Assay development and screening for inhibitors targeting Shiga toxin 2
针对志贺毒素 2 的抑制剂的检测开发和筛选
基本信息
- 批准号:10322373
- 负责人:
- 金额:$ 51.17万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2019
- 资助国家:美国
- 起止时间:2019-01-25 至 2024-12-31
- 项目状态:已结题
- 来源:
- 关键词:Active SitesAcute Kidney FailureAdenineAffinityAntibiotic TherapyAntibioticsAntidotesApoptosisBacteriophagesBindingBinding ProteinsBinding SitesBiochemicalBiological AssayCatalytic DomainCategoriesCategory B pathogenCellsChemicalsChildComplexCryoelectron MicroscopyCrystallizationCytolysisCytoprotectionDepurinationDevelopmentDiarrheaDiseaseDockingDrug TargetingEscherichia coliEscherichia coli EHECEscherichia coli InfectionsFDA approvedGlycine decarboxylaseGoalsHemolytic-Uremic SyndromeHemorrhagic colitisInfantInfectionKidney FailureKineticsLibrariesLifeMeasuresMolecularMolecular StructureMorbidity - disease rateNational SecurityOutcomePathogenesisPeptide FragmentsPeptidesPhage DisplayPharmaceutical ChemistryProtein Synthesis InhibitionPublic HealthRNA, Ribosomal, 28SResearchRibosomal InteractionRibosomesRicinRiskRoentgen RaysShiga ToxinShiga-Like Toxin IShiga-Like Toxin IIShigellaShigella InfectionsShigella dysenteriaeSiteSolubilityStructureTherapeuticToxic effectToxinTriageVaccinesVariantVirulence FactorsWorkWorld Health OrganizationX-Ray Crystallographyanalogassay developmentbasebiological adaptation to stresscytotoxicitydesigndrug discoveryfoodborne pathogeninhibitorinsightlipophilicitymortalitymutantnanomolarnovelnovel strategiespathogenphysical propertypreventpublic health prioritiesscreeningsmall molecule inhibitorstructural biologytherapeutic developmenttherapeutically effectivetool
项目摘要
Abstract
Shiga toxin (Stx) producing E. coli (STEC) and Shigella dysenteriae are foodborne pathogens
that can cause severe morbidity and mortality. STEC infections can progress to either
hemorrhagic colitis (HC) or life-threatening hemolytic-uremic syndrome (HUS). HUS is the most
common cause of acute renal failure in US children. Presently there are no FDA approved
vaccines or therapeutics against STEC or Shigella infection. Moreover, the use of antibiotics
exacerbates the disease. STEC and Shigella are classified as category B pathogens of national
security and public health risk. Shiga toxin has been a uniquely challenging drug target. Small
molecule inhibitors of Shiga toxin enzymatic activity with high potency have not been identified.
Interaction of A1 subunits with ribosomes has not been previously examined as a potential drug
target. The goal of this proposal is to fill this gap by developing novel screens to identify
fragment and peptide inhibitors that disrupt activity of Stx2 by inhibiting its interaction with the
ribosome. We identified P stalk as the ribosome docking site of the A1 subunits of Stx1 (Stx1A1)
and Stx2 (Stx2A1) and showed that an 11-mer peptide corresponding to the conserved last 11
residues of P proteins binds to Stx2A1 and inhibits its activity. These studies established toxin-
ribosome interactions as a new target for inhibitor discovery. We carried out a preliminary
fragment screen and identified fragments that bind to Stx2A1 with micromolar affinity. In aim 1
we propose to develop Biacore-based primary screens to identify fragments, which bind to
Stx2A1 with higher affinity. We will validate the hits using activity assays and verify binding and
selectivity of the inhibitors using ribosome binding and active site mutants. Medicinal chemistry
will be used to optimize the selected fragments into more potent leads based on their
experimental X-ray crystal structure with Stx2. In aim 2 we will develop phage displaying
multiple copies of the P protein peptide to determine if multivalent display of this peptide motif
will disrupt the interaction of Stx2A1 with the ribosome. We will screen random P7 phage
display library to identify novel peptides that can bind to Stx2A1 more strongly than the native P
protein peptide and inhibit its activity. In aim 3 we propose to solve the cryo-EM structure of
Stx2 in complex with the ribosome to identify the binding sites of the P proteins to facilitate
optimization of the inhibitors. We expect that our unique assays to dissect toxin-ribosome
interactions and toxin activity in combination with the medicinal chemistry and structural biology
expertise will lead to the development of novel tool compounds and peptides, which can provide
biochemical and mechanistic insight into STEC pathogenesis.
摘要
项目成果
期刊论文数量(0)
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科研奖励数量(0)
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Xiao-Ping Li其他文献
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{{ truncateString('Xiao-Ping Li', 18)}}的其他基金
Assay development and screening for inhibitors targeting Shiga toxin 2
针对志贺毒素 2 的抑制剂的检测开发和筛选
- 批准号:
9761636 - 财政年份:2019
- 资助金额:
$ 51.17万 - 项目类别:
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