细胞致死膨胀毒素(CDT),杜克雷嗜血杆菌,是最近被考虑用于靶向治疗的细菌毒素之一,特别是在癌症治疗中。CDT是A-B2外毒素。其催化亚基(CdtB)能够诱导DNA双链断裂,宿主真核细胞的细胞周期停滞和凋亡。序列比对表明CdtB在结构上与磷酸酶和脱氧核糖核酸酶I(DNA酶I)同源。最近,研究发现CdtB的毒性主要与其核酸酶活性有关。CDT的免疫原性可降低其在靶向治疗中的有效性。然而,如果其免疫原性显著降低,则该毒素可能非常有用。通过计算服务器检测热点异位残基,然后对其进行突变以消除B细胞表位是降低基于外源蛋白的治疗剂的免疫原性的有前途的方法。通过上述方法,在这项研究中,我们试图降低CdtB蛋白序列的免疫原性。这项研究最初筛选的CdtB残基是线性和构象的B细胞表位。通过将B细胞表位与排除的保守残基重叠,以及活性和酶位点,允许四个残基突变。有两个突变蛋白选项显示降低的抗原性概率。第一种选择是N19F,G74I,和S161F,VaxiJen评分为0.45,免疫表位数据库(IEDB)评分为1.80,选项二为N19F,G74I,和S161W,VaxiJen评分为0.45,IEDB评分为1.88。对所提出的序列的3D结构进行了评估和改进。通过分子动力学模拟获得天然和突变蛋白的结构稳定性。结果表明,突变体中的突变不会引起其结构稳定性的明显变化。然而,突变体1在模拟过程中显示出更多的热力学稳定性。本研究中应用的方法可用作在治疗性蛋白质中寻找热点免疫原区域的粗略指南。我们的结果提供了一个新版本的CdtB,由于免疫原性降低和稳定性增加,可用于以毒素为基础的药物如免疫毒素。
The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this
study, we try to reduce the immunogenicity of the CdtB- protein sequence. This
study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this
study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.