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.

Conformational flexibility of DENV NS2B/NS3pro: from the inhibitor effect to the serotype influence.

The dengue virus (DENV) has four well-known serotypes, namely DENV1 to DENV4, which together cause 50-100 million infections worldwide each year. DENV NS2B/NS3pro is a protease recognized as a valid target for DENV antiviral drug discovery. However, NS2B/NS3pro conformational flexibility, involving in particular the NS2B region, is not yet completely understood and, hence, a big challenge for any virtual screening (VS) campaign. Molecular dynamics (MD) simulations were performed in this study to explore the DENV3 NS2B/NS3pro binding-site flexibility and obtain guidelines for further VS studies. MD simulations were done with and without the Bz-nKRR-H inhibitor, showing that the NS2B region stays close to the NS3pro core even in the ligand-free structure. Binding-site conformational states obtained from the simulations were clustered and further analysed using GRID/PCA, identifying four conformations of potential importance for VS studies. A virtual screening applied to a set of 31 peptide-based DENV NS2B/NS3pro inhibitors, taken from literature, illustrated that selective alternative pharmacophore models can be constructed based on conformations derived from MD simulations. For the first time, the NS2B/NS3pro binding-site flexibility was evaluated for all DENV serotypes using homology models followed by MD simulations. Interestingly, the number of NS2B/NS3pro conformational states differed depending on the serotype. Binding-site differences could be identified that may be crucial to subsequent VS studies.