Inhibitors of HIV-1 Cell Fusion And Mycothiol-related En

LBC的天然产物组，NIDDK，活跃在两个研究领域，这两个领域都寻求确定和理解抑制剂的结构基础和作用机制：(1)真菌硫醇依赖的生物合成和解毒；(2)。艾滋病毒融合。1. 在真核生物中，谷胱甘肽是主要的硫醇，参与维持细胞间的还原环境，并保护细胞免受氧化应激和烷基化剂的影响。相比之下，许多原核生物（特别是革兰氏阳性细菌）使用特定细菌群特有的替代小分子量硫醇。在包括分枝杆菌在内的放线菌中，myo- d -肌酰基-2-（n -乙酰半胱氨酸）氨基-2-脱氧-a- d -葡萄糖吡喃苷（mycothiol， MSH） 1是唯一存在的硫醇，被认为在真核生物中具有与谷胱甘肽相似的功能。最近，从结核分枝杆菌（菌株Rv37）中发现了两种关键酶，它们既参与真菌硫醇的生物合成（rv1170编码AcCys-Ins脱乙酰酶），也参与真菌硫醇依赖性解毒（rv1082编码真菌硫醇- s偶联氨基酶，以下简称MCA）。鉴于真菌硫醇的独特结构和分布，以及rv1170或rv1082与公共数据库中可用的其他基因序列之间缺乏可检测的同源性，这些酶代表了新的革兰氏阳性特异性抗生素或抗结核药物的有价值的靶标。因此，我们的研究的一个方面旨在确定新的小分子量抑制剂结核分枝杆菌。去乙酰化酶和氨基酶，并最终确定真菌硫醇生物合成和抑制MCA的结构基础。为此，我们迄今已从不同的海洋植物、无脊椎动物和陆生真菌（NCI天然产物库）中筛选了约1500种有机提取物，以检测其抑制MCA的能力。我们选择这个文库的最初目标是获得具有抑制MCA能力的化学结构的概况。使用基于荧光的高效液相色谱法检测MSHmb的荧光双烷衍生物及其MCA裂解产物AcCys-mb的存在，我们已经确定了许多新的和已知的天然产物，这些产物在亚微摩尔到微摩尔浓度下抑制MCA，其中至少有一种已被报道对结核分枝杆菌是致命的。虽然rv1170和rv1082编码的基因具有30%的同源性，但对底物的偏好却有显著差异。我们目前正在研究MCA抑制剂对去乙酰化酶的抑制程度，以及对去乙酰化酶抑制剂的筛选。正在进行的合成工作包括合成假定的MSH前体，即GlcNAc-a（1?1）肌d -肌醇；mycothiol;最终每种物质的类似物将被用于研究底物特异性。考虑到迄今为止分离的抑制剂的化学结构和天然底物的结构，未来的研究将包括底物抑制剂的设计和合成。2. 为了使包膜病毒感染细胞，必须发生膜融合。在HIV的情况下，导致融合的事件包括HIV包膜糖蛋白gp120与主要受体CD4结合，随后与趋化因子受体CXCR4或CCR5结合，它们共同促进gp41的融合肽（即HIV包膜蛋白的跨膜亚基）插入宿主细胞膜，最终导致融合。目前的抗逆转录病毒药物仅针对HIV蛋白酶和逆转录酶（RT）。虽然我们在蛋白酶和逆转录酶抑制剂的设计和开发方面取得了显著的成功，但这些药物通常耐受性差，而且对许多不发达国家来说价格昂贵。我们研究的第二个方面集中在病毒进入的抑制剂上，包括以下项目：(i)鉴定HIV融合的小分子抑制剂，（ii）确定cyanovirin-N有效融合阻断活性的结构基础，以及（iii）设计HIV融合的蛋白抑制剂和/或抗原（下面没有讨论，参见参考文献7）。迄今为止，有效抑制HIV病毒进入的抑制剂仅限于含有HIV- env序列的肽或蛋白质，含有中和抗体的嵌合蛋白，或中等分子量（1-5 kDa）的化合物，这些化合物具有总体丰富的负电荷分子。在第三个例子中，与Env的结合被认为是非特异性的，并且通过与gp120上带正电的辅受体结合区相互作用而发生。因此，试图鉴定HIV融合的小分子抑制剂的动力不仅来自鉴定新型潜在抗病毒药物的愿望，而且还来自揭示模仿自然发生的蛋白质的新型支架。正如上文第1部分所述，迄今为止，我们已经在基于牛痘病毒的定量融合试验中筛选了约1500种提取物（Berger和同事），这些提取物忠实地再现了导致HIV-1包膜介导融合的事件，并鉴定了许多新的和已知的天然产物，其中一些在亚微摩尔浓度下抑制融合。这些包括新型双小角鲨烷倍半萜和胍类生物碱。二)。Cyanovirin-N （CVN）是一种11kDa的蓝藻蛋白，能有效抑制(IC50？1nM)所有HIV和SIV毒株通过高亲和力碳水化合物介导的gp120相互作用达到融合水平。通过使用融合实验和已知存在于哺乳动物细胞中表达的融合性gp120上的一组复杂和寡糖型低聚糖的一系列竞争实验，我们最近表明，CVN选择性地与D1D3异构体或寡糖-8 （Man8 D1D3）和寡糖-9 （Man9）结合，具有纳米摩尔亲和力，这些碳水化合物在纳米摩尔浓度下充当CVN的二价和三价配体。它们直接与融合性HIV-1 Env竞争，充分抑制CVN的融合阻断活性。由于Man8 D1D3和Man9的结构相对于其他高甘露糖低聚糖（不能与gp120竞争CVN结合），我们已经证明Mana(1-2)Mana，它代表低甘露糖全长D1和D3臂的末端二糖，也是CVN的高亲和力配体。利用核磁共振，我们跟踪了用Mana(1-2)Mana滴定的CVN的1H-15N相关谱，明确地证明CVN在伪对称蛋白的两端含有两个不同亲和的新型碳水化合物结合位点。我们还利用多维异核磁共振解析了1:2 CVN:Mana(1-2)Mana复合物的溶液结构，揭示了具有纳米摩尔亲和力的甘露糖特异性碳水化合物结合蛋白复合物的第一个结构。两个相反的碳水化合物结合位点的存在和位置，以及低寡甘露糖D1和D3臂间距所施加的几何限制，为CVN-gp120结合提供了一个简单的模型。

Carole Bewley LBC, NIDDK, NIH Annual Report, Fiscal Year 2001 The Natural Products Group of LBC, NIDDK, is active in two areas of research, both of which seek to identify and understand the structural basis and mechanisms of action of inhibitors to (1.) mycothiol-dependent biosynthesis and detoxification, and (2.) HIV fusion. 1. In eukaryotes, glutathione is the primary thiol involved in maintaining a reducing intercellular environment and in protecting cells from both oxidative stress and alkylating agents. In contrast, many prokaryotes (especially Gram-positive bacteria) employ alternative small molecular weight thiols that are unique to particular bacterial groups. In the case of actinomycetes, which include the mycobacteria, myo-D-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-a-D-glucopyranoside (mycothiol, MSH),1 , is the only thiol