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由DNA转录而来，经过加工，然后转运出细胞核并翻译成蛋白质。在感染 HIV-1 的细胞中，病毒 RNA 基因组必须未经加工就从细胞核中输出，以便将它们包装成新的病毒颗粒。为此，细胞必须结合细胞核中宿主 RNA 中自己的 RNA 基因组。这是通过使用识别病毒 RNA 中的 Rev 反应元件 (RRE) 的 HIV-1 Rev 蛋白来实现的。一旦与 RRE 结合，Rev 就会自缔合并结合其他宿主蛋白，形成从细胞核输出的多蛋白-RNA 复合物。我们目前的研究旨在描述该复合物的分子细节。除了提供有关病毒复制机制的基本信息外，这些研究还可能强调脆弱点，这些脆弱点可能是治疗干预的合适目标，包括 Rev 本身。 我们首先关注 Rev RNA 相互作用。使用缩短的非聚合形式的 Rev，其中包含氨基酸残基 1-93（野生型 Rev 为 1-115），并用单链可变片段 (scFv) 抗体进一步稳定，我们制备了与各种 RNA 制剂的复合物对应于 RRE 的区域。我们还使用了 RNA 适体，它们是折叠成 3 维构象并与其靶标（在本例中为 Rev）结合的 RNA。与 RRE 上的 Rev 结合位点相比，其结合亲和力更高的适体具有潜在的抗 HIV 活性。我们最近通过 X 射线晶体学确定了具有高亲和力结合适体的 Rev 的高分辨率结构。在该结构中，二聚体 Rev 桥接两个不连续的适体，这表明当它与 RRE 结合时，Rev 二聚体结合了由 RNA 构象共定位的两个 RNA 序列。使用 Rev 1-93 - scFv 作为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 Rev 进行结构研究的抗体片段 (Fab) 来自噬菌体展示抗体库。这种在细菌中表达的嵌合抗体（人框架和兔可变结构域）是人源化的，并且通过以非常高的亲和力与 Rev 结合而有效，从而防止其寡聚化。在之前的工作中，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 (CDR) 的环肽（长达 12 个氨基酸）可以与 Rev 结合，但我们尚未表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 共结晶，以获得复合物的高分辨率结构，这可能有助于设计或模拟与 Rev 具有改进（更强）结合的低分子量模拟物。 HIV-1 艾滋病与染色单体分离不当和非整倍性有关。我们之前已经证明 Rev 与微管蛋白以化学计量方式强烈相互作用，在体外形成双环复合物。我们还表明，用 Cryptophycin-45（癌症药物）处理微管蛋白会形成由八个微管蛋白异二聚体组成的高度稳定的单环，并且这些微管蛋白以化学计量与 Rev 结合也形成双环。使用冷冻电镜技术以 3.45 埃的分辨率确定了 Hela 微管蛋白与隐藻素复合物的第一个结构。我们还使用冷冻电镜技术以 3.85 埃的分辨率确定了与 HIV-Rev 蛋白复合的 Hela 微管蛋白-隐藻素环的结构。该结构显示在 Rev 存在的情况下形成微管蛋白双环。这些发现引起了癌症和微管蛋白研究界的兴趣。添加 Rev 时形成的双环也将提供对 Rev 结构和功能的进一步了解。一篇描述 HIV-Rev 解聚微管蛋白的新机制的手稿已提交出版。 SARS-cov-2 相关研究 (A-C) A. APN01：仍然迫切需要安全有效的 Covid19 治疗方法。我们假设，气溶胶施用可溶性人重组血管紧张素转换酶 2 (ACE2) 受体可以中和气道中的 SARS-CoV-2，限制肺部感染，并可能减轻肾素-血管紧张素通路中信号失调所造成的损害。在对该假设进行临床测试的前提下，我们使用当前的良好生产规范 (cGMP) 级形式评估了病毒结合活性（与 SARS-CoV-2 毒株 USA_WA1/2020）和雾化后 Ang II 裂解的酶活性。重组人ACE2。这两项活动均被保留。根据这些结果，计划在健康志愿者中进行 I 期临床试验，随后在 SARS-CoV-2 感染者中进行 II 期测试。与 Robert Shoemaker 博士 (NCI) 等人合作撰写的手稿已出版。 B. MAP30：MAP30 是一种多功能抗病毒和抗肿瘤蛋白，源自苦瓜 (Momordica charantia) 种子。 MAP30 先前已被证明具有多种生物活性，包括抑制蛋白质合成、导致病毒 DNA 拓扑失活的 DNA 腺苷糖苷酶活性以及抑制 HIV-1 整合酶。 MAP30 还被证明可以抑制 ACE2，并且含有一种化学合成的肽，该肽在化学合成时可以抑制 ACE2 并降低结构性高血压大鼠的血压。我们评估了 MAP30 对 SARS-CoV-2 的抑制作用。我们重复观察到，在低摩尔范围内，Vero E6 猴肾细胞中病毒诱导的细胞病变效应 (CPE) 降低了约 40%，且没有伴随毒性。由于 MAP30 不进入细胞，这表明 ACE2 介导的病毒进入受到抑制。然而，在 A549 人肺细胞（更合适的细胞类型）中进行的病毒复制测定中，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 端 TAT 肽（介导细胞进入）可将 Hill 系数从 1 增加到 2，而不会降低 CC50，从而扩大了有效范围。除了上述研究之外，我们还根据 MA​​P30 的结构以及 SARS-CoV-2 病毒刺突蛋白受体结合域，通过计算设计了一组环肽。总共 10 种肽已被商业合成，并将与上述重组 ACE2 蛋白以及一组参考化合物（包括瑞德西韦、氯喹和羟氯喹）一起在 Vero E6 CPE 测定中进行测试。与 Robert Shoemaker 博士 (NCI) 合作起草了一份题为 rMAP30（一种重组抗病毒和抗肿瘤药物）抑制 COVID-19 病毒 SARS-CoV-2 的手稿。 C. 治疗干预的一个有吸引力的目标是主要蛋白酶 (Mpro)，它是 SARS-CoV-2 复制所必需的二聚酶。迄今为止，开发 Mpro 抑制剂的大部分工作都集中在活性位点上。我们的工作（与 NCI 的 D. Davis 合作）揭示了通过二聚体界面半胱氨酸 (Cys300) 的谷胱甘肽化来调节 Mpro 活性的机制，这种作用可能发生在氧化应激下的细胞中。 Cys300 谷胱甘肽化通过阻断 Mpro 的二聚化来抑制 Mpro 活性。这为药物开发提供了一个新颖的可接近和反应性靶标。该工作发表在mBio杂志上（2021年7月，现已在线）。