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Acridine–O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity
European Journal of Medicinal Chemistry ( IF 6.0 ) Pub Date : 2021-10-09 , DOI: 10.1016/j.ejmech.2021.113909
Jaime Franco Pinto 1 , Alexandra Fillion 1 , Patricia Duchambon 1 , Sophie Bombard 1 , Anton Granzhan 1
Affiliation  

O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a–c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 μM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1–2 μM), similarly to acridine 8 (GI50 = 0.6 μM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2–0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.



中文翻译:

吖啶-O6-苄基鸟嘌呤杂化物:合成、DNA 结合、MGMT 抑制和抗增殖活性

O 6 -甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 是一种关键的 DNA 修复酶,通过直接修复药物诱导的 DNA 中的O 6 -甲基鸟嘌呤残基,参与对 DNA 烷基化抗癌药物如替莫唑胺 (TMZ) 的化学抗性。MGMT 底物类似物,例如O 6 -苄基鸟嘌呤 (BG),可在体外和细胞内有效地灭活 MGMT;然而,由于不良副作用,这些药物未能进入临床。在这里,我们设计了混合药物,将 BG 残基与 DNA 相互作用部分(6-氯-2-甲氧基-9-氨基吖啶)共价连接。具体来说,通过改变 BG ( N9鸟嘌呤与苄基)以及接头的长度和性质。紫外/可见光吸收和荧光数据表明,所有六种杂化物在很宽的温度范围内的水溶液中都采用分子内堆积的构象。所有杂合体都与双链 DNA 相互作用,分光光度滴定清楚地证明了这一点,没有吖啶环的嵌入,也不会诱导双链体的热稳定性。所有杂种以及参考 DNA 嵌入剂 (6-chloro-2-methoxy-9-aminoacridine 8 )在体外以可变的效率不可逆地抑制 MGMT,与 BG 相当。在耐多药胶质母细胞瘤细胞系 T98G 中,苄基连接的杂合体7a-cN 9- 连接的杂合体19b具有中等细胞毒性( 96 小时后GI 50 ≥ 15 μM),而N 9连接的杂合体19a19c具有强细胞毒性(GI 50  = 1-2 μM),类似于吖啶8(GI 50  = 0.6 μM ) )。在所有化合物中,杂合体19a19c与 BG 相似,在与亚毒性剂量的 TMZ 共同处理时显示出协同细胞毒性作用,组合指数 (CI) 值低至 0.2-0.3。与体外结果一致,化合物19a使细胞 MGMT 失活,但与 BG 不同,不会单独或与 TMZ 联合诱导显着水平的 DNA 损伤,如 γH2AX 免疫染色实验的结果所示。相反,与 BG 不同,化合物19a单独诱导 T98G 细胞的显着凋亡,与 TMZ 组合不会进一步增加。这些结果表明, 19a的细胞毒性及其与 TMZ 组合的分子机制与 BG 的不同。这种组合的强协同特性代表了治疗 TMZ 耐药性癌症的一个有趣的治疗机会。

更新日期:2021-10-28
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