Targeting RNA conformation for drug development

描述（由申请人提供）：传染病研究的挑战之一是找到方法，利用对疾病转化和进展机制的日益增长的知识来开发艾滋病的新治疗策略。靶向参与HIV-1增殖和存活的特异性RNA-蛋白质相互作用，如Tat-TAR或Rev-RRE，是一种有前途的方法。我们的初步结果显示了新配体稳定TAR RNA、在纳摩尔浓度下抑制Tat-TAR相互作用和抑制MT-2细胞中的HIV-1的能力。现在将建立这些初步结果以开发靶向Tat-TAR相互作用的缀合物文库，其以高亲和力和特异性结合TAR。拟议的研究将进一步帮助确定这种方法的有效性。这里提出的工作，一个多学科的努力，包括有机合成，生物物理化学和HIV发病机制描述了小分子介导的抑制Tat-TAR抑制剂作为HIV-1治疗剂的发展。这项工作的成功将是对目前艾滋病治疗和RNA靶向中可用的蛋白质特异性方法的重要补充。我们提出使用31 nt TAR靶序列来设计可用于抑制Tat-TAR相互作用的缀合物;为开发小分子RNA靶向HIV-1治疗剂打开了可能性。 公共卫生相关性：几十年来对RNA结构的研究表明，它是一个既定的药物靶点，众所周知是小分子抗生素的受体。尽管自20世纪40年代发现链霉素以来，细菌核糖体一直是抗生素阻断蛋白质合成的众所周知的受体，但迫切需要新的抗菌和抗病毒方法来对抗耐药性，这严重限制了当前抗生素的有效性。 为了研究基于小分子的特异性结合电荷/形状互补性的优点，我们已经启动了一个程序，开发了一种使用多聚体配体（由具有独立结合位点的配体组成）的方法，该方法可用于靶向特定的RNA。该提案的重点是小分子氨基糖（新霉胺）共轭物的发展，作为这种方法的一个例子。鉴定多种病原体中的基本药物靶标的综合方法可以与我们开发以特定方式与先前已知以及快速鉴定的新RNA靶标高亲和力结合的小分子的互补方法相结合。 达特/TAR相互作用的抑制促进HIV RNA转录，随后阻止HIV复制。本申请的中心假设是具有独立结合位点的两个配体的缀合可以与适当的接头缀合以提供高亲和力TAR特异性配体，其能够在纳摩尔浓度下抑制达特/TAR相互作用。此外，该测定适用于基于RNA的药物发现，其中可以组合具有独立结合位点的两个药效团以选择高亲和力配体。最终，TAR结合配体的发现与目前可用的分子相比具有改善的亲和力和特异性，这将为在对抗HIV的斗争中实施新靶标的潜在用途提供更好的理解。NUBAD有很好的设备来合成分子并进行抑制的生物物理测定。将检测从试验中鉴定的在纳摩尔Kd下抑制tat-TAR相互作用的选定化合物对HIV的抑制作用。

DESCRIPTION (provided by applicant): One of the challenges of research in infectious diseases is to find ways to use the increasing knowledge of the mechanisms underlying disease transformation and progression to develop novel therapeutic strategies for AIDS. Targeting specific RNA- protein interactions, such as Tat-TAR or Rev-RRE, which are involved in proliferation and survival of HIV-1 is a promising approach. Our preliminary results show the ability of novel ligands to stabilize TAR RNA, inhibit Tat-TAR interaction at nanomolar concentrations and inhibit HIV-1 in MT-2 cells. These preliminary results will now be built upon to develop a library of conjugates to target Tat-TAR interaction that bind with high affinity and specificity to TAR. Proposed studies will further help establish the efficacy of this approach. The work proposed here, a multidisciplinary effort encompassing organic synthesis, biophysical chemistry and HIV pathogenesis describes the development of small molecule mediated inhibition of Tat-TAR inhibitors as HIV-1 therapeutics. The success of the proposed work would be a significant addition to currently available protein- specific approaches in AIDS therapy and RNA targeting. We propose using a 31 nt TAR target sequences to design conjugates that can be employed to inhibit Tat-TAR interaction; opening possibilities for developing small molecule RNA targeted HIV-1 therapeutics. PUBLIC HEALTH RELEVANCE: Several decades of research on the RNA structure has shown it to be an established drug target, well known as a receptor for small molecule antibiotics. Though the bacterial ribosome has been a well known receptor for antibiotics blocking protein synthesis since the discovery of streptomycin in the 1940s, new antibacterial and antiviral approaches are urgently needed to combat drug resistance, which severely limits the effectiveness of current antibiotics. To investigate the advantage of small molecule-based specificity coupled with charge/shape complementarity, we have initiated a program in the development of a approaches using multimeric ligands (consisting of ligands with independent binding sites) that can be used to target a specific RNA. This proposal focuses on the development of small molecule aminosugars (neamine) conjugates as an example of this approach. A comprehensive approach to identifying essential drug targets in multiple pathogens can be combined with our complementary approach of developing small molecules that bind with high affinity in a specific fashion to previously known as well as rapidly identified, new RNA targets. The inhibition of the Tat/TAR interaction, which facilitates HIV RNA transcription subsequently arrests HIV replication. The central hypothesis of this application is that conjugation of two ligands with an independent binding sites can be conjugated with an appropriate linker to provide a high affinity TAR specific ligand, capable of inhibiting the Tat/TAR interaction at nanomolar concentrations. Furthermore, the assay is applicable to RNA based drug discovery where two pharmacophores with independent binding sites can be combined to select a high affinity ligand. Ultimately, the discovery of a TAR binding ligand with improved affinity and specificity over currently available molecules will provide a better understanding for the potential use of a novel target for implementation in the fight against HIV. NUBAD is well equipped to synthesize the molecules and carry out the biophysical assays for inhibition. Select compounds identified from the assay that inhibit tat-TAR interaction at nanomolar Kd will be tested for inhibition of HIV.