Lipid aldehyde hydrophobicity affects apo-SOD1 modification and aggregation.

Unsaturated lipids are oxidized by reactive oxygen species and enzymes, leading to the increased formation of lipid hydroperoxides and several electrophilic products. Lipid-derived electrophiles can modify macromolecules, such as proteins, resulting in the loss of function and/or aggregation. The accumulation of Cu,Zn-superoxide dismutase (SOD1) aggregates has been associated with familial cases of amyotrophic lateral sclerosis (ALS). The protein aggregation mechanisms in motor neurons remain unclear, although recent studies have shown that lipids and oxidized lipid derivatives may play roles in this process. Here, we aimed to compare the effects of different lipid aldehydes on the induction of SOD1 modifications and aggregation, in vitro. Human recombinant apo-SOD1 was incubated with 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE), 2-hexen-1-al (HEX), 2,4-nonadienal (NON), 2,4-decadienal (DEC), or secosterol aldehydes (SECO-A or SECO-B). High-molecular-weight apo-SOD1 aggregates dramatically increased in the presence of highly hydrophobic aldehydes (LogPcalc > 3). Notably, several Lys residues were modified by exposure to all aldehydes. The observed modifications were primarily observed on Lys residues located near the dimer interface (K3 and K9) and at the electrostatic loop (K122, K128, and K136). Moreover, HHE and HNE induced extensive apo-SOD1 modifications, by forming Schiff bases or Michael adducts with Lys, His, and Cys residues. However, these aldehydes were unable to induce large protein aggregates. Overall, our data shed light on the importance of lipid aldehyde hydrophobicity on the induction of apo-SOD1 aggregation and identified preferential sites of lipid aldehyde-induced modifications.

Virtual Screening Approach for the Identification of Hydroxamic Acids as Novel Human Ecto-5'-Nucleotidase Inhibitors.

Ecto-5'-nucleotidase (ecto-5'-NT, CD73) is a zinc-binding metallophosphatase that plays a key role in extracellular purinergic pathways, being implicated in several physiological and pathophysiological processes, such as immune homeostasis, inflammation, and tumor progression. As such, it has been recognized as a promising biological target for many diseases, including cancer, infections, and autoimmune diseases. Despite its importance, so far only a few inhibitors of this target enzyme are known, most of which are not suitable as drug candidates. Here, we aimed to search for hydroxamic acid-containing compounds as potential human ecto-5'-NT inhibitors, since this group is known to be a strong zinc chelator. To this end, we performed a hierarchical virtual screening (VS) search consisting of three consecutive steps (filtering for compounds bearing a hydroxamic acid group, shape-based matching, and docking followed by visual inspection), which were applied to screen the ZINC-14 database ("all purchasable subset"). Out of 25 compounds selected by this VS protocol, 12 were acquired and further submitted to enzymatic assays for VS experimental validation. Four of them (i.e., 33.3%) were found to inhibit human ecto-5'-NT in the low micromolar range. The most potent one showed an IC50 value of 6.2 ± 1.0 µM. All identified inhibitors satisfy drug-like criteria and provide novel scaffolds to be explored in further hit-to-lead optimization steps. Furthermore, to the best of our knowledge, they are the first hydroxamic acid-containing inhibitors of human ecto-5'-NT described so far.

Be Aware of Aggregators in the Search for Potential Human ecto-5'-Nucleotidase Inhibitors.

Promiscuous inhibition due to aggregate formation has been recognized as a major concern in drug discovery campaigns. Here, we report some aggregators identified in a virtual screening (VS) protocol to search for inhibitors of human ecto-5'-nucleotidase (ecto-5'-NT/CD73), a promising target for several diseases and pathophysiological events, including cancer, inflammation and autoimmune diseases. Four compounds (A, B, C and D), selected from the ZINC-11 database, showed IC50 values in the micromolar range, being at the same time computationally predicted as potential aggregators. To confirm if they inhibit human ecto-5'-NT via promiscuous mechanism, forming aggregates, enzymatic assays were done in the presence of 0.01% (v/v) Triton X-100 and an increase in the enzyme concentration by 10-fold. Under both experimental conditions, these four compounds showed a significant decrease in their inhibitory activities. To corroborate these findings, turbidimetric assays were performed, confirming that they form aggregate species. Additionally, aggregation kinetic studies were done by dynamic light scattering (DLS) for compound C. None of the identified aggregators has been previously reported in the literature. For the first time, aggregation and promiscuous inhibition issues were systematically studied and evaluated for compounds selected by VS as potential inhibitors for human ecto-5'-NT. Together, our results reinforce the importance of accounting for potential false-positive hits acting by aggregation in drug discovery campaigns to avoid misleading assay results.

Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches.

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

Nitroxides attenuate carrageenan-induced inflammation in rat paws by reducing neutrophil infiltration and the resulting myeloperoxidase-mediated damage.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and other cyclic nitroxides have been shown to inhibit the chlorinating activity of myeloperoxidase (MPO) in vitro and in cells. To examine whether nitroxides inhibit MPO activity in vivo we selected acute carrageenan-induced inflammation on the rat paw as a model. Tempol and three more hydrophobic 4-substituted derivatives (4-azido, 4-benzenesulfonyl, and 4-(4-phenyl-1H-1,2,3-triazol-1-yl)) were synthesized, and their ability to inhibit the in vitro chlorinating activity of MPO and carrageenan-induced inflammation in rat paws was evaluated. All of the tested nitroxides inhibited the chlorinating activity of MPO in vitro with similar IC(50) values (between 1.5 and 1.8 µM). In vivo, the attenuation of carrageenan-induced inflammation showed some correlation with the lipophilicity of the nitroxide at early time points but the differences in the effects were small (<2-fold) compared with the differences in lipophilicity (>200-fold). No inhibition of MPO activity in vivo was evident because the levels of MPO activity in rat paws correlated with the levels of MPO protein. Likewise, paw edema, levels of nitrated and oxidized proteins, and levels of plasma exudation correlated with the levels of MPO protein in the paws of the animals that were untreated or treated with the nitroxides. The effects of the nitroxides in vivo were compared with those of 4-aminobenzoic hydrazide and of colchicine. Taken together, the results indicate that nitroxides attenuate carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting MPO-mediated damage. Accordingly, tempol was shown to inhibit rat neutrophil migration in vitro.

MPO Inhibitors Selected by Virtual Screening.

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC50 values ranging from 1.0 to 2.8 µM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

Nucleotide conformational change induced by cationic bilayers.

The effects of cetyltrimethylammonium bromide (CTAB) micelles and dioctadecyldimethylammonium bromide (DODAB) bilayers on molecular conformation of 2'-deoxyadenosine 5'-monophosphate (dAMP) were evaluated from circular dichroism spectroscopy (CD) and molecular modeling of dAMP conformations of minimal energy upon varying torsion angles for the glycosidic bond (t(1)) for four different conditions of dielectric constant of the medium (E) and negative charge on the phosphate moiety (C), namely, E80_C2, E80_C0, E1_C2, and E1_C0. Upon decreasing medium polarity, a decreased intensity of the negative band over the 190-210 nm region for the dAMP CD spectrum was observed. Upon increasing relative proportion dAMP: DODAB, an increased intensity of the positive band over the 210-230 nm region plus a red shift were obtained that could be attributed to an increased nitrogenous base stacking, similar to A stacking in poly(A). Concomitant base stacking and insertion in the cationic aggregates were observed for DODAB bilayers but not for CTAB micelles. Thereby, the nucleotide extended, anti conformation in pure water typical for nucleotides in DNA was forced by the cationic bilayer to become syn. dAMP conformational modeling upon simultaneous changes in the nucleotide environment (from water to a hydrocarbon phase) and in the charge on phosphate moiety (-2 to zero) allowed to simulate dAMP conformation in the cationic bilayer/dAMP complex. Modeling confirmed the dAMP anti-to-syn conformational change experimentally characterized from CD spectroscopy. This nucleotide conformational change would possibly be at the root of DNA denaturation upon complexation with cationic lipids.