Targeting telomerase for melanoma therapeutics

项目总结 人类家族性和散发性高重复TERT(端粒酶催化亚单位)启动子突变 黑色素瘤导致TERT转录和端粒酶激活增加2-4倍，使端粒酶成为 黑色素瘤癌症治疗的有吸引力的靶点。高效小分子抗癌药物的研究进展 由于缺乏关于端粒酶的高分辨率结构数据，端粒酶一直受到阻碍。我们使用了TERT 用X射线结晶学测定结构，用电子方法筛选500,000个化合物。这 方法使我们能够识别出一组包含类似支架的小分子，这些小分子抑制了 端粒酶活性。我们已经得到了TERT的X射线共晶结构 这些化合物揭示了一个新的和意想不到的变构结合部位，即FVYL口袋，位于 在TERT拇指区域的表面。这是有史以来第一个配体-TERT共晶结构 到目前为止已经解决了。使用生化分析，我们发现FVYL Pocket与端粒酶RNA(TER)结合， 因此，我们最初的铅通过抑制TERT-TER结合和端粒酶RNP组装而起作用。 我们还发现了能够选择性抑制端粒酶阳性人类生长的化合物 黑色素瘤细胞株，但对端粒酶阴性癌和未转化细胞的生长抑制很小 在文化上。还检测了以前的端粒酶抑制剂BIBR-1532。我们确定了BIBR-1532- TERT共晶结构，表明它结合到相同的一般区域，但以略有不同的方式，如 做我们通过计算预筛选确定的配体。我们还发现，BIBR-1532具有 在杀死黑色素瘤细胞方面，延迟作用的效果比我们自己的顶击效果要差得多 学习。本建议的目的是(A)使用基于结构的设计方法，并结合医学 化学，以提高抑制端粒酶的效力，(B)评估生化活性和 在研究端粒酶抑制的细胞通路时端粒酶的特异性，以及(C) 探讨端粒酶抑制剂对黑色素瘤的体内溶瘤活性。我们具有得天独厚的优势 完成描述的工作，因为我们能够迭代地获得额外的配体-TERT共晶体 结构，以及我们在快速并行合成新化合物文库方面的专业知识，利用 现场提供20,000种试剂和起始材料(&gt；800种硼酸)。我们的研究可能会提供一种 基于结构设计端粒酶小分子抑制剂的新途径，并可能重新焕发活力 在小分子端粒酶抑制剂领域的研究，最终产生了治疗的新药 黑色素瘤单独或与现有的化疗或免疫疗法相结合。这个研究小组由一位 Wistar研究所的端粒酶(Skordalake)专家，以及经验丰富的制药研究人员 福克斯·蔡斯化学多样性中心医药(Reitz)和计算化学(Reynolds)。

PROJECT SUMMARY Highly recurrent TERT (the catalytic subunit of telomerase) promoter mutations in human familial and sporadic melanoma lead to a 2-4 fold increase in TERT transcription and telomerase activation, making telomerase an attractive target for melanoma cancer therapies. The development of effective small-molecule inhibitors of telomerase has been hindered by the lack of high-resolution structural data on telomerase. We used the TERT structure determined by X-ray crystallography to screen >500,000 compounds by in silico methods. This approach allowed us to identify a set of small molecules containing a similar scaffold that inhibited the enzymatic activity of telomerase. We have obtained the X-ray co-crystal structure of TERT bound to one of these compounds, revealing a novel and unexpected allosteric binding site, namely the FVYL pocket, located on the surface of the TERT thumb domain. This is the first-ever ligand-TERT co-crystal structure that has been solved to date. Using biochemical assays, we showed that the FVYL pocket binds telomerase RNA (TER), and therefore our initial lead acts by inhibiting the TERT – TER association and telomerase RNP assembly. We have also found compounds that can selectively inhibit the growth of telomerase-positive human melanoma cell lines, but show little growth inhibition of telomerase negative cancer and non-transformed cells in culture. The previous telomerase inhibitor BIBR-1532 was also examined. We determined the BIBR-1532- TERT co-crystal structure, showing that it binds to the same general area, but in a slightly different manner, as do the ligands that we identified by computational prescreening. We also found that BIBR-1532 had substantially less efficacy with a delayed action at killing melanoma cells than does the top hit from our own studies. The aims of this proposal are to (A) use structure-based design methods combined with medicinal chemistry to improve potency for the inhibition of telomerase, (B) evaluate the biochemical activity and specificity for telomerase while investigating the cellular pathways perturbed on telomerase inhibition, and (C) probe the in vivo melanoma oncolytic activity of the telomerase inhibitors. We are uniquely positioned to accomplish the work described because of our ability to iteratively obtain additional ligand-TERT co-crystal structures, and our expertise in the rapid parallel synthesis of new compound libraries, taking advantage of >20,000 reagents and starting materials available onsite (>800 boronic acids). Our research may provide a new approach for the structure-based design of small-molecule inhibitors of telomerase, and may reinvigorate research in the area of small-molecule telomerase inhibitors, eventually resulting in new drugs to treat melanoma alone or in combination with existing chemo- or immunotherapies. The research team is led by an expert in telomerase (Skordalakes) at The Wistar Institute, along with experienced pharma researchers in medicinal (Reitz) and computational chemistry (Reynolds) at the Fox Chase Chemical Diversity Center, Inc.