Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients.

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.

In-silico guided chemical exploration of KDM4A fragments hits.

BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.

Solution structure of recombinant Pvfp-5ß reveals insights into mussel adhesion.

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5ß, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5ß has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5ß is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.

Investigation on a MMACHC mutant from cblC disease: The c.394C>T variant.

The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Although missing part of the Cbl binding domain, p.R132X is associated to late-onset symptoms and, therefore, it is supposed to retain residual function. However, to our knowledge structural-functional studies on c.394C > T mutant aimed at verifying this hypothesis are still lacking. By using a biophysical approach including Circular Dichroism, fluorescence, Small Angle X-ray Scattering, and Molecular Dynamics, we show that the mutant protein MMACHC-R132X retains secondary structure elements and remains compact in solution, partly preserving its binding affinity for Cbl. Insights on the fragile stability of MMACHC-R132X-Cbl are provided.