Aim: The aim of this study was to design and examine a novel epidermal growth factor receptor (EGFR) inhibitor with apoptotic properties by utilizing the essential structural characteristics of existing EGFR inhibitors as a foundation. Method: The study began with the natural alkaloid theobromine and developed a new semisynthetic derivative (T-1-PMPA). Computational ADMET assessments were conducted first to evaluate its anticipated safety and general drug-likeness. Deep density functional theory (DFT) computations were initially performed to validate the three-dimensional (3D) structure and reactivity of T-1-PMPA. Molecular docking against the EGFR proteins was conducted to investigate T-1-PMPA’s binding affinity and inhibitory potential. Additional molecular dynamics (MD) simulations over 200 ns along with MM-GPSA, PLIP, and principal component analysis of trajectories (PCAT) experiments were employed to verify the binding and inhibitory properties of T-1-PMPA. Afterward, T-1-PMPA was semisynthesized to validate the proposed design and in silico findings through several in vitro examinations. Results: DFT studies indicated T-1-PMPA’s reactivity using electrostatic potential, global reactive indices, and total density of states. Molecular docking, MD simulations, MM-GPSA, PLIP, and ED suggested the binding and inhibitory properties of T-1-PMPA against the EGFR protein. The in silico ADMET predicted T-1-PMPA’s safety and general drug-likeness. In vitro experiments demonstrated that T-1-PMPA effectively inhibited EGFR^WT and EGFR^790m, with IC₅₀ values of 86 and 561 nM, respectively, compared to Erlotinib (31 and 456 nM). T-1-PMPA also showed significant suppression of the proliferation of HepG2 and MCF7 malignant cell lines, with IC₅₀ values of 3.51 and 4.13 μM, respectively. The selectivity indices against the two cancer cell lines indicated the overall safety of T-1-PMPA. Flow cytometry confirmed the apoptotic effects of T-1-PMPA by increasing the total percentage of apoptosis to 42% compared to 31, and 3% in Erlotinib-treated and control cells, respectively. The qRT-PCR analysis further supported the apoptotic effects by revealing significant increases in the levels of Casp3 and Casp9. Additionally, T-1-PMPA controlled the levels of TNFα and IL2 by 74 and 50%, comparing Erlotinib’s values (84 and 74%), respectively. Conclusion: In conclusion, our study’s findings suggest the potential of T-1-PMPA as a promising apoptotic anticancer lead compound targeting the EGFR.

中文翻译：

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具有针对 EGFR 蛋白的抗癌潜力的新型可可碱凋亡类似物：计算和体外研究

目的：本研究的目的是利用现有 EGFR 抑制剂的基本结构特征作为基础，设计和检验一种具有凋亡特性的新型表皮生长因子受体 (EGFR) 抑制剂。方法：研究从天然生物碱可可碱开始，开发出一种新的半合成衍生物（T-1-PMPA）。首先进行计算 ADMET 评估，以评估其预期安全性和一般药物相似性。最初进行深密度泛函理论 (DFT) 计算以验证T-1-PMPA的三维 (3D) 结构和反应性。针对 EGFR 蛋白进行分子对接，以研究T-1-PMPA的结合亲和力和抑制潜力。采用超过 200 ns 的额外分子动力学 (MD) 模拟以及 MM-GPSA、PLIP 和轨迹主成分分析 (PCAT) 实验来验证T-1-PMPA的结合和抑制特性。随后，T-1-PMPA被半合成，以验证所提出的设计和通过多次体外检查的计算机结果。结果：DFT 研究使用静电势、全局反应指数和总态密度表明了T-1-PMPA的反应性。分子对接、MD 模拟、MM-GPSA、PLIP 和 ED 表明T-1-PMPA对 EGFR 蛋白具有结合和抑制特性。计算机ADMET预测了T-1-PMPA的安全性和一般药物相似性。体外实验表明，与厄洛替尼（31 和 456 nM）相比，T-1-PMPA有效抑制 EGFR ^WT和 EGFR ^790m，IC _{50值分别为 86 和 561 nM。} T-1-PMPA还显示出对 HepG2 和 MCF7 恶性细胞系增殖的显着抑制，IC ₅₀值分别为 3.51 和 4.13 μM。针对两种癌细胞系的选择性指数表明T-1-PMPA的总体安全性。流式细胞术证实了T-1-PMPA的细胞凋亡作用，将厄洛替尼处理细胞和对照细胞的细胞凋亡总百分比分别提高到 42%，而厄洛替尼处理细胞和对照细胞的细胞凋亡总百分比分别为 31% 和 3%。 qRT-PCR 分析显示 Casp3 和 Casp9 水平显着增加，进一步支持了细胞凋亡效应。此外，与厄洛替尼的值（84% 和 74%）相比， T-1-PMPA将 TNFα 和 IL2 水平分别控制了 74% 和 50%。结论：总而言之，我们的研究结果表明了T-1-PMPA作为一种有前途的针对 EGFR 的细胞凋亡抗癌先导化合物。