Jiang Jiewei, Liang Taimeng, Solberg Jonathan, Chan Alice, Kalra Prakriti, Shi Rui, Pomerantz William C K, Hawkinson Jon E, Schönbrunn Ernst, Georg Gunda I
Department of Medicinal Chemistry and Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, 717 Delaware Street, MN, 55414, USA.
Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA.
Eur J Med Chem. 2025 Jun 5;290:117504. doi: 10.1016/j.ejmech.2025.117504. Epub 2025 Mar 16.
Starting from dihydropyridopyrimidine benzyl ester 1, we pursued a macrocyclization strategy by linking its two aryl rings, hypothesizing that decreasing the conformational flexibility of the ester side chain would increase bromodomain and extra-terminal (BET) protein affinity and selectivity. We prepared 14 analogs and tested them in our fluorescent polarization (FP) assay for BRDT-1 and BRD4-1 affinity. Based on their K values, we selected compounds 6b (BRDT-1 K = 1.05 μM and BRD4-1 K = 0.68 μM) and 6d (BRDT-1 K = 0.86 μM and BRD4-1 K = 0.70 μM) for further testing. Differential Scanning fluorimetry (DSF) experiments with the BD1 and BD2 proteins of BRD4 and BRDT showed that the most significant increases in the melting temperatures occurred for BRDT-2 for both compounds (13 °C for 6b and 8.9 °C for 6d). Preferential binding to the second bromodomain of BRDT-2 was further confirmed by protein-observed fluorine NMR with the tandem bromodomain of BRDT. A BROMOscan showed that both compounds are pan-BET-BD2 selective (K = 33-160 nM). A bromoMAX assay with 32 bromodomains verified BET bromodomain selectivity for 6d. The co-crystal structure between macrocyclic analogs 6b and 6d and BRD4-1 shows that the two molecules adopt almost identical conformations despite different spacer lengths. We posit that the increased BD2 selectivity could result from pi-stacking (and additional H-bonds) between the inhibitors and a His residue that is conserved across BET-BD-2 but is absent in BET-BD-1. Compound 6d inhibited MM.1S cancer cell growth with an IC of 2.6 μM. The study exemplifies how constraining conformational flexibility can impart target selectivity. The results indicate that the macrocyclization strategy achieved an increase in pan-BD1-affinity and comparable pan-BD2-affinity compared to lead compound 1.
从二氢吡啶嘧啶苄酯1开始，我们通过连接其两个芳环采用了一种大环化策略，推测降低酯侧链的构象灵活性会增加溴结构域和额外末端（BET）蛋白的亲和力和选择性。我们制备了14种类似物，并在我们的荧光偏振（FP）测定中测试了它们对BRDT-1和BRD4-1的亲和力。根据它们的K值，我们选择了化合物6b（BRDT-1 K = 1.05 μM，BRD4-1 K = 0.68 μM）和6d（BRDT-1 K = 0.86 μM，BRD4-1 K = 0.70 μM）进行进一步测试。对BRD4和BRDT的BD1和BD2蛋白进行的差示扫描荧光法（DSF）实验表明，两种化合物对BRDT-2的熔解温度升高最为显著（6b为13℃，6d为8.9℃）。通过对BRDT串联溴结构域进行蛋白观察氟核磁共振进一步证实了对BRDT-2第二个溴结构域的优先结合。BROMOscan显示两种化合物都是泛BET-BD2选择性的（K = 33 - 160 nM）。用32个溴结构域进行的bromoMAX测定验证了6d对BET溴结构域的选择性。大环类似物6b和6d与BRD4-1之间的共晶体结构表明，尽管间隔长度不同，但这两个分子采用几乎相同的构象。我们认为，BD2选择性的增加可能是由于抑制剂与一个在BET-BD-2中保守但在BET-BD-1中不存在的His残基之间的π-堆积（以及额外的氢键）。化合物6d以2.6 μM的IC抑制MM.1S癌细胞生长。该研究例证了限制构象灵活性如何赋予目标选择性。结果表明，与先导化合物1相比，大环化策略实现了泛BD1亲和力的增加和相当的泛BD2亲和力。
