SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

Synthesis and In silico Studies of N-acylhydrazone Derivatives as hnRNP K Ligands with Potential Anti-cancer Activity

Background: A green and efficient synthetic methodology for a wide family of Nacylhydrazones (yields: 42-76%) using microwave irradiation is described, as well as their full characterization. Their potential antineoplastic activity was evaluated in vitro via EMSA by testing protein- DNA interactions. Among the 11 compounds tested, N-acylhydrazone derivative 5 bearing a hydroxyl group, showed the highest affinity to bind and inhibit the hnRNP K KH3 domain. Docking simulations of compound 5 showed three possible modes of interaction between the KH3 domain of hnRNP K protein and compound predict. : The N-acylhydrazones are knows as powerful chemical entities for Medicinal Chemistry, since it has been identified in a huge number of hit and lead compounds that act on various types of molecular targets, including in tumorigenesis processes. Objective: We evaluated their potential ability to inhibit the KH3 domain of the hnRNP K protein binding to single stranded DNA (ssDNA). Furthermore, a docking simulation was performed for the newly synthetized compounds to evaluate their interactions between proteins and N-acylhydrazine derivative. Methods: The N-acylhydrazone derivatives were synthetized through three reaction steps, from a simple and commercial substrate, using microwave irradiation as a green energy source. The N-acylhydrazone derivatives ability to bind with the hnRNP K protein was evaluated via EMSA by testing protein-DNA interactions. The docking simulations were performed in a Gold 5.2.2 software using 100 conformers, 10.000 operations, 95 mutations and 95 crossovers. Results: Eleven new N-acylhydrazone derivatives were synthetized using microwave showing yields between 42% and 76%. Among the eleven compounds tested, compound 5 was shown to be most capable to prevent the natural binding of hnRNP K protein to the oligonucleotide. Regarding the docking simulation, compound 5 can bind to the main binding residues of KH3 domain and compete with the natural ligand ssDNA of this protein. Conclusion: A green and efficient synthetic methodology for a wide family of N-acylhydrazones (yields: 42-76%) using microwave irradiation is described, as well as their full characterization. Their potential antineoplastic activity was evaluated in vitro via EMSA by testing protein-DNA interactions. Among the 11 compounds tested, N-acylhydrazone derivative 5 bearing a hydroxyl group, showed the highest affinity to bind and inhibit the hnRNP K KH3 domain. Docking simulations of compound 5 showed three possible modes of interaction between the KH3 domain of hnRNP K protein and compound predict.

MOLECULAR MODELING OF THE INTERACTIONS BETWEEN HISTONE DEACETYLASE 8 AND INHIBITORS

Inhibitors of histone deacetylases (HDACs) have become an attractive class of anticancer agent. To understand the interaction between HDAC8 and inhibitors, including "pan-" inhibitors that inhibit many HDACs isoforms and selective inhibitors with no linker domain, docking and molecular dynamics simulation were conducted. Docking results showed the presence of π-π interactions between "linkerless" inhibitors and the aromatic amino acid residues of HDAC8 in the active site. Binding between HDAC8 and inhibitors was also stabilized by hydrogen bond and hydrophobic interaction. In molecular dynamics simulations, the zinc ion was shown to coordinate one more atom of HDAC8-"linkerless" inhibitor complexes than HDAC8-"pan-" inhibitor complexes. Persistent hydrogen bonds also existed between Tyr306 of HDAC8 and some inhibitors. When inhibitors with large cap groups bound to the active pocket of HDAC8, Phe152 and Met274 shifted from their initial positions and the entrance of the active pocket became more open, resulting in the formation of sub-pocket. Hydrophobic interactions contributed most favorably to the binding free energy between HDAC8 and inhibitors. Lys33, Asp178, Asp267, Tyr306 and Leu308 of HDAC8 were favorable for binding with all inhibitors.

Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase

A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

The C1 domains in protein kinase C (PKC) isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and for mediating translocation to membranes. Previous studies revealed that the C1 domain in α- and β-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the endoplasmic reticulum/Golgi protein p23/Tmp21. Here, we found that p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β2-chimaerin. Glu227 and Leu248 in the β2-chimaerin C1 domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 domains from individual PKC isozymes differentially interact with p23/Tmp21. For PKCε, it interacts with p23/Tmp21 specifically via its C1b domain; however, this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 domain-containing proteins, and it plays an essential role in β2-chimaerin relocalization. Our study also highlights the relevance of C1 domains in protein–protein interactions in addition to their well-established lipid-binding properties.