Abstract：
Antimicrobial resistance (AMR) is a global health threat. The dramatic increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections emphasizes the need to find new anti-infective agents with a novel mode of action. The Caseinolytic protease (ClpP) is a central virulence factor in stress survival, virulence, and antibiotic resistance of MRSA. Here, we found ayanin, a flavonoid isolated from Callicarpa nudiflora, was an inhibitor of MRSA ClpP with an IC50 of 19.63 μM. Using quantitative real-time PCR, ayanin reduced the virulence of Staphylococcus aureus (S. aureus) by down-regulating the level of some important virulence factors, including agrA, RNAⅢ, hla, pvl, psmα and spa. The results of cellular thermal shift assay and thermal shift assay revealed a binding between ayanin and ClpP. Molecular docking showed that ASP-168, ASN-173 and ARG-171 were the potential binding sites for ClpP binding to ayanin. ClpP mutagenesis study further indicated that ARG-171 and ASN-173 were the main active sites of ClpP. The affinity constant (KD) value of ayanin with ClpP was 3.15 × 10-5 M measured by surface plasmon resonance. In addition, ayanin exhibited a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo, especially in combination with vancomycin. This is the first report of ayanin with in vivo and in vitro efficacy against S. aureus infection. In conclusion, ayanin is a promising therapeutic agent to combat MRSA infections by targeting ClpP.
摘要：
抗菌素耐药性(AMR)是一个全球性的健康威胁。耐甲氧西林金黄色葡萄球菌（MRSA）感染的急剧增加强调了寻找具有新型作用方式的新型抗感染药的必要性。酪蛋白分解蛋白酶（ClpP）是应激生存的中心毒力因子，毒力,和MRSA的抗生素耐药性。这里,我们找到了ayanin,从裸花紫珠中分离出的一种类黄酮，是MRSAClpP的抑制剂,IC50为19.63μM。使用定量实时PCR，ayanin降低金黄色葡萄球菌的毒力(S.金黄色葡萄球菌)通过下调一些重要毒力因子的水平，包括agrA,RNAⅢ,hla,pvl,PSMα和水疗中心。细胞热移位测定和热移位测定的结果揭示了ayanin和ClpP之间的结合。分子对接显示ASP-168、ASN-173和ARG-171是ClpP与ayanin结合的潜在结合位点。ClpP诱变研究进一步表明，ARG-171和ASN-173是ClpP的主要活性位点。通过表面等离子体共振测得的阿赖宁与ClpP的亲和常数（KD）值为3.15×10-5M。此外,在小鼠体内对金黄色葡萄球菌引起的肺炎感染有显著的治疗作用,尤其是与万古霉素合用。这是首次报道在体内和体外对金黄色葡萄球菌感染具有功效的ayanin。总之,ayanin是通过靶向ClpP来对抗MRSA感染的有前途的治疗剂。
