Smadbeck James, Peterson Meghan B, Zee Barry M, Garapaty Shivani, Mago Aashna, Lee Christina, Giannis Athanassios, Trojer Patrick, Garcia Benjamin A, Floudas Christodoulos A
PLoS One. 2014 Apr 16;9(4):e95535. doi: 10.1371/journal.pone.0095535. eCollection 2014.
Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.
组蛋白是一类小分子蛋白质，对于细胞核中DNA的高效包装至关重要。当DNA缠绕在组蛋白表面时，会形成被称为核小体的DNA-蛋白质复合物。zeste同源物2（EZH2）对组蛋白残基的甲基化作用可在连续的细胞世代中维持基因抑制状态。EZH2的过表达会使重要的肿瘤抑制基因沉默，导致多种癌症的侵袭性增加。这使得抑制EZH2成为癌症治疗药物开发中的一个重要靶点。我们采用了一种三阶段的从头计算肽设计方法来设计EZH2的抑制肽。该方法包括一个序列选择阶段以及两个针对折叠特异性和近似结合亲和力的验证阶段。序列选择阶段由一个整数线性优化模型组成，通过求解该模型可生成在结合肽-EZH2结构中稳定性增加的氨基酸序列的排名列表。通过计算设计肽的折叠特异性和近似结合亲和力来验证这些序列。在此，我们报告了使用从头肽设计方法发现新型EZH2抑制肽的情况。通过剂量滴定和作用机制酶促试验在体外对计算发现的肽进行了实验验证。体外反应最强的肽SQ037，使用基于定量质谱的蛋白质组学在细胞核内进行了验证。该肽的IC50为13.5 mM，与天然对照肽和K27A突变对照肽相比，作为抑制剂表现出更强的效力，并且对肽底物表现出竞争性抑制作用。此外，与其他组蛋白甲基转移酶相比，该肽对EZH2靶点具有高度特异性。经过验证的这些肽是首批直接抑制EZH2的通过计算设计的肽。这些抑制剂对于进一步的染色质生物学研究应该是有用的。
