Combined Substrate Polymerase Inhibitors

描述(申请人提供)：DNA聚合酶和逆转录酶的联合底物抑制剂被研究为治疗病毒性疾病(如肝炎、艾滋病等)的更特异和毒性更低的治疗方法的潜在途径，可能是为了改进目前的癌症化疗，也可能是为了寻找暴露于生物恐怖生物(包括病毒和细菌)的一线治疗方法。这类新的抑制剂是基于两种抑制剂探针，即DNA聚合酶抑制剂2-S-4-叠氮苯酰基-2‘-脱氧腺苷-5’-三磷酸(1)和RT抑制剂2-S-4-叠氮苯基-硫代-1，N6-亚乙基-2‘-脱氧腺苷-5’-三磷酸(2)。这两个结构类别(第三个探针不包括在本项目中)一起包围了腺苷碱基的小凹槽一侧周围的整个空间，当三磷酸与催化三联体配位时，将侧链放置在适当的位置以与模板结合位点相互作用。这些抑制剂将在多种酶上进行测试，包括HIV-1、M-MLV、AMV和Rav2逆转录酶，大肠杆菌DNA聚合酶I、T4、T7和Taq DNA聚合酶，以及DNA依赖的RNA聚合酶和末端转移酶，全面描述与合成和天然模板/引物和所有dNTPs相比的抑制机理。对于侧链长度增加和减少的两种抑制剂类型，将生成一个文库，然后通过苯环替换或环烷基环替换，该库中确定的每个酶的特定抑制剂将进一步多样化。这一建议是基于这样的假设，即尽管前导1、2和3的结合底物抑制剂性质将在整个聚合酶中保持，但特定的抑制性质将取决于单个聚合酶，从而允许识别非常特定的抑制模式和抑制剂结合部位。

DESCRIPTION (provided by applicant): Combined substrate inhibitors of DNA polymerases and reverse transcriptases are investigated as potential avenues to more specific and less toxic therapies for treating viral diseases, e.g., hepatitis, AIDS, etc., perhaps to improve current cancer chemotherapies, and perhaps to find frontline treatments for exposure to bio-terror organisms, including viruses and bacteria. These new classes of inhibitors are based on two inhibitor probes, i.e., the DNA polymerase inhibitor 2-S-4-azidophenacyl-thio-2'- deoxyadenosine 5'-triphosphate (1) and the RT inhibitor 2-S-4-azidophenacyl-thio-1, N6-etheno-2 '- deoxyadenosine 5'-triphosphate (2). These two structural classes (with a third probe not included in this project) together envelop the entire space surrounding the minor groove side of the adenosine base, placing the side chain in position to interact with template binding sites when the triphosphate is coordinated to the catalytic triad. These inhibitors will be tested on a large variety of enzymes, including HIV-1, M-MLV, AMV, and RAV2 reverse transcriptase, E. coli DNA polymerase I, T4, T7 and Taq DNA polymerases, and DNA-dependent RNA polymerase and terminal transferase for selectivity, with complete characterization of inhibition mechanism versus synthetic and natural template/primers and all dNTPs. A library will be generated for the two-inhibitor types where the length of the side chain is increased and decreased, and then specific inhibitors for each enzyme identified in this library will be further diversified by phenyl ring substitutions or replacement with cycloalkyl rings. This proposal is based on the hypothesis that although the combined substrate inhibitor properties of leads 1, 2, and 3 will be maintained across the polymerases, specific inhibitory properties will depend on individual polymerases, thus allowing identification of very specific inhibition patterns and inhibitor binding sites.