Antimicrobial peptides (AMPs), which hold tremendous promise in overcoming the emergence of drug resistance, are limited in wide clinical applications due to their instability, especially against trypsin. Herein, we designed six peptide mutants based on the cathelicidin CATHPb2, followed by screening. Pb2-1, which showed the best activity against drug-resistant bacteria among these mutants, was selected to be combined with the trypsin inhibitory loop ORB-C to obtain two hybrid peptides: PCL-1 and Pb2-1TI. Notably, both of the hybrid peptides exhibited a remarkable enhancement in trypsin resistance compared with Pb2-1. The tests showed that PCL-1 displayed broad-spectrum antimicrobial activity that was superior to that of Pb2-1TI. In addition, PCL-1 had relatively lower cytotoxicity than Pb2-1TI towards the L02 and HaCaT cell lines and negligible hemolysis, as well as tolerance to high concentrations of salt, extreme pH, and temperature variations. In vivo, PCL-1 effectively improved the survival rate of mice that were systemically infected with drug-resistant Escherichia coli through efficient bacterial clearance from the blood and organs. With regard to mode of action, PCL-1 damaged the integrity of the bacterial cell membrane and attached to the membrane surface while bound to bacterial genomic DNA to eventually kill the bacteria. Altogether, the trypsin-resistant peptide PCL-1 is expected to be a candidate for the clinical treatment of bacterial infections.

抗菌肽 (AMPs) 在克服耐药性的出现方面具有巨大的前景，但由于其不稳定性，尤其是对胰蛋白酶的不稳定性，在广泛的临床应用中受到限制。在此，我们设计了六种基于cathelicidin CATHPb2 的肽突变体，然后进行了筛选。选择在这些突变体中对耐药菌表现出最佳活性的 Pb2-1 与胰蛋白酶抑制环 ORB-C 结合得到两种杂合肽：PCL-1 和 Pb2-1TI。值得注意的是，与 Pb2-1 相比，这两种杂合肽都表现出显着增强的胰蛋白酶抗性。测试表明，PCL-1 显示出优于 Pb2-1TI 的广谱抗菌活性。此外，与 Pb2-1TI 相比，PCL-1 对 L02 和 HaCaT 细胞系的细胞毒性相对较低，溶血作用可忽略不计，并且对高浓度盐、极端 pH 值和温度变化具有耐受性。在体内，PCL-1有效提高了全身感染耐药小鼠的存活率大肠杆菌通过从血液和器官中有效清除细菌。在作用方式上，PCL-1破坏细菌细胞膜的完整性并附着在膜表面，同时与细菌基因组DNA结合，最终杀死细菌。总之，胰蛋白酶抗性肽PCL-1有望成为临床治疗细菌感染的候选药物。