Optimization of dipeptide-linked benzimidazole topoisomerase 1 poisons

描述(申请人提供)：抑制拓扑异构酶I(Top1)活性的喜树碱(CPT)衍生物已被证明在各种癌症的治疗中具有临床实用价值。然而，CPT药物性质的限制促使人们寻找针对Top1蛋白-DNA切割复合体的新的CPT样毒剂。我们假设，同时与Top1蛋白的DNA小沟和鼻锥区结合的双功能制剂将是具有优于CPT同系物的药学特性的有效毒物。这些化合物还应该对对CPT结构类毒药产生抗药性的肿瘤有效，并且通常与CPT类药物(如拓扑替康)相比，毒性应该较低。从TOP1DNA晶体结构中获得的结构信息被用来设计一个连接到脱氧核糖核酸小槽结合蛋白Hoechst 33258的N-乙酰二肽的小文库。对文库进行体外实验筛选和随后的文库去卷积得到了高亲和力(EC50~50 NM)的试剂LL217，它也导致了几种肿瘤细胞系的生长停滞。随后用苯基取代的单苯并咪唑取代Hoechst，提供了具有与LL217相似的肿瘤细胞系杀伤活性的试剂MB1，但大大减少了非靶向效应。对LL217和MB1进行的生化分析表明，肿瘤细胞系的主要作用方式是由于抑制Top1活性而导致细胞周期停滞。我们的长期目标是将MB1转化为临床有效的药物。我们的近期目标是生产一种精制的铅化合物，具有足够的组织培养和小鼠异种移植数据，以支持进一步的开发。近期的目标将通过四个具体目标来实现：(1)MB1的结构将被略微修改以便于溶液相合成，(2)MB1衍生物的体外和细胞活性将被检测并与Topotecan进行比较，(3)MB1的潜在治疗效果将在小鼠移植瘤模型中与Topotecan进行比较，以及(4)MB1和Topotecan的药学特性将被评估。公共卫生相关性：癌症最近已超过心血管疾病，成为美国发病率和死亡率的主要原因。虽然需要新类型的化疗药物，但也需要开发具有更好的药学性能的药物，以临床验证的靶点为靶点。临床上使用的喜树碱类药物专门针对拓扑异构酶1酶。然而，它们的溶解性和稳定性较差，需要使用大剂量，这增加了对患者产生不良反应的机会。这里描述的药物也针对拓扑异构酶1，但它们有望具有更高的生物利用度，因此，提供了对喜树碱类药物的改进。由于这些药物在结构上也是不同的，预计它们将在临床上对对喜树碱产生抗药性的癌症有效。

DESCRIPTION (provided by applicant): Derivatives of camptothecin (CPT) that inhibit Topoisomerase I (Top1) activity have demonstrated clinical utility in the treatment of various cancers. However, limitations in CPT pharmaceutical properties have driven the search for new CPT-like poisons that target the Top1 protein-DNA cleavage complex. We hypothesized that bifunctional agents that simultaneously bind to the DNA minor groove and the nose-cone region of the Top1 protein would be effective poisons with pharmaceutical properties that are superior to the CPT congeners. Such compounds also should be effective against tumors that are otherwise resistant to the CPT structural class of poisons and should generally exhibit reduced toxicity compared to the CPT- class of agents such as topotecan. Structural insights from Top1-DNA crystal structures were used to design a small library of N-acetyl dipeptides linked to Hoechst 33258, a DNA minor groove binder. Screening of the library against a in vitro assays and subsequent library deconvolution afforded a high affinity (EC50 ~ 50 nM) agent, LL217, that also caused growth arrest of several tumor cell lines. Subsequent replacement of the Hoechst with a phenyl- substituted monobenzimidazole provided the agent MB1 that exhibits tumor cell line killing activity similar to LL217 but with substantially reduced off-target effects. Biochemical assays performed with LL217 and MB1 demonstrate that the primary modality of action in tumor cell lines is cell cycle arrest due to inhibition of Top1 activity. Our long-term goal is to translate MB1 into a clinically efficacious drug. Our immediate goal is to produce a refined lead compound with sufficient tissue culture and murine xenograft data to support further development. The immediate goals will be achieved via four specific aims: (1) the structure of MB1 will be slightly modified to facilitate solution phase syntheses, (2) the in vitro and cellular activities of the MB1 derivatives will be assayed and compared to topotecan, (3) the potential therapeutic efficacy of MB1 will be evaluated in murine xenograft tumor models as compared to topotecan, and (4) the pharmaceutical properties of MB1 and topotecan will be evaluated. PUBLIC HEALTH RELEVANCE: Cancer has recently surpassed cardiovascular disease as a major cause of morbidity and mortality in the United States. Although new classes of chemotherapeutic agents are needed, it is also desirable to develop agents with improved pharmaceutical properties that target clinically validated targets. The camptothecin class of agents used in the clinic specifically target the Topoisomerase 1 enzyme. However, their poor solubilities and stabilities require the use of large doses, which increases the chances for adverse effects to the patient. The agents described here also target Topoisomerase 1, but they are expected to be more bioavailable and, thus, offer an improvement to the camptothecin class of agents. Because these agents are also structurally distinct, it is expected that they will be clinically efficacious against cancers that have developed resistance to the camptothecins.