Targeting ROS production through inhibition of NADPH oxidases.

NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.

A lonely electron blocks incoming pairs.

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.

Engineering stability in NADPH oxidases: A common strategy for enzyme production.

NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19 °C increase in the apparent melting temperature (app Tm) and a much tighter binding of the FAD cofactor, which allowed the crystallization and structure determination of the domain holo-form. Here, we analyze the transferability of this mutation onto prokaryotic and eukaryotic full-length NOX enzymes. We found that the mutation exerts a significative stabilizing effect on the full-length NOX5 from both Cylindrospermum stagnale (app Tm increase of 8 °C) and Homo sapiens (app ΔTm of 2 °C). Enhanced thermal stability resulted in more homogeneous preparations of the bacterial NOX5 with less aggregation problems. Moreover, we also found that the mutation increases the overall expression of recombinant human NOX4 and NOX5 in mammalian cells. Such a 2-5-fold increase is mainly due to the lowered cell toxicity, which leads to higher biomasses. Because of the high sequence identity of the catalytic core within this family of enzymes, this strategy can be a general tool to boost the production of all NOXs.

Impact of ancestral sequence reconstruction on mechanistic and structural enzymology.

Ancestral sequence reconstruction (ASR) provides insight into the changes within a protein sequence across evolution. More specifically, it can illustrate how specific amino acid changes give rise to different phenotypes within a protein family. Over the last few decades it has established itself as a powerful technique for revealing molecular common denominators that govern enzyme function. Here, we describe the strength of ASR in unveiling catalytic mechanisms and emerging phenotypes for a range of different proteins, also highlighting biotechnological applications the methodology can provide.

Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency.

Reactive oxygen species are unstable molecules generated by the partial reduction of dioxygen. NADPH oxidases are a ubiquitous family of enzymes devoted to ROS production. They fuel an array of physiological roles in different species and are chemically demanding enzymes requiring FAD, NADPH and heme prosthetic groups in addition to either calcium or a various number of cytosolic mediators for activity. These activating partners are exclusive components that partition and distinguish the NOX members from one another. To gain insight into the evolution of these activating mechanisms, and in general in their evolutionary history, we conducted an in-depth phylogenetic analysis of the NADPH oxidase family in eukaryotes. We show that all characterized NOXs share a common ancestor, which comprised a fully formed catalytic unit. Regarding the activation mode, we identified calcium-dependency as the earliest form of NOX regulation. The protein-protein mode of regulation would have evolved more recently by gene-duplication with the concomitant loss of the EF-hands motif region. These more recent events generated the diversely activated NOX systems as observed in extant animals and fungi.

A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action.

NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs' flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs.