COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

Background: As a result of the paucity of treatment, Leishmaniasis continues to provoke about 60,000 deaths every year worldwide. New molecules are needed, and drug discovery research is oriented toward targeting proteins crucial for parasite survival. Among them, trypanothione reductase (TR) is of remarkable interest owing to its vital role in Leishmania species protozoan parasite life. Our previously identified compound 1 is a novel chemotype endowed with a unique mode of TR inhibition thanks to its binding to a formerly unknown but druggable site at the entrance of the NADPH binding cavity, absent in human glutathione reductase (hGR). Methods: We designed and synthesized new 3-amino-1-arylpropan-1-one derivatives structurally related to compound 1 and evaluated their potential inhibition activity on TR from Leishmania infantum (LiTR). Cluster docking was performed to assess the binding poses of the compounds. Results: The newly synthesized compounds were screened at a concentration of 100 μM in in vitro assays and all of them proved to be active with residual activity percentages lower than 75%. Conclusions: Compounds 2a and 2b were the most potent inhibitors found, suggesting that an additional aromatic ring might be promising for enzymatic inhibition. Further structure–activity relationships are needed to optimize our compounds activity.

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.

Infantile-onset Ascending Hereditary Spastic Paralysis, Juvenile Primary Lateral Sclerosis and Juvenile Amyotrophic Lateral Sclerosis are all motor neuron diseases related to mutations on the ALS2 gene, encoding for a 1657 amino acids protein named Alsin. This ~185 kDa multi-domain protein is ubiquitously expressed in various human tissues, mostly in the brain and the spinal cord. Several investigations have indicated how mutations within Alsin’s structured domains may be responsible for the alteration of Alsin’s native oligomerization state or Alsin’s propensity to interact with protein partners. In this review paper, we propose a description of differences and similarities characterizing the above-mentioned ALS2-related rare neurodegenerative disorders, pointing attention to the effects of ALS2 mutation from molecule to organ and at the system level. Known cases were collected through a literature review and rationalized to deeply elucidate the neurodegenerative clinical outcomes as consequences of ALS2 mutations.