Carbocyclic Nucleoside Isosteres as Potential Antitumor

该项目的主要目标是系统地探测核苷/核苷酸结合酶与构象刚性底物的集合，以了解它们的首选结合模式。这是通过在双环3.1.0己烷模板上构建碳环核苷类似物来实现的，该模板通过将糖的构象锁定在两种优选的天然构象（北或南）之一中而基本上去除了糖部分的固有柔性。该项目的第二个目标是构建修饰的核酸，其掺入许多锁定的核苷酸单元以分别增强或破坏与南构象和北构象相关的典型B-或A-DNA构象。主要发现是：1）与去年公开的锁定腺苷类似物（North-MCdA）的5'-三磷酸一样，新合成的胸腺嘧啶类似物（North-MCT）也非常有效地抑制聚合酶位点以外的HIV RT，并对所有通过切除机制发挥作用的HIV耐药株表现出极大的有效性。这种方法，这提供了一种新的和有前途的方法来克服艾滋病毒的耐药性，是成功的治疗艾滋病毒感染的HOS细胞转染疱疹激酶基因。2）合成了一系列与EcoR 1识别序列ds（5'-CGCGAATTCGCG-3 '）相对应的短寡脱氧核苷酸（ODNs），其中中间的T被锁胸苷取代，并通过NMR、CD和量热法对其结构进行了解析。在丝状噬菌体各向异性介质中的NMR研究允许测量残留的偶极耦合，这表明添加剂诱导的弯曲的DNA与锁定的胸苷类似物的数量相当。3)该系列中最令人兴奋的化合物North-MCT被发现对人类疱疹病毒8（一种γ-2疱疹病毒和卡波西肉瘤的病原体）非常有效，并且两种体外模型预测了对天花的活性。今年启动了对这两项发现的知识产权保护。4)在以双环3.1.0己烷为模板构建的胞嘧啶的两个锁定类似物中，South类似物是胞苷脱氨酶的首选底物。该结果与机理相似的腺苷脱氨酶所显示的偏好相反，其中明显偏好北腺嘌呤类似物。

The main objective of this project has been the systematic probing of nucleoside/nucleotide binding enzymes with sets of conformationally rigid substrates to learn about their preferred binding mode. This has been accomplished by constructing carbocyclic nucleoside analogues on a bicyclo3.1.0hexane template which essentially removes the inherent flexibility of the sugar moiety by locking the conformation of the sugar in one of the two preferred natural conformations (North or South). A secondary objective of this project has been the construction of modified nucleic acids that incorporate a number of locked nucleotide units to either reinforce or disrupt the typical B- or A-DNA conformations associated with South and North conformations, respectively. The major findings are:1) As with the 5'-triphosphate of the locked adenosine analogue (North-MCdA) disclosed last year, the newly synthesized thymine analogue (North-MCT) was also very effective in inhibiting HIV RT beyond the polymerase site and showed great effectiveness against all HIV-resistant strains that function by the excision mechanism. This approach, which offers a novel and promising way to overcome HIV resistance, was successful in treating HIV-infected HOS cells that were transfected with the herpes kinase gene. An effective mean to deliver the monophosphate pro-drug of either the adenine or thymine analogues are being investigated to avoid the use of gene transfection.2) A series of short oligodeoxynucleotides (ODNs) corrresponding to the self-complementary EcoR1 recognition sequence ds(5'-CGCGAATTCGCG-3') where the middle T's were replaced by locked thymidines were synthesized and the structures solved by NMR, CD and calorimetry. NMR studies in a filamentous phage anisotropic medium permitted the measurement of residual dipolar couplings which demonstrated an additive-induced bending of the DNA commensurate with the number of locked thymidine analogues present. 3) The most exciting compound of this series, North-MCT was found to be very active against human herpesvirus 8, a gamma-2 herpesvirus and causative agent of Kaposi's sarcoma, and two in vitro models predictive of activity against small-pox. Intellectual protection on these two findings was initiated this year. 4) Of the two locked analogues of cytosine built on a bicyclo3.1.0hexane template, it was the South analogue the preferred substrate for cytidine deaminase. This result is opposite to the preference shown by the mechanistically similar adenosine deaminase enzyme where the preference is clearly for the North adenine analogue.