Design and Synthesis of HIV Integrase as Potential Anti-AIDS Drugs

针对HIV-1生命周期中两个不同点的抑制剂正在研制中。这些抑制剂旨在作为潜在的新疗法和药理学探针，以研究病毒复制的生化机制。研究的两个领域是：（1）HIV-1整合酶（IN），其中抑制剂可能破坏病毒cDNA掺入宿主基因组;（2）HIV p6Gag蛋白与人Tsg101蛋白的结合，其中抑制剂可能破坏病毒组装和出芽;（1）HIV-1 IN抑制剂：这项工作是与Yves Pompek（CCR，NCI）、Steven Hughes（CCR，NCI）和Peter Cherepanov（帝国理工学院，伦敦）博士合作完成的。已经报道了大量的IN抑制剂。其中许多具有共同的关键结构特征。这些特征包括螯合镁离子的杂原子的共面排列。与抑制剂的螯合部分连接的卤素取代的芳族官能团也已显示与病毒DNA碱基和IN-DNA复合物中的蛋白质之间形成的区域相互作用。这类IN抑制剂被认为通过在IN催化位点内螯合Mg 2+离子而起作用，在IN催化位点中，它们选择性地抑制链转移（ST）反应而不是3-加工（3-P）反应。我们已经开发了2，3-二氢-6，7-二羟基-1H-异吲哚-1-酮和4，5-二羟基-1H-异吲哚-1，3（2H）-二酮，它们是结构简单的IN抑制剂，在Mg2+辅因子的存在下在体外表现出良好的效力和ST选择性。我们基于这些类似物开发新抑制剂的努力受到与原始泡沫病毒（PFV）整合酶的IN-DNA复合物结合的4，5-二羟基-1H-异吲哚-1，3（2H）-二酮的共晶体结构的指导（与Peter Cherepanov博士（帝国学院，伦敦）合作完成）。最近，我们已经制备了2，3-二氢-6，7-二羟基-1H-异吲哚-1-酮平台的含磺酰胺的变体，其在体外测定和使用病毒载体的抗病毒测定中表现出针对HIV-1 IN的低纳摩尔IC 50值。这些数据与Raltegravir相比是有利的，Raltegravir是目前唯一FDA批准的HIV IN抑制剂。然而，这些化合物的潜在治疗效用受到不可接受的细胞毒性的限制，所述细胞毒性可能由嵌入的儿茶酚官能团引起。为了去除这种儿茶酚功能，我们设计了一系列含羟基吡咯并吡啶三酮的类似物。这些化合物的有效合成依赖于亚氨基亚砜的Pummerer环化、去质子化环加成级联反应以及异醌的[3 + 2]环加成反应的应用。尽管这些抑制剂的效力不如原始的2，3-二氢-6，7-二羟基-1H-异吲哚-1-酮，但代表性抑制剂保留了其对三种主要的雷特格韦抗性突变体IN酶G140 S/Q148 H、Y143 R和N155 H的大部分抑制效力。在使用编码这些IN突变体的病毒载体的抗病毒试验中，该化合物对G140 S/Q148 H和Y143 R突变的影响分别比雷特格韦低约200倍和20倍。针对N155H突变，该化合物的影响比雷特格韦小约10倍。后一系列化合物代表了一种新的结构类型，可以进一步开发以克服对雷特格韦的耐药性，特别是在G140 S/Q148 H突变的情况下。(2)Tsg101结合抑制剂：HIV p6Gag蛋白与人Tsg101蛋白的结合已被证明是病毒出芽所必需的，并且涉及p6蛋白的关键9-mer P-E-P-T-A-P-P-E-E序列。在与Eric Freed博士（CCR，NCI，NIH）的合作中，我们正在制备这种9聚体序列的肽和肽模拟变体，作为Tsg101结合拮抗剂，可能导致一类新的病毒出芽抑制剂。指导我们的方法的统一原则是将氨基-氧基官能团掺入肽残基中，所述肽残基可以在单个步骤中容易地官能化以提供肟衍生物的文库。这种方法导致了几个低微摩尔亲和力Tsg101结合拮抗剂的鉴定。通过基于与Tsg101蛋白结合的我们的高亲和力抑制剂的共晶体结构的计算机建模来指导进一步的肟库多样化。该结构是通过与James Hurley博士（NIDDK，NIH）合作获得的。荧光标记已被引入到高亲和力结合拮抗剂之一中，以产生一种试剂，该试剂将用于在Doug Auld博士的指导下在NIH化学基因组学中心通过小化合物文库的高通量筛选来鉴定Tsg101结合拮抗剂。在平行的工作中，Tsg101与肽配体相互作用的化学生物学正在探索使用构象约束的肽大环。这些通过肽-类肽前体上的闭环复分解反应形成。与母体开链肽相比，这些大环化合物表现出更高的Tsg101结合亲和力和更好的细胞生物利用度。

Inhibitors directed against two distinct points in the HIV-1 life cycle are being prepared. These inhibitors are intended to serve as potential new therapeutics and as pharmacological probes to investigate biochemical mechanisms of viral replication. The two areas of investigation are: (1) HIV-1 integrase (IN), where inhibitors may disrupt incorporation of viral cDNA into the host genome; (2) Binding of HIV p6Gag protein to human Tsg101 protein, where inhibitors may disrupt viral assembly and budding (1) HIV-1 IN inhibitors: This work is being done in collagoration with Drs. Yves Pommier (CCR, NCI) Steven Hughes (CCR, NCI) and Peter Cherepanov (Imperial College, London). A large number of IN inhibitors have been reported. Many of these exhibit common key structural features. These features include a co-planar arrangement of heteroatoms that chelate magnesium ions. Halogen-substituted aromatic functionality linked to the chelating portion of the inhibitors has also been shown to interact with a region formed between a viral DNA base and the protein in the IN-DNA complex. This class of IN inhibitors is thought to function by chelating Mg2+ ions within the IN catalytic site, where they selectively inhibit strand transfer (ST) reactions over 3-processing (3-P) reactions. We have developed 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one and 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones, which are structurally simple IN inhibitors that exhibit good potency and ST selectivity in vitro in the presence of Mg2+ cofactor. Our efforts to develop new inhibits based on these analogues are being guided by co-crystal structures of our 