Data Sheet 1_Computational advances in the design and discovery of artemis inhibitors for radiosensitization in cancer therapy.docx

IntroductionArtemis is a key scaffold repair protein involved in the non-homologous end-joining (NHEJ) DNA repair pathway and is encoded by the DCLRE1C gene in humans. Its inhibition disrupts double-strand break (DSB) repair, sensitizing cancer cells to ionizing radiation (IR). However, no Artemis-targeted inhibitors are currently available for therapeutic use. This study aims to identify and characterize novel small-molecule Artemis inhibitors that act as potential radiosensitizers in cancer treatment.
MethodsMicronuclei formation was assessed in Artemis-deficient (CJ179), proficient (1BR3), and mutant (48BR) cell lines following 1 Gy IR exposure. Initial in vitro screening identified HMAD as a potential Artemis inhibitor. A focused virtual screening of 69 compounds was performed using AutoDock4 and Glide to evaluate binding affinity to Artemis. The top 16 compounds (ΔG < −8.0 kcal/mol) were further analyzed. Density Functional Theory (DFT) calculations at the B3LYP/6−311+G(d,p) level were used to assess frontier molecular orbitals and reactivity. ADMET profiling was conducted to evaluate pharmacokinetic properties. Compounds 42 and 51 were subjected to 100 ns molecular dynamics (MD) simulations with MMGBSA binding free energy calculations, PCA, and FEL analysis.
ResultsCJ179 cells exhibited significantly higher micronuclei post-irradiation, confirming Artemis’s role in DNA repair. Among the top hits, compound 42 showed a highly stable binding profile, with a favorable MMGBSA binding energy of −36.94 kcal/mol. ADMET analysis indicated optimal drug-like properties. MD simulations revealed stable interaction trajectories, hydrogen bonding, and a narrow binding pocket. PCA and FEL analysis further supported the dynamic stability of compound 42.
DiscussionThis study identifies compound 42 as a promising Artemis inhibitor with potential as a radiosensitizing agent. The integrated in vitro and computational findings offer a foundation for further preclinical development, contributing to more effective radiotherapy strategies in cancer treatment.

引言 阿耳忒弥斯（Artemis）是一种关键的支架修复蛋白，参与非同源末端连接（NHEJ）DNA修复通路，在人类中由DCLRE1C基因编码。抑制阿耳忒弥斯会破坏双链断裂（DSB）修复过程，使癌细胞对电离辐射（IR）敏感。然而，目前尚无靶向阿耳忒弥斯的抑制剂可用于临床治疗。本研究旨在鉴定并表征新型小分子阿耳忒弥斯抑制剂，其可作为癌症治疗中潜在的放射增敏剂。方法 采用1 Gy电离辐射处理阿耳忒弥斯缺陷型（CJ179）、野生型（1BR3）及突变型（48BR）细胞系，评估微核形成情况。初始体外筛选鉴定出HMAD作为潜在的阿耳忒弥斯抑制剂。利用AutoDock4与Glide对69种化合物开展聚焦虚拟筛选，以评估其与阿耳忒弥斯的结合亲和力。选取结合自由能ΔG < −8.0 kcal/mol的前16种化合物进行后续分析。采用B3LYP/6−311+G(d,p)水平下的密度泛函理论（DFT）计算，评估前沿分子轨道与反应活性。开展ADMET属性分析以评价药代动力学特性。对化合物42与51进行100 ns分子动力学（MD）模拟，并辅以MMGBSA结合自由能计算、主成分分析（PCA）及自由能面（FEL）分析。结果 辐射后，CJ179细胞的微核数量显著升高，证实了阿耳忒弥斯在DNA修复中的核心作用。在筛选得到的候选化合物中，化合物42展现出高度稳定的结合模式，其MMGBSA结合能为−36.94 kcal/mol，特性优良。ADMET分析显示其具备理想的成药属性。分子动力学模拟结果表明，化合物42与靶点的相互作用轨迹稳定，存在特异性氢键结合，且结合口袋较为狭窄。主成分分析与自由能面分析进一步验证了化合物42的动态稳定性。讨论 本研究鉴定出化合物42是一种极具潜力的阿耳忒弥斯抑制剂，有望作为放射增敏剂应用于癌症治疗。本研究整合体外实验与计算生物学的研究结果，为后续临床前开发奠定了坚实基础，有助于开发更高效的癌症放射治疗策略。