, Phox-I1, targeting the interactive site of p67(phox) with Rac GTPase, which is a necessary step of the signaling leading to NOX2 activation. Phox-I1 binds to p67(phox) with a submicromolar affinity and abrogates Rac1 binding and is effective in inhibiting NOX2-mediated superoxide production dose-dependently in human and murine neutrophils without detectable toxicity. Medicinal chemistry characterizations have yielded promising analogs and initial information of the structure-activity relationship of Phox-I1. Our studies suggest the potential utility of Phox-I class inhibitors in NOX2 oxidase inhibition and present an application of rational targeting of a small GTPase-effector interface.
Article: Expression and translocation of fluorescent-tagged p21-activated kinase-binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis.[show abstract] [hide abstract]
ABSTRACT: Neutrophils are key cells of the innate immune system; they are terminally differentiated and therefore difficult to genetically manipulate and study in vitro. In the present study, we describe a protocol to transiently express two fluorescent markers, the PH domain of protein kinase B fused to red fluorescent protein and the p21-activated kinase-binding domain fused to a yellow fluorescent protein, in primary neutrophils. Using this approach, we are able to achieve a transfection efficiency of approximately 30%. The expression of the transfected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neutrophil activation pathways at the leading edge of migrating cells. We describe here a transfection protocol for primary neutrophils, which preserves fMLP-mediated cell polarization and cytoskeleton reorganization with simultaneous accumulation of PI-3K products and active Rac at the leading edge. The visualization and analysis of transfected fluorescent markers in primary neutrophils are a powerful technique to monitor chemotaxis signaling pathways in real time.Journal of Leukocyte Biology 10/2007; 82(3):559-66. · 4.99 Impact Factor
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ABSTRACT: Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.Molecular Biology of the Cell 05/1999; 10(4):1163-78. · 4.94 Impact Factor