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones bound to the IN-DNA complex of the primitive foamy virus (PFV) integrase (done in collaboration with Dr. Peter Cherepanov (Imperial Colleage, London). Most recently, we have prepared sulfonamide-containing variants of the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one platform that exhibit low nanomolar IC50 values against HIV-1 IN in in vitro assays, and in antiviral assays employing viral vectors. These data compare favorably to Raltegravir, the only current FDA-aproved HIV IN inhibitor. However, the potential therapeutic utility of these compounds is limited by unacceptable cytotoxicity that may arise from embedded catechol functionality. In order to remove this catechol functionality, we designed a series of hydroxy-pyrrolopyridine-trione-containing analogues. The efficient syntheses of these compounds relies on the application of Pummerer cyclization deprotonation cycloaddition cascades of imidosulfoxides as well as [3+2] cycloadditions of isomnchnones. Although the potency of these inhibitors was not as great as the original 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-ones, a representative inhibitor retained most of its inhibitory potency against the three major raltegravir-resistance mutant IN enzymes, G140S/Q148H, Y143R and N155H. In antiviral assays employing viral vectors coding these IN mutants, compound this compound was approximately 200-fold and 20-fold less affected than raltegravir against the G140S/Q148H and Y143R mutations, respectively. Against the N155H mutation the conpound was approximately 10-fold less affected than raltegravir. This latter series of compounds represent a novel structural class that may be further developed to overcome resistance to raltegravir, particularly in the case of the G140S/Q148H mutations. (2) Tsg101-binding inhibitors: Binding of the HIV p6Gag protein to human Tsg101 protein has been shown to be necessary for viral budding and to involve a critical 9-mer P-E-P-T-A-P-P-E-E sequence of the p6 protein. In a collaboration with Dr. Eric Freed (CCR, NCI, NIH) we are preparing peptide and peptide mimetic variants of this 9-mer sequence as Tsg101-binding antagonists that may lead to a new class of viral budding inhibitors. A unifying principal guiding our approach is the incorporation of amino-oxy functionality into peptide residues that can be easily functionalized in a single step to provide a library of oxime derivatives. This approach has resulted in the identification of several low micromolar affinity Tsg101 binding antagonists. Further oxime library diversification is being guided by in silico modeling based on a co-crystal structure of our high affinity inhibitor bound to Tsg101 protein. This structure was obtained through a collaboration with Dr. James Hurley (NIDDK, NIH). Fluorescent labeling has been introduced into one of the high affinity-binding antagonists to produce a reagent that will be used for identification of Tsg101-binding antagonists by high throughput screening of small compound libraries in the NIH Chemical Genomics Center under the direction of Dr. Doug Auld. In parallel work, the chemical biology of Tsg101 interactions with peptide ligands is being explored using conformationally-constrained peptide macrocycles. These were formed by ring-closing metathesis reactions on peptide-peptoid precursors. These macrocycles exhibit higher Tsg101-binding affinity and better cellular bioavailability than the parent open-chain peptides.