The maintenance of redox homeostasis in the brain is critical for the prevention of the development of neurodegenerative diseases. Drugs acting on brain redox balance can be promising for the treatment of neurodegeneration. For more than four decades, dimethyl fumarate (DMF) and other derivatives of fumaric acid ester compounds have been shown to mitigate a number of pathological mechanisms associated with psoriasis and relapsing forms of multiple sclerosis (MS). Recently, DMF has been shown to exert a neuroprotective effect on the central nervous system (CNS), possibly through the modulation of microglia detrimental actions, observed also in multiple brain injuries. In addition to the hypothesis that DMF is linked to the activation of NRF2 and NF-kB transcription factors, the neuroprotective action of DMF may be mediated by the activation of the glutathione (GSH) antioxidant pathway and the regulation of brain iron homeostasis. This review will focus on the role of DMF as an antioxidant modulator in microglia processes and on its mechanisms of action in the modulation of different pathways to attenuate neurodegenerative disease progression.

The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum “physiological” lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD–ACE2 binding, bringing about a measurable, up to 300-fold increase of the K_D value relative to RBD–ACE2 complex formation.

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.

Cytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed—catalytic competent—forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect.