Production Of HIV And HIV Related Proteins For Structural Studies

用于结构研究的 HIV 和 HIV 相关蛋白的生产

• 批准号: 10707808
• 负责人: PAUL T WINGFIELD
• 金额: $ 130.09万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707808
• 关键词: 2019-nCoV; 3-Dimensional; A549; ACE2; Acquired Immunodeficiency Syndrome; Active Sites; Adenosine; Aerosols; Affinity; Amino Acids; Aneuploidy; Angiotensins; Antibodies; Antineoplastic Agents; Bacteria; Binding; Binding Sites; Biological; Biological Assay; Biosynthetic Proteins; C-terminal; COVID-19 therapeutics; Cell Nucleus; Cells; Chemicals; Chloroquine; Chromatids; Collaborations; Communities; Complex; Cryoelectron Microscopy; Cryptophycin; Crystallization; Cyclic Peptides; Cysteine; DNA; Dimerization; Drug Design; Drug Targeting; Enzymes; Genome; Glycoside Hydrolases; HIV; HIV-1; HIV-1 integrase; Hela Cells; Human; Hydroxychloroquine; Immunoglobulin Fragments; In Vitro; Individual; Infection; Journals; Label; Life Cycle Stages; Lung; Malignant Neoplasms; Manuscripts; Mediating; Melons; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Molecular Weight; Momordica charantia; Monkeys; Multidimensional NMR Techniques; Nuclear Magnetic Resonance; Oryctolagus cuniculus; Oxidative Stress; Pathway interactions; Peptide Hydrolases; Peptides; Phage Display; Phase I Clinical Trials; Preparation; Process; Production; Protein Synthesis Inhibition; Proteins; Publications; Publishing; RNA; RNA Conformation; RNA Folding; RNA Sequences; Rattus; Recombinant DNA; Recombinants; Renin; Research; Resolution; Response Elements; Role; SARS-CoV-2 infection; Seeds; Signal Transduction; Structure; System; Techniques; Testing; Therapeutic Intervention; Toxic effect; Translating; Tubulin; Viral; Virus; Virus Inactivation; Virus Replication; Work; X ray diffraction analysis; X-Ray Crystallography; antibody libraries; antitumor agent; aptamer; base; blood pressure reduction; cell type; chimeric antibody; depolymerization; design; dimer; drug development; healthy volunteer; hypertensive; improved; inhibitor; insight; interest; kidney cell; novel; particle; phase 2 testing; prevent; protein structure; receptor; receptor binding; remdesivir; research clinical testing; rev Protein; stable isotope; targeted treatment; therapeutically effective; three dimensional structure; viral DNA; viral RNA

In uninfected cells, RNA is transcribed from DNA, processed, and then transported out of the nucleus and translated into protein. In cells infected with HIV-1, the viral RNA genomes must be exported out of the nucleus without being processed so they can be packaged into new viral particles. To do this, the cell must bind its own RNA genome from among the host RNA in the nucleus. This is achieved using the HIV-1 Rev protein that recognizes a Rev response element (RRE) in the viral RNA. Once bound to RRE, Rev self-associates and binds other host proteins, forming a multiprotein-RNA complex that is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. In addition to contributing fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability that may be suitable targets for therapeutic intervention including Rev itself. We have focused first on Rev RNA interactions. Using a shortened and non-polymerizing form of Rev that incorporates amino acid residues 1-93 (wild type Rev is 1-115) and is further stabilized with a single chain variable fragment (scFv) antibody, we have prepared complexes with various RNA preparations corresponding to regions of the RRE. We have also used RNA aptamers, which are RNAs that fold into 3-dimensional conformations that bind to their targets (in this case Rev). Aptamers that bind with higher affinity than Rev-binding sites on RRE have potential anti-HIV activity. We have recently determined a high-resolution structure by X-ray crystallography of Rev with a high affinity binding aptamer. In this structure, dimeric Rev bridges two discontinuous aptamers, suggesting when it binds to RRE the Rev dimer is binding two RNA sequences co-localized by the RNA conformation. Using the Rev 1-93 - scFv as a proven crystallization platform we are extending structural studies to solve interactions with other high affinity aptamers. The antibody fragment (Fab) used for stabilizing Rev for structural studies was derived from a phage display antibody library. This chimeric antibody (human framework and rabbit variable domains), expressed in bacteria, was humanized and was effective by binding to Rev with a very high affinity, thereby preventing its oligomerization. In previous work we showed that this antibody had anti-HIV-1 activity. We also showed that cyclic peptides (up to 12 amino acids long) from the antibody