CryoEM guided enhancement of ribosome-targeting antibiotics
CryoEM guided enhancement of ribosome-targeting antibiotics
批准号:
10219931
负责人:
David John Lee
金额:
$0.59万
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-08-01 至 2021-08-02
关键词:
ATP HydrolysisAcetylationAcetyltransferaseAddressAntibiotic ResistanceAntibioticsBacteriaBacterial InfectionsBar CodesBindingBiological AssayCellular StructuresClinicalCollaborationsComplementCoupledCryoelectron MicroscopyDevelopmentDissociationEconomicsElementsEngineeringEnterococcus faecalisEscherichia coliEvaluationFellowshipFutureGoalsGrantGrowthHydrogen BondingLaboratoriesLibrariesMeasuresMinimum Inhibitory Concentration measurementModern MedicineModernizationModificationMolecularMolecular ConformationMutateMutationNatural ProductsNeedlesOutcome StudyPeptidyltransferasePharmaceutical PreparationsProteinsRNAResistanceResistance profileResolutionRibosomal ProteinsRibosomesRoentgen RaysSocietiesStreptograminsStreptomycesStructureSubstrate SpecificitySurvivorsTechniquesTranslatingTranslationsTransmembrane DomainUrsidae FamilyVariantanalogantimicrobialappendagebaseclinically relevantcombatdeep sequencingdesignefflux pumpemerging antibiotic resistanceexperimental studyinhibitor/antagonistinnovationinsightmutation screeningnext generationresistance mechanismresistant strainscreeningsmall moleculetraining opportunitytranslation assay
中文摘要
项目摘要/摘要-低温电镜引导下核糖体靶向抗生素的增强
英文摘要
Project Summary/Abstract - CryoEM Guided Enhancement of Ribosome-Targeting Antibiotics
The continued emergence of antibiotic resistance materially threatens modern medicine and by
extension, modern society. A dearth of newly developed or discovered antibiotics has made the situation even
more dire, as pan-resistant strains have emerged. Retooling and modifying existing classes of antibiotics offers
an opportunity to study and counteract resistance mechanisms while producing truly rationally-designed small
molecule drug leads. Iterative rounds of rapid structural characterization via cryoelectron microscopy (CryoEM)
and synthetic modification offers a rational approach to such retooling efforts. One of the most prolific targets
for antibiotics is the bacterial ribosome, which is inhibited by streptogramin antibiotics produced by several
species of Streptomyces. Streptogramin A (SA) binds at the peptidyl transferase center of the ribosome.
Streptogramins are of limited value clinically because of resistance mechanisms including inactivation by
acetyltransferases such as VatA and molecular interference by ribosomal protection proteins which displace
the inhibitor. In collaboration with the Seiple laboratory at UCSF, we have access to a wide variety of
streptogramin analogs. These are produced using modular synthesis, to allow rapid access to variations in
structural and hydrogen bonding elements. Initially, we will use rounds of Minimum Inhibitory Concentration
screening and structural characterization to increase the efficacy of streptogramin analogs for the E. coli
ribosome. These will be followed by studies tuning the activity of streptogramin A analogs against bacteria
expressing VatA, the acetyltransferase. Analogs with inhibitory effects will be characterized by CryoEM and
acetylation rates will be measured to complement the resistance profiles. To probe the rise of
acetylation-based resistance, we will then passage E. coli expressing VatA in sub-MIC levels of SA analogs
and sequence survivors. We will complement this by comprehensive mutation of VatA by deep mutational
scanning, performing parallel competitive growth and deep sequencing. Together, these approaches will
identify mutations that act as global stabilizers and mutations that provide substrate specificity, and guide
efforts to enhance steric clashes between the SA analog and “unmutable” residues identified. Following these
studies, we will apply similar techniques to explore the structural basis of a ribosomal protection protein, EfrCD.
Such proteins bear homology to efflux pumps, but lack the transmembrane domains required for cellular efflux.
Explorations of the structural space within the ribosome in the presence of such protection proteins will help
probe the poorly understood mechanisms of such resistance. Ultimately, the explosive growth of cryoEM
coupled to modern modular synthesis provides an opportunity to retool antibiotic classes with limited clinical
relevance by specifically modifying them to combat and understand mechanisms of antibiotic resistance.
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