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准备中。这些抑制剂有望作为潜在的新疗法和药理学探针来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），其抑制剂可能会破坏病毒cDNA与宿主基因组的结合；(2) HIV p6Gag蛋白与人类Tsg101蛋白的结合，其抑制剂可能会破坏病毒的组装和出芽(1)HIV-1 IN抑制剂：这项工作正在与dr。Yves Pommier (CCR， NCI) Steven Hughes （CCR， NCI）和Peter Cherepanov（伦敦帝国理工学院）。大量的IN抑制剂已被报道。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共面排列。与抑制剂螯合部分相连接的卤素取代芳族功能也被证明与in -DNA复合体中病毒DNA碱基和蛋白质之间形成的区域相互作用。这类IN抑制剂被认为是通过在IN催化位点螯合Mg2+离子起作用，在那里它们选择性地抑制链转移（ST）反应而不是3-加工（3-P）反应。我们开发了2,3-二氢-6,7-二羟基- 1h -异吲哚-1- 1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+辅助因子存在下，在体外表现出良好的效价和ST选择性。我们与Peter Cherepanov博士（伦敦帝国学院）合作，开发了基于这些类似物的新抑制剂，这些抑制剂是基于我们与原始泡沫病毒（PFV）整合酶的in - dna复合体结合的4,5-二羟基- 1h -异吲哚-1,3(2H)-二酮的共晶结构。最近，我们制备了含有磺胺的2,3-二氢-6,7-二羟基- 1h -异吲哚-1- 1平台的变体，在体外试验和采用病毒载体的抗病毒试验中对HIV-1 IN表现出低纳摩尔IC50值。这些数据与目前唯一获fda批准的HIV IN抑制剂雷替格拉韦（Raltegravir）相比具有优势。然而，这些化合物的潜在治疗效用受到嵌入儿茶酚功能可能产生的不可接受的细胞毒性的限制。为了去除这种儿茶酚的功能，我们设计了一系列含有羟基吡咯吡啶-三酮的类似物。这些化合物的高效合成依赖于亚胺基亚砜的Pummerer环化、去质子化、环加成级联以及异甲酮的[3+2]环加成的应用。虽然这些抑制剂的效力不如最初的2,3-二氢-6,7-二羟基- 1h -异吲哚-1抑制剂那么大，但有一种代表性抑制剂保留了对三种主要抗替地韦突变体IN酶G140S/Q148H、Y143R和N155H的大部分抑制效力。在使用编码这些In突变的病毒载体进行的抗病毒实验中，该化合物对G140S/Q148H和Y143R突变的影响分别比雷替格拉韦低约200倍和20倍。对于N155H突变，该化合物的影响比雷替格拉韦小约10倍。后一系列化合物代表了一种新的结构类别，可以进一步开发以克服对雷替重力韦的耐药，特别是在G140S/Q148H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，并涉及p6蛋白的一个关键的9-mer p - e - p - p - 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-mer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。指导我们方法的统一原则是将氨基-氧功能结合到肽残基中，可以在一个步骤中轻松地功能化，以提供肟衍生物库。这种方法已经鉴定出几种低微摩尔亲和力的Tsg101结合拮抗剂。进一步的肟库多样化是由基于我们的高亲和力抑制剂结合Tsg101蛋白的共晶结构的硅建模指导。该结构是通过与James Hurley博士（NIDDK， NIH）合作获得的。在Doug Auld博士的指导下，在NIH化学基因组学中心通过高通量筛选小化合物文库，将荧光标记引入到一种高亲和结合拮抗剂中，以生产一种试剂，用于鉴定tsg101结合拮抗剂。在平行工作中，Tsg101与肽配体相互作用的化学生物学正在利用构象约束的肽大环进行探索。它们是由肽-类肽前体的合环复分解反应形成的。与母体开链肽相比，这些大环具有更高的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.