A Novel Conformation of the ZIKV Protease Provides a Unique 3D Scaffold for Discovering Allosteric Protease Inhibitors as Direct Anti-virals, via HT and Virtual Screening, and Protein Engineering

ZIKV 蛋白酶的新颖构象为通过 HT 和虚拟筛选以及蛋白质工程发现变构蛋白酶抑制剂作为直接抗病毒药物提供了独特的 3D 支架

• 批准号: 9757689
• 负责人: SUMIT K CHANDA
• 金额: $ 24.38万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-08 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9757689
• 关键词: 3-Dimensional; Active Sites; Acute; Adopted; Adult; Aedes; Affinity; Animal Testing; Antibodies; Antibody-Dependent Enhancement; Antiviral Agents; Binding; Biological Assay; Brazil; C-terminal; Cells; Chronic; Cleaved cell; Clinic; Clinical; Complex; Computer Simulation; Crystallization; Culicidae; Dengue; Development; Disease; Distal; Distant; Drug Kinetics; Drug Targeting; Elements; Engineering; Enzymatic Biochemistry; Exhibits; Flaviviridae; Flavivirus; Flavivirus Infections; Genome; Guillain-Barré Syndrome; Hepatitis C virus; Hydrophobicity; In Vitro; Individual; Infection; Japanese encephalitis virus; Libraries; Life Cycle Stages; Ligands; Membrane; Microcephaly; Minority; Molecular Conformation; N-terminal; Nature; Neurologic; Outcome; Pathway interactions; Peptide Hydrolases; Peptides; Persons; Polyproteins; Population; Powder dose form; Protease Inhibitor; Protein Engineering; Proteins; RNA Helicase; Reporting; Risk; Seminal fluid; Sexual Transmission; Specificity; Structure; Surface; Symptoms; Temperature; Testing; Therapeutic; Toxic effect; Vaccines; Viral; Virtual Library; West Nile virus; Work; ZIKV infection; Zika Virus; base; biophysical techniques; conformer; cytotoxicity; cytotoxicity test; design; drug discovery; druggable target; human pathogen; infection risk; inhibitor/antagonist; innovation; melting; mutant; neonatal infection; novel; pathogen; preference; prenatal; scaffold; screening; small molecule; small molecule therapeutics; success; therapeutic development; vaccine development; virtual

Project Summary Zika flavivirus (ZIKV) is an emerging pathogen globally and in the US. ZIKV infection usually causes mild symptoms; however, ZIKVBR infection has in some individuals resulted in serious neurological sequelae, including Guillain-Barré syndrome in adults, and microcephaly in prenatally-infected infants. No treatments for flaviviral infections are available. Vaccine strategies for DENV flavivirus have been disappointing, possibly due to Antibody-Dependent Enhancement of infection (ADE); and it has been demonstrated that antibodies to DENV result in ADE for ZIKV. Furthermore, ZIKV is present at very high levels in semen for up to six months post-infection. Since only ~20% of infected persons exhibit visible signs of infection, the risk of sexual transmission is greatly increased. These issues highlight the need for therapeutic small-molecule approaches that directly target the viral life-cycle. The flaviviral NS2B-NS3 protease is required for polyprotein cleavage and viral infectivity and represents an attractive target. The major innovation of this proposal arises from our recent crystallographic observation of a novel “third” conformation of ZIKV NS2B-NS3 protease, which we call the “double-open” state. It is distinguished from the “closed” and “single-open” states by a radical reorganization of the C-terminal substrate-binding β-hairpin of NS3PRO that is incompatible with protease activity. This double- open state displays a new surface-exposed, deep hydrophobic pocket, distal to the active site, which appears highly druggable, since it is highly conserved among flaviviruses, and lined with elements of the reorganized β- hairpin. Thus, small molecules that bind tightly to this pocket should stabilize this inactive state, and act as allosteric inhibitors. We propose to test this hypothesis starting with both real and virtual libraries. In principle, allosteric inhibitors should have much greater specificity compared with active-site inhibitors, since many host proteases have very similar, or identical target recognition sequences. We further hypothesize that our unique scaffold should provide a superior pathway to refinement of inhibitory hits by generating co-crystal structures to guide further design, an element that has been problematic for the development of more conventional allosteric inhibitors that target shallow pockets at the labile NS2B-NS3 interface. The nature of our novel pocket should expedite co-crystallization. We will screen compound libraries using both conventional HT approaches and pocket-directed in silico screening. The primary screen is PTS, with mutant protease locked into the “double- open” state. Small-molecule pocket binders should raise the melting temperature. Host proteases with similar target preferences will be used as counter-screens. Protease inhibition by surviving hits will be tested using a HT fluorescent peptide screen. HepaRG cells will be used to test for cytotoxicity and Huh-7.5 cells for inhibition of infection by ZIKV Brazil strain. We will validate allosteric binding using biophysical techniques, co- crystallization and enzymology; and direct inhibitor optimization by co-crystallization. We anticipate obtaining ~5 inhibitors suitable for further therapeutic development.

项目摘要 Zika Flavivirus（ZIKV）是全球和美国的新兴病原体。 ZIKV感染通常会导致中期 症状;但是，Zikvbr感染在某些个体中导致了严重的神经系统后遗症， 包括成年人的Guillain-Barré综合征和产前感染的婴儿的小头畸形。没有治疗 可以使用黄病毒感染。 DENV黄病毒的疫苗策略令人失望，可能 依赖抗体的感染增强（ADE）；并且已经证明了 DENV为ZIKV带来了ADE。此外，ZIKV的精液非常高至六个月 感染后。由于只有约20％的感染者暴露了可见的感染迹象，因此性行为风险 传输大大增加。这些问题突出了需要治疗小分子方法的需求 直接针对病毒生命周期。多蛋白裂解和 病毒感染，代表一个有吸引力的靶标。该提议的主要创新是我们的最新 ZIKV NS2B-NS3蛋白酶的新型“第三”构象的晶体学观察，我们称之为 “双开”状态。通过根本重组，它与“封闭”和“单开”状态区分开 NS3PRO的C末端底物结合β-脱发与蛋白酶活性不相容。这个双人 开放状态显示出一个新的表面暴露，深疏水的口袋，距离活性部位远端 高度可吸毒，因为它在黄病毒之间是高度保守的，并且衬有重组β-的元素 簪。那就是，与这个口袋紧密结合的小分子应稳定这种无效状态，并充当 变构抑制剂。我们建议从实际和虚拟库开始检验这一假设。原则， 与活性位点抑制剂相比，变构抑制剂应具有更大的特异性，因为许多宿主 蛋白酶具有非常相似或相同的目标识别序列。我们进一步假设我们的独特 脚手架应通过产生共晶体结构来提供抑制性命中的较高途径 指导进一步的设计，这是对开发更常规变构的问题的元素 靶向不稳定的NS2B-NS3界面的较浅口袋的抑制剂。我们新颖的口袋的本质应该 加快共结晶。我们将使用常规的HT方法筛选复合库，并且 袖珍指导在硅筛选中。主要屏幕是PTS，突变蛋白锁定在“双 - 开放的“状态。小分子袖珍粘合剂应升高熔化温度。宿主蛋白酶具有相似的 目标偏好将用作反屏幕。幸存的命中蛋白酶抑制作用将使用 HT荧光肽筛查。 HEPARG细胞将用于测试细胞毒性和HUH-7.5细胞以抑制 ZIKV巴西菌株感染。我们将使用生物物理技术（共同）来验证变构结合 结晶和酶学；和通过共结晶的直接抑制剂优化。我们预计会获得 〜5抑制剂适合进一步的治疗发育。