PO.ET09.10 · 实验与分子治疗
通过片段重组设计具有肉瘤选择性细胞毒性的双重VEGFR2/TIE2抑制剂
Fragment-recombination design of a dual VEGFR2/TIE2 inhibitor with sarcoma-selective cytotoxicity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
肉瘤除细胞毒性化疗外，系统性治疗选择有限。以VEGFR为靶点的多激酶抑制剂（如瑞戈非尼regorafenib）提供的获益有限，而其广泛的RTK抑制作用又限制了治疗窗。由于VEGFR2和TIE2是肿瘤血管生成和血管稳定性的核心调控因子，我们假设选择性双重VEGFR2/TIE2抑制可在提高效力的同时降低脱靶毒性。我们采用对已验证的RTK抑制剂骨架进行片段重组，结合双重VEGFR2/TIE2对接和表型筛选，设计出在效力-选择性平衡上更优、适用于肉瘤治疗的候选抑制剂。我们通过片段重组以计算方式设计了约300个双重VEGFR2/TIE2候选物，并根据对接和结合位点分析优先合成了其中15个。在人骨肉瘤细胞系（143B、MG63、KHOS/NP、U-2 OS）和小鼠巨噬细胞（RAW264.7）中，于24和48小时通过MTS实验评估抗增殖活性（2.5-10 μM），以评估对髓系细胞的毒性。将先导化合物与帕唑帕尼（pazopanib）、瑞戈非尼和索拉非尼（sorafenib）进行比较。在2.5 μM（48小时）时，帕唑帕尼、瑞戈非尼和索拉非尼在所有肉瘤细胞系中均维持≥100%的活力。在10 μM时，帕唑帕尼和瑞戈非尼显示≥80%的活力，而索拉非尼将143B活力降至55%，但对其他细胞系无影响（≥80%）。在合成的候选物中，SMC-101-008和SMC-101-011在2.5 μM时表现出早期活性，将143B和MG63活力降至57-65%（24小时）和47-84%（48小时）。然而，在10 μM（48小时）时，这两种化合物也消灭了RAW264.7（活力1%），表明在较高剂量下丧失选择性。相比之下，SMC-101-017表现出时间和剂量依赖的肉瘤选择性活性。在2.5 μM时，24小时观察到极小活性（活力≥88%），但到48小时，KHOS/NP和143B活力分别降至39%和19%，而RAW264.7不受影响（>100%）。在10 μM时，SMC-101-017显示中等程度的早期抑制（24小时时所有细胞系活力66-72%），但到48小时选择性地清除了KHOS/NP和143B（分别为25%和7%），同时保持RAW264.7活力（>100%）。SMC-101-017在4倍剂量范围内对骨肉瘤表现出强效、时间依赖的细胞毒性，同时不影响髓系细胞，从而区别于已获批的多激酶TKI。这些发现推荐SMC-101-017进行靶点验证和体内评估，并支持基于片段的设计用于开发具有更佳治疗窗的激酶抑制剂。
查看英文原文 English abstract
Sarcomas have limited systemic options beyond cytotoxic chemotherapy. VEGFR-directed multi-kinase inhibitors such as regorafenib provide modest benefit, but their broad RTK inhibition constrains the therapeutic margin. Because VEGFR2 and TIE2 are central regulators of tumor angiogenesis and vascular stability, we hypothesized that selective dual VEGFR2/TIE2 inhibition would improve potency while reducing off-target toxicity. We applied fragment recombination of validated RTK inhibitor scaffolds, combined with dual VEGFR2/TIE2 docking and phenotypic screening, to design candidate inhibitors with an improved potency-selectivity balance for sarcoma therapy. We computationally designed ~300 dual VEGFR2/TIE2 candidates via fragment recombination and synthesized 15 prioritized by docking and binding-site analyses. Antiproliferative activity was evaluated by MTS assay at 24 and 48 h (2.5-10 µM) in human osteosarcoma lines (143B, MG63, KHOS/NP, U-2 OS) and murine macrophages (RAW264.7) to assess myeloid cell toxicity. Lead compounds were compared with pazopanib, regorafenib, and sorafenib. At 2.5 µM (48 h), pazopanib, regorafenib, and sorafenib maintained ≥100% viability across all sarcoma lines. At 10 µM, pazopanib and regorafenib showed ≥80% viability, while sorafenib reduced 143B viability to 55% but spared other lines (≥80%). Among synthesized candidates, SMC-101-008 and SMC-101-011 showed early activity at 2.5 µM, reducing 143B and MG63 viability to 57-65% (24 h) and 47-84% (48 h). However, at 10 µM (48 h), both compounds also ablated RAW264.7 (1% viability), indicating loss of selectivity at higher doses. In contrast, SMC-101-017 exhibited time- and dose-dependent sarcoma-selective activity. At 2.5 µM, minimal activity was observed at 24 h (≥88% viability), but by 48 h, KHOS/NP and 143B viability dropped to 39% and 19%, respectively, while RAW264.7 remained unaffected (>100%). At 10 µM, SMC-101-017 showed moderate early suppression (66-72% viability across all lines at 24 h) but selectively eliminated KHOS/NP and 143B by 48 h (25% and 7%, respectively) while preserving RAW264.7 viability (>100%) SMC-101-017 demonstrated potent, time-dependent cytotoxicity against osteosarcoma while sparing myeloid cells across a 4-fold dose range, distinguishing it from approved multi-kinase TKIs. These findings nominate SMC-101-017 for target validation and in vivo evaluation, and support fragment-based design for developing kinase inhibitors with improved therapeutic windows.
利益披露 Disclosure
Y. Lee, None..
B. Kim, None..
J. Choi, None..
M. Kim, None..
S. Seo, None.