Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.

Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein.

The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5'-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.