Targeting telomerase for melanoma therapeutics

项目摘要 家族性和散发性端粒酶催化亚单位启动子突变的研究 黑色素瘤导致端粒酶转录和端粒酶激活增加2-4倍，使端粒酶成为一种新的肿瘤抑制剂。 黑色素瘤癌症治疗的有吸引力的靶点。有效的小分子抗肿瘤药物的研究进展 由于缺乏端粒酶的高分辨率结构数据，端粒酶的研究受到阻碍。我们用了四叔丁基醚 通过X射线晶体学确定结构，通过计算机方法筛选> 500，000种化合物。这 这种方法使我们能够鉴定出一组含有类似支架的小分子， 端粒酶的酶活性我们已经获得了与以下物质结合的TERT的X射线共晶结构： 这些化合物，揭示了一种新的和意想不到的变构结合位点，即FVYL口袋，位于 在TERT拇指域的表面上。这是有史以来第一个配体-TERT共晶体结构， 解决至今。使用生物化学测定，我们表明FVYL口袋与端粒酶RNA（TER）结合， 因此我们最初的先导化合物通过抑制TERT-TER结合和端粒酶RNP装配而起作用。 我们还发现了可以选择性抑制端粒酶阳性的人巨噬细胞生长的化合物， 黑色素瘤细胞系，但对端粒酶阴性癌和非转化细胞几乎没有生长抑制作用 在文化中。还检查了先前的端粒酶抑制剂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 做我们通过计算机预筛选鉴定的配体。我们还发现，BIBR-1532 在杀死黑色素瘤细胞的延迟作用下， 问题研究该提案的目的是（A）使用基于结构的设计方法结合医学 （B）评价生物化学活性， 在研究对端粒酶抑制扰动的细胞途径时对端粒酶的特异性，和（C） 探测端粒酶抑制剂的体内黑素瘤溶瘤活性。我们有独特的优势， 由于我们能够迭代地获得另外的配体-TERT共晶体， 结构，以及我们在快速并行合成新化合物库方面的专业知识， 现场可提供> 20，000种试剂和起始物料（>800种硼酸）。我们的研究可以提供一个 这是一种基于结构设计端粒酶小分子抑制剂的新方法， 研究领域的小分子端粒酶抑制剂，最终导致新的药物来治疗 黑素瘤单独或与现有的化疗或免疫疗法组合。该研究小组由一名 Wistar研究所的端粒酶（Skordalakes）专家，沿着经验丰富的制药研究人员， 医学（雷茨）和计算化学（雷诺兹）在福克斯大通化学多样性中心，公司。

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.