Mechanisms of influenza NA drug resistance

流感NA耐药机制

• 批准号: 7928788
• 负责人: JENNIFER L MCKIMM-BRESCHKIN
• 金额: $ 25.46万
• 依托单位: COMMONWEALTH SCIENTIFIC & INDUST RES ORG
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2011-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928788
• 关键词: Active Sites; Address; Affect; Affinity Chromatography; Antibodies; Asia; Baculovirus Expression System; Baculoviruses; Binding; Biological; Carbohydrates; Cells; Characteristics; Cleaved cell; Clinical; Complex; Crystallization; Data; Development; Disease Outbreaks; Dose; Drug Kinetics; Drug resistance; Engineering; Enzymes; Epidemic; Europe; Fab Immunoglobulins; Future; Gel Chromatography; Genes; Genotype; Growth; Head; Human; Image; Immunocompetent; In Vitro; Influenza; Influenza A Virus, H5N1 Subtype; Insecta; Kinetics; Length; Lung; Membrane; Minor; Molecular; Monoclonal Antibodies; Mus; Mutate; Mutation; Neuraminidase; Neuraminidase inhibitor; Oseltamivir; Patients; Pharmaceutical Preparations; Phenotype; Play; Population; Predisposition; Production; Pronase; Proteins; Recombinant Proteins; Recombinants; Reporting; Resistance; Robot; Roentgen Rays; Role; Site; Structure; Surveillance Program; System; Technology; Tokyo; Trypsin; Vaccines; Variant; Virion; Virus; Virus Replication; Work; anti-influenza drug; base; c-myc Genes; drug sensitivity; egg; fitness; in vivo; influenzavirus; inhibitor/antagonist; monoclonal antibody production; mouse model; mutant; pandemic disease; protein function; resistance mutation; sugar; thermostability; transmission process; zanamivir; zanamivir resistance

DESCRIPTION (provided by applicant): There are two specific anti-influenza drugs which are effective against all strains of influenza, zanamivir and oseltamivir. Since they bind to highly conserved residues in the active site of the virus neuraminidase (NA) they are effective against all strains of influenza. With the spread of the highly pathogenic H5N1 avian influenza in Asia and now across into Europe, the possibility of a pandemic strain emerging is more likely. Since it will take several months to produce a vaccine the NA inhibitors, especially oseltamivir, are being stockpiled as the first line of defense. While resistance to zanamivir has not yet been seen in immunocompetent patient, there are 3 common mutations in conserved residues in the NA active site which confer oseltamivir resistance. However, these mutations do not confer resistance in all NA subtypes. Resistance rates of up to 18% have now been seen after oseltamivir treatment, and there are already reports of H5N1 oseltamivir resistant viruses, hence there are increasing concerns about drug resistance. Through the Neuraminidase Inhibitor Susceptibility Network (NISN) global surveillance program we have also identified several other viruses which have mutations in non-conserved residues outside of the active site. There must therefore be secondary structural effects outside the active site which can impact on drug binding. Hence our central hypothesis is that "Additional secondary structural characteristics in the NA play a critical role in determining drug sensitivity and whether a mutation confers drug resistance". In addition to the NA mutations, we also have HA mutations in one of the isolates which confer resistance in vitro. Hence we also have a minor hypothesis that "HA mutations in clinical isolates can play a role in resistance to the NA inhibitors in vivo". The long term aim of this project is to use structural and biological studies to understand the mechanisms of influenza resistance which contribute to differences in N1/N2 drug sensitivity, N1/N2 subtype specific resistance, how mutations in non-conserved residues outside the active site affect inhibitor binding, as well as the role of HA mutations in resistance in vivo. We aim to determine the structures of the wild type and mutant NAs from either egg-grown or baculovirus expressed protein. We will assess the impacts of the NA and HA mutations on growth and drug sensitivity in vitro and in vivo as an indication of the "fitness" of the viruses , to understand the likelihood of them being transmissible and drug resistant in the clinical situation. Relevance Understanding the mechanisms of drug resistance, either in naturally occurring or drug selected variants, is critical for appropriate management of epidemic and pandemic outbreaks when resistant virus is circulating, and for the potential long term development of alternative inhibitors.

描述（由申请人提供）：有两种特定的抗炎性药物，可有效抵抗所有流感，Zanamivir和Oseltamivir的菌株。由于它们与病毒神经酶酶（NA）活性部位中高度保守的残基结合，它们对所有流感菌株有效。随着高度致病的H5N1鸟类流感在亚洲和现在进入欧洲的传播，出现大流行菌株的可能性更有可能。由于将需要几个月的时间才能将NA抑制剂（尤其是Oseltamivir）作为第一道防线。尽管在免疫能力患者中尚未看到对Zanamivir的耐​​药性，但在NA活性部位保守残基有3个常见突变，可以赋予Oseltamivir耐药性。但是，这些突变并非在所有NA亚型中赋予抗性。奥塞达米维尔治疗后，现在已经看到了高达18％的耐药率，并且已经有H5N1 oseltamivir耐药病毒的报道，因此人们对耐药性的担忧越来越多。通过神经氨酸酶抑制剂易感性网络（NISN）全球监测计划，我们还确定了其他几种病毒，这些病毒在活性部位以外的非保存残基中具有突变。因此，在活性部位之外必须有次要结构效应，这可能会影响药物结合。因此，我们的中心假设是“ NA中的其他二级结构特征在确定药物敏感性以及突变是否同时抗药性方面起着关键作用”。除了NA突变外，我们还具有在体外赋予耐药性的一种分离株中具有HA突变。因此，我们也有一个较小的假设：“临床分离株中的HA突变可以在体内对Na抑制剂的抗性中发挥作用”。该项目的长期目的是使用结构和生物学研究来了解流感耐药性的机制，这有助于N1/N2药物敏感性的差异，N1/N2亚型特异性耐药性，如何在活性位点影响抑制剂结合的活性位点影响之外的非保存残基中的突变以及HA突变在VIVO中的突变。我们旨在确定卵生长或杆状病毒表达蛋白质的野生型和突变体NA的结构。我们将评估Na和HA突变对体外和体内的生长和药物敏感性的影响，以表明病毒的“适应性”，以了解它们在临床情况下可传播和抗药性的可能性。相关性理解在自然发生的或选择的变体中，耐药性的机制对于在耐药病毒循环时的流行病和大流行暴发以及替代抑制剂的潜在长期发展时，至关重要。

项目成果

Taking down the FLAG! How insect cell expression challenges an established tag-system.

• DOI: 10.1371/journal.pone.0037779
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Schmidt PM;Sparrow LG;Attwood RM;Xiao X;Adams TE;McKimm-Breschkin JL
• 通讯作者: McKimm-Breschkin JL

Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal.

用于比较流感野生型和抗性突变体去除抑制剂后的神经氨酸酶重新激活率的固相测定。

• DOI: 10.1016/j.antiviral.2014.05.006
• 发表时间: 2014
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Barrett,Susan;McKimm-Breschkin,JenniferL
• 通讯作者: McKimm-Breschkin,JenniferL

Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses.

• DOI: 10.1371/journal.pone.0023627
• 发表时间: 2011
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Barrett S;Mohr PG;Schmidt PM;McKimm-Breschkin JL
• 通讯作者: McKimm-Breschkin JL

Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses.

• DOI: 10.1021/jm100621s
• 发表时间: 2010-09-09
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Oakley AJ;Barrett S;Peat TS;Newman J;Streltsov VA;Waddington L;Saito T;Tashiro M;McKimm-Breschkin JL
• 通讯作者: McKimm-Breschkin JL