variable regions (CDRs) could bind to Rev but we have not yet shown whether they also have anti-HIV-1 activity. In addition, we are attempting to co-crystallize the peptides with Rev in order to obtain high-resolution structures of the complexes, which may help design or model low-molecular weight mimics with improved (stronger) binding to Rev. HIV-1 AIDS is associated with improper chromatid separation and aneuploidy. We have previously shown that Rev interacts strongly and stoichiometrically with tubulin to form double-ring-like complexes in vitro. We have also shown that treatment of tubulin with Cryptophycin-45 (cancer drug) forms highly stable single-rings composed of eight tubulin heterodimers, and that these bind Rev stoichiometrically to also form double-rings. The first structure of Hela tubulin in complex with cryptophycin was determined at 3.45 Angstrom resolution using cryo-EM techniques. We have also determined structure of Hela tubulin-cryptophycin rings in complex with HIV-Rev protein at 3.85 Angstrom resolution using cryo-EM techniques. The structure shows tubulin double rings formed in the presence of Rev. These findings are of interest to both the cancer and tubulin research communities. The double-rings formed when Rev is added will also provide further insights into Rev structure and function. A manuscript describing a novel mechanism for tubulin depolymerization by HIV-Rev has been submitted for publication. SARS-cov-2 related studies (A-C) A. APN01: There remains an urgent need for safe and effective therapeutics for Covid19. We hypothesized that aerosol administration of soluble human recombinant angiotensin-converting enzyme 2 (ACE2) receptor would neutralize SARS-CoV-2 in the airways, limiting infection in the lung and potentially also mitigate damage caused by deregulated signaling in the renin-angiotensin pathway. Prerequisite to a clinical test of this hypothesis, we evaluated both virus binding activity (with SARS-CoV-2 strain USA_WA1/2020) and enzymatic activity for cleavage of Ang II following aerosolization using a current Good Manufacturing Practices (cGMP)-grade form of recombinant human ACE2. Both activities were retained. Based on these results, a Phase I clinical trial in healthy volunteers is planned, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. A manuscript written in collaboration with Dr Robert Shoemaker (NCI) and others has been published. B. MAP30: MAP30 is a multifunctional antiviral and antitumor protein derived from the seeds of bitter melon (Momordica charantia). MAP30 has previously been shown to possess several biological activities including inhibition of protein synthesis, DNA adenosine-glycosidase activity that leads to topological inactivation of viral DNAs, and inhibition of HIV-1 integrase. MAP30 has also been shown to inhibit ACE2, and to contain a peptide that when chemically synthesized inhibits ACE2 and reduces blood pressure in constitutively hypertensive rats. We have assessed MAP30 for inhibition of SARS-CoV-2. We have reproducibly observed about a 40% reduction of virus-induced cytopathic effect (CPE) in Vero E6 monkey kidney cells in the low molar range without concomitant toxicity. As MAP30 does not enter cells, this suggests inhibition of ACE2-mediated viral entry. However, in a viral replication assay in A549 human lung cells, a more appropriate cell type, we reproducibly observe 100% viral inhibition (IC50 = 0.27 M, CC50 = 4.17 M). Furthermore, appending a C-terminal TAT peptide (to mediate cell entry) increases the Hill coefficient from 1 to 2 without lowering the CC50, thereby broadening the effective range. In addition to the studies above, we have computationally designed a set of cyclic peptides, based on the structure of MAP30, and also on the SARS-CoV-2 viral spike protein receptor binding domain. A total of 10 peptides have been commercially synthesized and will be tested in a Vero E6 CPE assay together with the recombinant ACE2 protein described above and a set of reference compounds that includes Remdesivir, Chloroquine, and Hydroxychloroquine. A manuscript titled Inhibition of the COVID-19 virus, SARS-CoV-2 by rMAP30, a recombinant anti-viral and anti-tumor agent has been drafted in collaboration with Dr. Robert Shoemaker (NCI). C. One attractive target for therapeutic intervention is the main protease (Mpro), a dimeric enzyme necessary for SARS-CoV-2 replication. Most work to date developing Mpro inhibitors has focused on the active site. Our work (in collaboration D. Davis, NCI) has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. This work been published in the journal mBio (July 2021 and is now online).

