Three-Dimensional HIV RNA Genome Targeted Drug Leads

描述（由申请人提供）：该项目的总体目标是使用新的药物设计范式开发有前途的抗艾滋病药物先导。使用基本RNA元素作为开发小型治疗药物的靶标是一种很少使用的策略，尽管有先例表明许多抗生素是通过结合rRNA上的特定位点起作用的。我们的实验室和其他实验室已经确定了一些关键的HIV-1 RNA基因组元件的3D结构，这些元件可以作为计算筛选可用化合物数据库的目标。对于RNA靶向，我们继续开发两种对接程序，DOCK和MORDOR，后者非常新颖，因为它允许假设的配体和靶标具有灵活性，以实现真正的诱导配合；这种灵活的拟合对蛋白质靶标的虚拟筛选很有用，但对RNA靶标非常重要，因为它们通常具有相当的延展性。候选化合物是水溶性、非肽、非核苷酸的有机化合物，通常分子量<500道尔顿，电荷不超过+2（一般为0或+1）。计算“命中”将通过核磁共振和表面等离子体共振与RNA靶结合而不是脱靶RNA对照进行实验测试。那些结合的将作为相似搜索的模型，以识别其他潜在的结合物。核磁共振将用于绘制RNA上的结合位点，并测量使用两个对照RNA的最佳命中的Kd值，以确定选择性。核磁共振将用于模拟最有前途的粘合剂的结构；这样的结构将定义后续有机合成的支架（这里没有明确提出），以提高亲和力和选择性。在这个简短的研究项目中，我们建议继续进行所需的方法开发。对新目标的应用将说明该方法的实用性，并有望获得有希望的命中结果。主要靶点是HIV-1 RNA基因组5'-UTR包装序列中茎环1 （SL1）的富含g的内环（GRIL），我们发现它基本上是100%保守的（这表明可能避免耐药性）。更重要的是，人类的rRNA和trna没有任何类似于GRIL的结构基序。GRIL对于将基因组包装成新的感染性病毒粒子至关重要，但考虑到5'UTR其他部分缺乏序列保守性，它可能还发挥着另一个未知的关键作用。我们已经确定了SL1的结构，将用于靶向。由于我们已经确定了与包装位点结合的另一种配体，研究将集中在我们对该配体的了解上，试图确定HIV-1复制的抑制剂。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop promising anti-AIDS drug leads using a new drug design paradigm. Using essential RNA elements as targets for developing small therapeutic drugs is a little-used strategy, despite the precedent that many antibiotics work by binding specific sites on rRNA. Our lab and others have determined 3D structures of some crucial HIV-1 RNA genome elements that can serve as targets for computational screening of databases of available compounds for binding. For RNA targeting, we continue to develop two docking programs, DOCK and MORDOR, the latter quite novel in that it permits flexibility in both putative ligand and target for a true induced fit; such flexible fitting is useful for virtual screening of protein targets but is very important for RNA targets, as they are typically quite malleable. Candidate compounds are water-soluble, nonpeptide, nonnucleotide organic compounds generally with molecular weight <500 daltons and a charge of no more than +2 (generally 0 or +1). Computational "hits" will be tested experimentally using NMR and surface plasmon resonance for binding to the RNA target but not off-target RNA controls. Those that bind will serve as models for similarity searches to identify other potential binders. NMR will be used to map the binding site on the RNA and to measure Kd values for the best hits using two control RNAs to determine selectivity. NMR will be used to model the structure of the most promising binders; such structures will define scaffolds for subsequent organic synthesis (not explicitly proposed here) to enhance affinity and selectivity. In this short research project, we propose a continuation of needed methodology development. Application to a novel target will illustrate the utility of the methodology with the prospect that promising hits will result. The primary target is the G-rich internal loop (GRIL) of stem-loop 1 (SL1) in the packaging sequence in the 5'-UTR of the HIV-1 RNA genome, which we discovered to be essentially 100% conserved (suggesting the possibility to avert drug resistance). More importantly, human rRNA and tRNAs do not have any structural motifs similar to GRIL. GRIL is critical for packaging of the genome into new infectious virions but, considering the lack of sequence conservation in other parts of the 5'UTR, it may play another unknown crucial role as well. We have determined the structure of SL1, which will be used for targeting. As we have identified another ligand that binds to the packaging site, studies focused on our knowledge of that ligand will attempt to identify inhibitors of HIV-1 replication. PUBLIC HEALTH RELEVANCE: Screening a large database of drug-like compounds by computer for binding to a 3D RNA or protein structure important for HIV-1 activity followed by experimental verification of the top predicted compounds should be a fast, cost effective way to find compounds that might become drug leads. We have developed software for screening that is a significant improvement over existing software, especially for targeting crucial sites on RNA, which has largely been ignored compared with the typical protein targets. We propose to improve the software and to use it to find promising compounds that bind to an important conserved portion of the RNA genome of HIV-1, where the sequence conservation suggests evolution of drug resistance will be minimized, and ultimately to inhibit HIV-1 replication.