Rational design of indole compounds as HIV fusion inhibitors

描述（由申请方提供）：拟开发基于羧苄基取代吲哚支架的低分子量HIV融合抑制剂。早期的先导化合物显示出对HIV-1 gp 41卷曲螺旋上的深疏水腔的有希望的结合亲和力，与对细胞-细胞和病毒细胞融合的抑制活性相关。成功抑制gp 41为HIV初始感染提供了一个通用屏障，并已被证明通过防止感染扩散到旁观者细胞而在感染个体中具有重要的免疫保护作用。目前，融合抑制剂限于衍生自gp 41 C-七肽重复序列的肽，其虽然高度有效，但具有药理学限制，例如给药途径、半衰期和成本。HIV融合的小分子抑制一直是一个长期追求但难以实现的目标，在这一领域的成功将标志着可用疗法的显著改进以及在杀微生物制剂中预防的潜力。我们计划合成50 - 100种新化合物，以破译抑制参与gp 41 N-C-肽相互作用的蛋白质-蛋白质相互作用所需的药效团，依赖于相对简单的化学方法对基本支架进行修饰。我们有特定的生物化学测定来确定我们的化合物对gp 41中已知的疏水腔的亲和力，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测定将结合活性与生物活性相关联。我们还开发了一种新的NMR方法的结合配体的结构测定的基础，这将扩大使用的高亲和力的配体正在开发的这一建议。从具有55 M结合亲和力和95 M融合抑制IC 50的起始化合物，我们已经合成了几种下一代抑制剂，并获得了10倍的活性改善。我们的目标是获得比先导化合物提高至少100倍的抑制剂，其融合抑制的IC 50 <100 nM。因此，我们的具体目标是：1）利用我们目前对结构-活性关系的理解来设计一组新的含吲哚的抑制剂，并使用结合、细胞-细胞融合和病毒-细胞融合试验来确认对HIV的活性、机制和特异性; 2）开发NMR程序以获得用于结合配体的计算结构测定的实验约束，作为改进的抑制剂的合理设计的辅助。该分子库的构建和测试有望产生低分子量抑制剂，其对HIV融合的生物活性大大提高，并且具有明确的作用机制。这些化合物可以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。 公共卫生相关性：艾滋病毒感染的流行仍然是一个重大的公共卫生问题，这是由于病毒株的发展对目前的治疗产生了抗药性，以及由于病毒从不知道自己被感染的个人传播。该提案旨在应用结构辅助药物设计来开发低分子量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。这类药物可用于杀微生物剂以预防感染，或者可以口服治疗感染，作为目前使用的融合抑制剂Enfuvirtide（R）的替代品，Enfuvirtide（R）必须静脉注射。

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.