在未感染的细胞中，RNA从DNA转录，处理，然后从细胞核中运出并转化为蛋白质。在感染HIV-1的细胞中，必须将病毒RNA基因组从细胞核中输出而不会被处理，以便可以将它们包装到新的病毒颗粒中。为此，细胞必须从核中的宿主RNA中结合其自己的RNA基因组。这是使用HIV-1 REV蛋白来实现的，该蛋白识别病毒RNA中的Rev Respent元件（RRE）。一旦与RRE结合，REV会自缔合并结合其他宿主蛋白，形成从细胞核中导出的多蛋白-RNA复合物。我们目前的研究旨在描述该复合物的分子细节。除了贡献有关病毒复制机制的基本信息外，这些研究还可能突出脆弱性点，这可能是包括Rev本身在内的治疗干预措施的合适靶标。 我们首先关注RNA相互作用。使用缩短和非聚合形式的Rev形式，该形式结合了氨基酸残基1-93（野生型Rev为1-115），并用单个链可变片段（SCFV）抗体进一步稳定，我们已经准备了与RRE区域相对应的各种RNA制剂。我们还使用了RNA适体，它们是折叠成3维构象的RNA，与其靶标结合（在本例中为Rev）。与RRE上的Rev-Binding位点结合的适体具有潜在的抗HIV活性。我们最近通过具有高亲和力的适体的Rev的X射线晶体学确定了高分辨率结构。在这种结构中，Dimeric Rev桥接了两个不连续的适体，这表明它与RRE时，Rev Dimer何时结合了通过RNA构象共定位的两个RNA序列。使用REV 1-93 -SCFV作为一个经过验证的结晶平台，我们正在扩展结构研究，以解决与其他高亲和力适体的相互作用。 用于稳定结构研究的REV的抗体片段（FAB）源自噬菌体显示抗体库。这种嵌合抗体（人类框架和兔可变结构域）在细菌中表达，是人性化的，并且通过具有很高亲和力的Rev结合而有效，从而防止其低聚。在先前的工作中，我们表明该抗体具有抗HIV-1活性。我们还表明，来自抗体可变区域（CDR）的环状肽（长达12个氨基酸长）可以与REV结合，但我们尚未证明它们是否也具有抗HIV-1活性。此外，我们正在尝试将肽与Rev共结合，以获得复合物的高分辨率结构，这可能有助于设计或模拟低分子量的模拟物，并通过改进（更强）与Rev的结合进行模拟。 HIV-1 AIDS与染色单体分离不当和非整倍性有关。我们先前已经表明，Rev与微管蛋白强烈和化学相互作用，体外形成了双环状复合物。我们还表明，用隐藻蛋白-45（癌症）对微管蛋白进行处理形成高度稳定的单环，由八个微管蛋白异二聚体组成，并且这些结合了Rev stoichiometrementic，也形成双环。使用冷冻EM技术以3.45的Angstrom分辨率确定HeLa微管蛋白在复合物中的第一个结构。我们还确定了使用冷冻EM技术以3.85 Angstrom分辨率下的HIV-REV蛋白在复合物中的HeLa微管蛋白 - 晶状体环的结构。该结构表明，在Rev存在的情况下形成的小管蛋白双环。这些发现对癌症和微管蛋白研究群落都很感兴趣。添加Rev时形成的双环还将提供对Rev结构和功能的进一步见解。已经提交了一种用HIV-REV来描述微管蛋白去聚合的新机制的手稿，已提交出版。 SARS-COV-2相关研究（A-C） A. APN01：迫切需要对Covid19的安全有效治疗。我们假设，可溶性人类重组血管紧张素转换酶2（ACE2）受体的气溶胶给药会中和气道中的SARS-COV-2，从而限制了肺部的感染，并可能减轻肾蛋白 - 血管紧张素途径中的信号传导引起的损害。对该假设进行临床检验的先决条件，我们使用当前的良好制造实践（CGMP） - 重组形式的重组人ACE2评估了病毒结合活性（具有SARS-COV-2菌株USA_WA1/2020）和酶活性，用于裂解ANG II的裂解。两项活动都保留了。基于这些结果，计划在健康志愿者中进行I期临床试验，随后在SARS-COV-2感染的个体中进行了II期测试。与罗伯特·鞋匠（NCI）和其他人合作的手稿已出版。 B. MAP30：MAP30是一种源自苦瓜种子（Momordica Charantia）的多功能抗病毒和抗肿瘤蛋白。先前已显示MAP30具有多种生物学活性，包括抑制蛋白质合成，DNA腺苷 - 糖苷酶活性，导致病毒DNA拓扑失活以及HIV-1整合酶的抑制。 MAP30还显示出抑制ACE2，并包含一种肽，该肽是化学合成时抑制ACE2并降低组成性高血压大鼠的血压的。我们已经评估了MAP30抑制SARS-COV-2。我们已经可重复地观察到在低摩尔范围内的VERO E6猴肾细胞中，病毒诱导的细胞质效应（CPE）的降低约为40％，而无需毒性。由于MAP30未进入细胞，这表明抑制ACE2介导的病毒进入。但是，在A549人肺细胞中的病毒复制测定中，我们可重复地观察到100％病毒抑制（IC50 = 0.27 m，CC50 = 4.17 m）。此外，附加C末端TAT肽（介导细胞进入）将山系数从1增加到2，而无需降低CC50，从而扩大了有效范围。除上述研究外，我们还根据MAP30的结构以及SARS-COV-2病毒尖峰蛋白受体结合结构域设计了一组循环肽。总共有10种肽已商业合成，并将在Vero E6 CPE测定中与上述重组ACE2蛋白以及包括Remdesivir，氯喹和羟基氯喹的一组参考化合物一起进行测试。由RMAP30（重组抗病毒和抗肿瘤剂）与Robert Shoemaker（NCI）合作起草的一项手稿，该手稿是RMAP30对COVID-19病毒的SARS-COV-2。 C.治疗干预的一个有吸引力的靶标是主要蛋白酶（MPRO），这是SARS-COV-2复制所需的二聚体酶。迄今为止，开发MPRO抑制剂的大多数工作都集中在活跃部位上。我们的工作（NCI的D. Davis合作）揭示了通过二聚体界面处半胱氨酸（CYS300）的谷胱甘肽化（Cys300）的调节机制，该机制可以在氧化应激下发生在细胞中。 Cys300谷胱甘肽通过阻断其二聚化抑制MPRO活性。这为药物开发提供了一种新颖的可访问和反应性目标。这项工作发表在《 MBIO》杂志上（2021年7月，现已在线）。