Mandal Pijus K, Ren Zhiyong, Chen Xiaomin, Kaluarachchi Kumar, Liao Warren S-L, McMurray John S
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Int J Pept Res Ther. 2013 Mar;19(1):3-12. doi: 10.1007/s10989-012-9313-0.
Signal transducer and activator of transcription 3 (Stat3) transmits signals from growth factors and interleukin-6 family cytokines by binding to their receptors via its Src homology 2 (SH2) domain. This results in phosphorylation of Tyr705, dimerization, translocation to the nucleus, and regulation of transcription of downstream genes. Stat3 is constitutively activated in several human cancers and is a target for anti-cancer drug design. We have shown previously phosphorylation of Tyr705 in intact cancer cells can be inhibited with prodrugs of phosphopeptide mimics targeting the SH2 domain. In a series of prodrugs consisting of bis-pivaloyloxymethyl esters of 4'-phosphonodifluoromethyl cinnamoyl-Haic-Gln-NHBn, appending methyl group to the -position of the cinnamate increased potency ca. twofold, which paralleled the increase in affinity of the corresponding phosphopeptide models. However, dramatic increases in potency were observed when the C-terminal C(O)NHBn of Gln-NHBn was replaced with a simple methyl group. In this communication we continue to explore the effects of structural modifications of prodrugs on their ability to inhibit Tyr705 phosphorylation. A set of 4-substituted prolines incorporated into -methyl-4-phosphocinnamoyl-leucinyl-Xaa-4-aminopentamide model peptides exhibited affinities of 88-317 nM by fluorescence polarization (Pro IC = 156 nM). In corresponding prodrugs, Pro inhibited constitutive Stat3 phosphorylation at 10 μM in MDA-MB-468 breast tumor cells. However, 4,4-difluoroproline and 4,4-dimethylproline resulted in complete inhibition at 0.5 μM. These results suggest that the prodrug with native proline undergoes metabolism that those with substituted prolines do not. In conclusion, changes in structure with minimal impact on intrinsic affinity can nevertheless have profound effects on the cellular potency of prodrug inhibitors of Stat3.
信号转导子和转录激活子3（Stat3）通过其Src同源2（SH2）结构域与生长因子和白细胞介素-6家族细胞因子的受体结合来传递信号。这导致Tyr705磷酸化、二聚化、转位至细胞核，并调控下游基因的转录。Stat3在多种人类癌症中持续激活，是抗癌药物设计的靶点。我们之前已表明，靶向SH2结构域的磷酸肽模拟物前药可抑制完整癌细胞中Tyr705的磷酸化。在一系列由4'-膦酰二氟甲基肉桂酰-Haic-Gln-NHBn的双新戊酰氧基甲酯组成的前药中，在肉桂酸酯的α位附加甲基使效力提高约两倍，这与相应磷酸肽模型亲和力的增加相平行。然而，当Gln-NHBn的C末端C(O)NHBn被一个简单甲基取代时，效力显著增加。在本通讯中，我们继续探索前药结构修饰对其抑制Tyr705磷酸化能力的影响。一组掺入α-甲基-4-膦酰肉桂酰-亮氨酰-Xaa-4-氨基戊酰胺模型肽中的4-取代脯氨酸通过荧光偏振显示出88 - 317 nM的亲和力（Pro IC50 = 156 nM）。在相应前药中，Pro在10 μM时抑制MDA-MB-468乳腺肿瘤细胞中组成型Stat3磷酸化。然而，4,4-二氟脯氨酸和4,4-二甲基脯氨酸在0.5 μM时导致完全抑制。这些结果表明，具有天然脯氨酸的前药会发生代谢，而具有取代脯氨酸的前药则不会。总之，对内在亲和力影响最小的结构变化仍可能对Stat3前药抑制剂的细胞效力产生深远影响。
