Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, 24341, Republic of Korea.
Biomedical Manufacturing Technology Center, Korea Institute of Industrial Technology, Yeongcheon, 38822, Republic of Korea.
Biochem Biophys Res Commun. 2019 Jan 8;508(2):570-575. doi: 10.1016/j.bbrc.2018.11.080. Epub 2018 Nov 30.
Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ∼100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.
恩替莫德（CBLB502）是一种鞭毛衍生的辐射对策，目前正在临床试验中。恩替莫德通过直接与先天免疫受体 TLR5 相互作用发挥放射防护活性，利用鞭毛的保守结构域。恩替莫德被设计为包含一个非药物效应相关的人为引入的 N 端区域，可能会在人类中引发意想不到的毒性免疫原性反应。为了改进恩替莫德的药物设计，我们通过去除其辅助区域将恩替莫德工程化为 KMRC011。通过生物物理和细胞分析评估了 KMRC011 与 TLR5 的结合和激活能力。KMRC011 以 1:1 的摩尔比与 TLR5 形成异常稳定的复合物，平衡解离常数约为 100 pM，并能有效激活 TLR5。此外，丙氨酸扫描诱变鉴定出 KMRC011 的 R90 和 E114 残基是 TLR5 激活的热点。进一步的比较分析表明，KMRC011 以类似于恩替莫德的方式与 TLR5 结合并激活 TLR5。因此，我们提出 KMRC011 可以替代恩替莫德作为第二代辐射对策，不会产生源自恩替莫德辅助区域的免疫原性副作用。