present and is thought to function similarly to glutathione in eukaryotes. Two key enzymes from Mycobacterium tubercuolsis (strain Rv37) that are involved in either the biosynthesis of mycothiol (rv1170 coding for AcCys-Ins deacetylase), or in mycothiol-dependent detoxification (rv1082 coding for mycothiol-S-conjugate amidase, hereafter referred to as MCA), have been identified recently. Given the unique structure and distribution of mycothiol, along with the absence of detectable homology between rv1170 or rv1082 and other gene sequences available in public databases, these enzymes represent worthy targets for new classes of Gram-positive-specific antibiotics or antituberculars. Thus, one aspect of our research aims to identify novel small-molecular weight inhibitors to the M. tuberculosis. deacetylase and amidase enzymes, and ultimately to determine the structural basis for the biosynthesis of mycothiol and inhibition of MCA. To this end we have thus far screened ~ 1500 organic extracts from diverse collections of marine plants, invertebrates, and terrestrial fungi (NCI Natural Products Repository) for their ability to inhibit MCA. Our initial goal in selecting this library was to obtain a profile of chemical structures that have the ability to inhibit MCA. Using a flouroescence-based HPLC assay that detects the presence of a fluorescent bimane derivitive of MSHmb and its MCA cleavage product AcCys-mb, we have identified a number of novel and known natural products that inhibit MCA at submicromolar to micromolar concentrations, at least one of which has been reported to be lethal to M. tuberculosis. Although the genes encoded by rv1170 and rv1082 exhibit ~30% homology, the substrate preferences are significantly different. We are currently investigating the degree of inhibition of the MCA inhibitors on the deacetylase, as well as screening for inhibitors of the deacetylase. Ongoing synthetic work includes the synthesis of a putative precursor to MSH, namely GlcNAc-a(1?1)myo-D-inositol; of mycothiol; and eventually of analogs of each that will be used to study substrate specificity. Mindful of both the chemical structures of the inhibitors isolated thus far and the structures of the natural substrates, future studies will include the design and synthesis of substrate-based inhibitors. 2. In order for enveloped viruses to infect cells, membrane fusion must occur. In the case of HIV, the events leading to fusion include binding of the HIV envelope glycoprotein gp120 to the primary receptor CD4, and subsequent binding to the chemokine receptors CXCR4 or CCR5, which together facilitate insertion of the fusion peptide of gp41 (i.e. the transmembrane subunit of the HIV envelope protein) into the host cell membrane, ultimately leading to fusion. Current anti-retrovirals target exclusively HIV protease and reverse transcriptase (RT). While we have witnessed remarkable success in the design and development of protease and RT inhibitors, these drugs are often poorly tolerated and prohibitively expensive for much of the underdeveloped world. The second aspect of our research focuses on inhibitors of viral entry, and encompasses the following projects: (i.) identification of small-molecule inhibitors to HIV fusion, (ii.) determination of the structural basis of the potent fusion-blocking activity of cyanovirin-N, and (iii.) the design of proteinaceous inhibitors and/or antigens of HIV fusion (not addressed below, see Ref. 7). i.) To date potent inhibitors to viral entry by HIV are limited to peptides or proteins comprising sequences from HIV-Env, to chimeric proteins incorporating neutralizing antibodies, or to compounds of modest molecular weight (1-5 kDa) that bear an overall abundance of negatively charged moeities. In the 3rd example, binding to Env is thought to be nonspecific and to occur through interactions with the positively charged coreceptor-binding regions on gp120. Thus, the impetus for attempting to identify small-molecule inhibitors to HIV fusion stems not only from the desire to identify new classes of potential antivirals, but also to reveal novel scaffolds that mimic naturally occurring proteins. As in Part 1 above, we have thus far screened ~1500 extracts in a quantitative vaccinia virus-based fusion assay (Berger and coworkers) that faithfully reproduces the events leading to HIV-1 Envelope-mediated fusion , and have identified a number of new and known natural products, several of which inihibit fusion at submicromolar concentrations. These include novel bishomoscalarane sesquiterpenes and guanidinium alkaloids. ii.) Cyanovirin-N (CVN) is an 11kDa cyanobacterial protein that potently inhibits (IC50?1nM) all strains of HIV and SIV at the level of fusion via high affinity carbohydrate-mediated interactions with gp120. Through a series of competition experiments using the fusion assay and a panel of complex and oligomannose-type oligosaccharides known to be present on fusogenic gp120 expressed in mammalian cells, we have recently shown that CVN selectively binds to the D1D3 isomer or oligomannose-8 (Man8 D1D3) and to oligomannose-9 (Man9) with nanomolar affinity, that these carbohydrates act as divalent and trivalent ligands to CVN at nanomolar concentrations, and that they directly compete with fusogenic HIV-1 Env to fully inhibit the fusion-blocking activity of CVN. Owing to the structures of Man8 D1D3 and Man9 relative to other high-mannose oligosaccharides (which cannot compete with gp120 for CVN binding), we have shown that Mana(1-2)Mana, which represents the terminal disaccharide of the full-length D1 and D3 arms of oligomannose, is also a high affinity ligand for CVN. Using NMR we followed the 1H-15N correlation spectrum of CVN titrated with Mana(1-2)Mana to unambiguously demonstrate that CVN contains two novel carbohydrate binding sites of differing affinities located at opposite ends of the pseudo-symmetrical protein. We have also solved the solution structure of a 1:2 CVN:Mana(1-2)Mana complex using multi-dimensional heteronuclear NMR to reveal the first structure of a complex of a mannose-specific carbohydrate-binding protein with nanomolar affinity. The presence and location of two opposing carbohydrate binding sites in conjunction with the geometrical restraints imposed by the spacing of the D1 and D3 arms of oligomannose presents a simple model for CVN-gp120 binding.