Studies On Small Molecule Inhibitors Of Staphylococcus Aureus Sortase A

Staphylococcus aureus can cause local tissue purulent infections, pneumonia, pericarditis, sepsis and even life-threatening diseases. Emergence of the multi-drug resistant bacterial strains such as Methicillin resistant Staphylococcus aureus（MRSA） and Vancomycin resistant Staphylococcus aureus（VRSA）in hospital and community results from the abuse of the antibiotics. Recently, the bacterial resistance has become a serious global public health problem, so the urgent thing that we must do is to deal with the problem of the drug-resistant pathogens by researching the new antibacterial drugs.Traditional antibiotics are mainly directly kill the Staphylococcus aureus to cure the infection disease. But they also kill the mutually beneficial symbiotic bacteria with the host leading to the destruction of host microbial groups. Anti-virulence therapies as a new stratigy treating with anti-bacterial infection interfere with the pathogenic ability of bacteria rather than directly killing bacteria.Sortase A not the necessary for survival and growth in Staphylococcus aureus and other gram-positive bacterial is responsible for cataslying the covalent anchoring of the surface protein to the staphylococcus aureus cell envelope.Therefore, the inhibitors of SaSrtA will not bring about the survival pressure of pathogens and enormously reduce the probability of emergence of resistant strains in clinical. In a word, SaSrtA is a potential candidate target for anti-virulence factors.Partners synthesized about 200 derivatives that taken Benzoquinone and pyran lactone ring Participating in the process of biological reduction alkylation as a mother skeleton in the structure of natural products Pyranonaphthoquinone. We found 16 compounds as promising hits with IC₅₀ between 6μM and 80μM based on the fluorescence resonance energy transfer. Based on the physicochemical property and in vitro inhibitory activity of compounds, we select the compound SOMCL-14-283 and the enantiomer SOMCL-14-284 to further study:（1） using SDS-PAGE assay, we found that Sortase cleavage of sorting signals was blocked in a dose-dependent manner by compounds SOMCL-14-283 and SOMCL-14-284 with the relativity of IC₅₀. The inhibitory activity of compound SOMCL-14-283 was about 3 times better than that of SOMCL-14-284.（2） Furthermore, LC-MS experiments demonstrated its ability to bind the active center with high velocity in an irreversible fashion. These experiments also confirmed the reactivity toward the Cys184 side chain, which forms a covalent bond with the sulfur atom of the active site cysteine.（3） Addition of 400μM compound SOMCL-14-283 to purified SrtAΔN24 caused minor changes in secondary structure content, indicating that the inhibitor does not promote protein aggregation by Circular dichroism（CD）. Then, using FITC-IGg and western blot assay, we found that treatment of S. aureus cultures with the sortase inhibitor SOMCL-14-283 and SOMCL-14-284 reduced the incorporation of SpA into the bacterial envelope. However, the addition of compound SOMCL-14-283 and SOMCL-14-284 concentrations inhibitory for Srt A（up to 150 μM） to staphylococcal cultures did not affect the growth of S. aureus Newman. Using the microtiter broth dilution method, we measured the minimal inhibitory concentration of compound SOMCL-14-283 and SOMCL-14-284 to be >200 μM. These results indicate that compound SOMCL-14-283 selectively inhibits sortase activity and does not function as an antibiotic for S. aureus. Last but not the most, SOMCL-14-283 and SOMCL-14-284 inhibit sortase enzymes from different Gram-positive bacteria.This paper based on new target and mechanism of novel antibacterial infection active compounds as the goal, found a clear inhibition of srtA active compounds SOMCL-14-283, and further study the molecular mechanism of inhibiting the activity of SaSrtA for closely related with pathogenicity of srtA by high-throughput screening evaluation and modify the structure of cooperative research. Therefore, this study provides a meaningful attempt for the development of new antibacterial drugs different from traditional antibiotics and a new direction for the design and selection of SrtA covalent inhibitors.