Selective chemical inhibition provides a powerful tool for dissecting complex physiological functions mediated by structurally-similar cellular enzymes. In particular, selective inhibition of NADPH oxidase (Nox) family members has the distinct advantage of enabling acute, reversible modulation of molecular function, while circumventing the developmental compensations that can arise in gene deletion studies. We have targeted this approach to elucidate the biological functions mediated by the Nox1 member of the NADPH oxidase family and have identified a novel nanomolar small-molecule Nox1 inhibitor. Importantly, we demonstrate that this chemical probe can be used to clarify the role of Nox1-dependent ROS generation in the pathogenesis of colon cancer.
The NADPH oxidase family, consisting of the homologous enzymes Nox1-4 and the more distantly related Nox5, Duox1 and Duox2, catalyzes the regulated formation of reactive oxygen species (ROS) (1
). Among all seven Nox isoforms, the Nox1-4 enzymes share the highest level of structural similarities (2
). Their basic catalytic subunit contains a C-terminal dehydrogenase domain featuring a binding site for NADPH and a bound flavin adenine nucleotide (FAD), as well as an N-terminal domain consisting of six transmembrane alpha helices that bind two heme groups. On activation, cytosolic NADPH transfers its electrons to the FAD, which in turn passes electrons sequentially to the two hemes and ultimately to molecular oxygen on the opposing side of the membrane, to form the superoxide anion (3
). Although all Nox1-4 isoforms catalyze the reduction of molecular oxygen and are expressed in a complex with p22phox
subunit, they differ in both tissue distributions and mechanisms by which their activity is regulated (4
). Nox2 is expressed by phagocytic leukocytes and its activity is triggered by inflammatory mediators which induce the assembly of four cytosolic regulatory proteins (p40phox
and Rac2-GTPase) with the Nox2 core enzyme to stimulate superoxide formation. Nox1 and Nox3 are highly expressed in the colon epithelium and in the inner ear respectively and their activity is also regulated by Rac1-GTPase and by related cytosolic adaptors, known as the activator subunit NoxA1 (homologous to p67phox
) and the organizer subunits NoxO1, Tks4 and Tks5 (homologues of p47phox
). Nox4 is widely distributed in the kidney, bone and vascular cells and its activity is independent of Rac-GTPase.
All Nox enzymes have been implicated in physiological and pathophysiological processes (5
). Particularly, Nox1-dependent ROS generation has been shown to play a pivotal role in cell signaling, cell growth, angiogenesis, motility and blood pressure regulation (6
). Interestingly, ROS generated via Nox1 have been reported to contribute to a growing number of diseases, including cancer, atherosclerosis, hypertension, neurological disorders and inflammation (9
). In keeping with a role of Nox1 in colon cancer, we have recently shown that in human colon cancer cells Nox1-derived ROS are necessary for the formation of extracellular matrix (ECM)-degrading, actin-rich cellular structures known as invadopodia (13
), whose presence directly correlate with the ability of cells to invade the surrounding tissues (14
Since it has been reported that ROS can also be produced by other cellular enzymes such as xanthine oxidase (XO), cytochrome P450, and mitochondrial oxidases (16
), dissecting of the contribution to these pathologies of Nox1-derived ROS in comparison to other ROS generators has been complicated by the lack of potent, selective and specific Nox1 inhibitors. Currently, only a few non-specific inhibitors (that also target other Nox isoforms) have been identified (i.e diphenylene iodonium [DPI] and apocynin) (19
). Several issues of selectivity, specificity, potency and toxicity limit the value of these compounds as Nox1 inhibitors both as research and clinical tools (20
). In the case of the most widely used Nox inhibitor DPI, because of its chemical mechanism of inhibition which involves accepting an electron from flavin, followed by covalently reacting with the enzymes or its prosthetic group, DPI rapidly and irreversibly blocks not only all Nox isoforms but also many other flavin-dependent enzymes such as XO (see also ).
Table 1 Schematic representation of the screening cascade and table summarizing IC50 values of 2-(trifluoromethyl)-phenothiazine (parental hit), ML171 (after-SAR hit), chlorpromazione (negative control) and DPI (positive control) are reported. (a) A schematic (more ...)
Here, by using high-throughput screening, we identify a sub-set of phenothiazines, specifically 2-acetylphenothiazine (here referred to as ML171) (and its related 2-(trifluoromethyl)-phenothiazine) as nanomolar, cell-active and specific Nox1 inhibitors that potently block Nox1-dependent ROS generation, with only marginal activity on other cellular ROS-producing enzymes and receptors including the other Nox isoforms. ML171 also blocks the ROS-dependent formation of ECM-degrading invadopodia in colon cancer cells. Such effects can be reversed by over-expression of Nox1 protein, which is possibly suggestive of a selective mechanism of inhibition of Nox1 by this compound. These results elucidate the relevance of Nox1-dependent ROS generation in mechanisms of cancer invasion, and define ML171 as a useful Nox1 chemical probe with potential therapeutic insights.