Rational design of indole compounds as HIV fusion inhibitors

描述(由申请人提供)：基于羧基取代吲哚支架的低分子HIV融合抑制剂的开发被提出。早期的先导化合物表现出与HIV-1 gp41卷曲线圈上的深疏水空腔的良好结合亲和力，与对细胞-细胞和病毒细胞融合的抑制活性相关。成功地抑制gp41为艾滋病毒的初始感染提供了一种普遍的屏障，并已被证明通过防止感染扩散到旁观者细胞，对感染者具有重要的免疫保护作用。目前，融合抑制剂仅限于来源于gp41C-7重复序列的多肽，虽然高效，但有药理限制，如给药途径、半衰期和成本。抑制艾滋病毒融合的小分子一直是人们追逐的目标，但在这一领域难以实现的目标和成功将标志着现有治疗方法的重大改进以及在杀微生物剂配方中预防的可能性。我们计划合成50-100个新化合物，以破译抑制gp41 N-C肽相互作用中的蛋白质-蛋白质相互作用所需的药效团，依靠相对简单的化学对基本支架进行修饰。我们有特定的生化分析来确定我们的化合物与gp41中已知疏水空腔的亲和力，并将在存在或不存在添加剂(如血清、精液和宫颈液)的情况下，使用细胞-细胞和病毒细胞融合分析将结合活性与生物活性关联起来。我们还为一种新的确定结合配体结构的核磁共振方法奠定了基础，该方法将被扩展用于本提案中开发的高亲和力配体。从一个具有55M结合亲和力和95M融合抑制IC50的起始化合物，我们合成了几个下一代抑制剂，活性提高了10倍。我们的目标是获得至少比领先水平提高100倍的抑制剂，具有融合抑制作用的100 nm IC50。因此，我们的具体目标是：1)利用我们目前对构效关系的理解来设计一套新的含有吲哚的抑制剂，并使用结合、细胞-细胞融合和病毒-细胞融合实验来确认HIV的活性、机制和特异性；2)开发核磁共振程序，获得用于确定结合配体的计算结构的实验限制条件，以帮助合理设计改进的抑制剂。这个分子文库的构建和测试有望产生低分子量抑制剂，具有极大的抗HIV融合的生物活性和明确的作用机制。这种化合物可以被开发成廉价的非肽融合抑制剂，这将彻底改变全球每年发生的数百万新感染的治疗选择和预防措施。 与公共卫生相关：艾滋病毒感染的流行仍然是一个重大的公共卫生问题，这是由于对当前治疗方法产生抗药性的病毒株的发展，以及由于病毒从不知道自己被感染的个人身上传播。这项提议寻求应用结构辅助药物设计来开发低分子HIV融合抑制剂，以防止病毒进入和导致免疫缺陷的细胞间传播。这类药物将在杀菌剂中用于预防感染，或者可以口服治疗感染，作为目前使用的融合抑制剂Enfuvitid(R)的替代药物，后者必须静脉给药。

DESCRIPTION (provided by applicant): Development of low molecular weight HIV fusion inhibitors based on a carboxybenzyl-substituted indole scaffold is proposed. Early lead compounds show promising binding affinity to a deep hydrophobic cavity on the coiled coil of HIV-1 gp41, correlated with inhibitory activity against cell-cell and viral cell fusion. Successful inhibition of gp41 provides a universal barrier to initial infection by HIV, and has been shown to have important immune-protective effects in an infected individual by preventing the spread of infection to bystander cells. Currently, fusion inhibitors are limited to peptides derived from the gp41 C-heptad repeat, which, while highly efficacious, have pharmacological limitations such as route of administration, half-life and cost. Small molecule inhibition of HIV fusion has been a long sought after but elusive goal and success in this area would mark a significant improvement in available therapies as well as potential for prophylaxis in a microbicidal formulation. We plan synthesis of 50 - 100 new compounds to decipher the pharmacophore required to inhibit the protein - protein interaction involved in the gp41 N - C-peptide interaction, relying on relatively simple chemistry to make modifications to the basic scaffold. We have specific biochemical assays to determine affinity of our compounds for the known hydrophobic cavity in gp41, and will correlate binding activity with biological activity using cell-cell and viral cell fusion assays in the presence or absence of additives such as serum, seminal and cervical fluid. We have also developed the foundation for a novel NMR method for structure determination of bound ligands, which will be expanded for use with the high affinity ligands being developed in this proposal. From a starting compound with 55M binding affinity and 95M IC50 for fusion inhibition, we have synthesized several next generation inhibitors, and have obtained a 10-fold improvement in activity. Our goal is to obtain inhibitors that are at least 100-fold improved over the lead, having < 100nM IC50 for fusion inhibition. Thus our specific aims are 1) to utilize our current understanding of structure-activity relationships to design a new set of indole containing inhibitors, and to use binding, cell-cell fusion and viral-cell fusion assays to confirm activity, mechanism and specificity for HIV; 2) to develop NMR procedures to obtain experimental restraints for computational structure determination of bound ligands, as an aid to the rational design of improved inhibitors. Construction and testing of this molecular library promises to result in low molecular weight inhibitors with greatly improved biological activity against HIV fusion and with a well-defined mechanism of action. Such compounds could be developed into inexpensive non-peptide fusion inhibitors, which would revolutionize both treatment options and prophylaxis for the millions of new infections that occur worldwide annually. PUBLIC HEALTH RELEVANCE: The prevalence of HIV infection remains a significant public health problem due to the development of viral strains resistant to current treatments, and due to spread of the virus from individuals who are unaware they are infected. This proposal seeks to apply structure-aided drug design to develop low molecular weight HIV fusion inhibitors which prevent viral entry and the cell-to-cell spread that causes immune deficiency. Drugs in this class would be useful in microbicides to prevent infection, or could be given orally to treat infection as an alternative to the currently used fusion inhibitor Enfuvirtide(R), which must be given intravenously.