Carbocyclic Nucleoside Isosteres as Potential Antitumor

这个项目的主要目标是系统地探索核苷/核苷酸结合酶与一组构象刚性底物的结合，以了解它们首选的结合模式。这是通过在双环3.1.0正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个优选的自然构象(北方或南方)之一，基本上消除了糖部分的固有柔性。该项目的第二个目标是构建含有许多锁定核苷酸单元的修饰核酸，以增强或破坏分别与南、北构象相关的典型的B-DNA或A-DNA构象。1)与去年公布的锁定腺苷类似物(North-MCDA)的5‘-三磷酸一样，新合成的胸腺嘧啶类似物(North-MCT)也非常有效地抑制了多聚酶以外的HIV RT，并对所有通过切除机制发挥作用的HIV耐药株表现出了巨大的有效性。这种方法为克服HIV耐药性提供了一种新的和有前途的方法，在治疗感染HIV的HOS细胞方面取得了成功，这些细胞都是转疱疹病毒激酶基因的。2)合成了一系列与自身互补的EcoR1识别序列DS(5‘-CGAATTCGCG-3’)相对应的短链寡核苷酸(ODN)，其中中间T被锁定的胸苷取代，并用核磁共振、圆二色谱和量热法进行了结构解析。在丝状噬菌体各向异性介质中的核磁共振研究允许测量残留的偶极偶联，这表明添加剂诱导的DNA弯曲与存在的锁定胸苷类似物的数量相称。3)这一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非常有效，人类疱疹病毒8是一种伽马-2疱疹病毒，也是卡波西肉瘤的病原体，两种体外模型预测了对天花的活性。对这两项发现的知识产权保护工作已于今年启动。4)在构建在双环3.1.0正己烷模板上的两个胞嘧啶类似物中，南方类似物是胞苷脱氨酶的首选底物。这一结果与机械上相似的腺苷脱氨酶表现出的偏好相反，后者明显偏爱北腺嘌呤类似物。

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.