Novel antimicrobials targeting type IV pilus and type 2 secretion systems

针对 IV 型菌毛和 2 型分泌系统的新型抗菌药物

• 批准号: 9089860
• 负责人: MICHAEL S DONNENBERG
• 金额: $ 4.57万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089860
• 关键词: Acinetobacter; Acinetobacter baumannii; Active Sites; Adherence; Animal Model; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Aspartic Acid; Bacteria; Bacterial Infections; Binding; Biogenesis; Biological Assay; Characteristics; Chemicals; Chimeric Proteins; Complex; Disease; Drug Targeting; DsRed; Energy Transfer; Enzymes; Equipment; Escherichia coli; Fimbriae Proteins; Fluorescence; Fluorescence Anisotropy; Fluorescence Polarization; Formulation; Fostering; Goals; Growth; Health; Human; Immunoblotting; In Vitro; Infection; Lead; Libraries; Maryland; Medicine; Membrane; Modeling; Molecular Machines; Molecular Models; Mutation; N-terminal; Neisseria gonorrhoeae; Organism; Pathogenesis; Peptide Hydrolases; Peptide Signal Sequences; Performance; Pharmaceutical Chemistry; Phase; Physicians; Pilum; Pneumonia; Preclinical Drug Evaluation; Preclinical Testing; Prevention; Probability; Property; Protease Inhibitor; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa pneumonia; Quality Control; Recombinant Proteins; Resistance; Resources; Series; Specificity; Structure; Surface; System; Testing; Therapeutic; Toxin; Universities; Validation; Virulence; analog; antimicrobial; antimicrobial drug; appendage; drug development; drug discovery; efficacy testing; enteropathogenic Escherichia coli; experience; improved; in vitro activity; in vivo; inhibitor/antagonist; innovation; killings; molecular modeling; mouse model; mutant; novel; novel therapeutics; pathogen; prevent; protein S precursor; screening; small molecule; success; therapeutic target

 DESCRIPTION (provided by applicant): Bacterial pathogens have conserved complex molecular machines that are required to cause disease. Among these machines are those that make up a type IV pilus (T4P) biogenesis apparatus and the related type II secretion (T2S) machine. T4Ps are surface appendages essential for adherence and host colonization by many important bacterial pathogens; T2S systems export toxins that are critical for many diseases. Acinetobacter baumanii, Pseudomonas aeruginosa, and Neisseria gonorrhoeae all produce T4Ps. Among the components common to both machines is a pre-pilin peptidase enzyme that specifically removes the characteristic N-terminal signal sequence from pre-pilin proteins and N-methylates the nascent amino terminus. These enzymes are highly conserved, absolutely required for T4P and T2S function, and represent a novel class of neutral pH aspartic acid peptidases that do not resemble mammalian enzymes. As such, they are ideal targets for new drug discovery. We have devised an in vivo Föster resonance energy transfer (FRET) system comprised of three recombinant proteins expressed in E. coli: the pre-pilin peptidase, the pre-pilin protein fused to enhanced yellow fluorescent protein (eYFP), and an essential assembly protein to which the pre-pilin binds fused to dsRed. Cleavage of the pre-pilin fusion protein by the pre-pilin peptidase releases eYFP and prevents FRET with dsRed. If the pre-pilin peptidase is inactivated by mutation, then FRET occurs. We will adapt this extremely sensitive system and a related even simpler system that relies on fluorescence anisotropy to develop in vitro high-throughput small molecule screens to identify pre-pilin peptidase inhibitor candidates. Secondary assays will establish specificity and spectrum of activity. The initial effort will provie candidates that will subsequently subjected to further chemical refinement to enhance potency, specificity and activity against whole bacteria. Finally, (a) lead compound(s) will be advanced to preclinical testing using an established murine model of P. aeruginosa pneumonia in which T4Ps are required for disease. Ultimately, the goal is to discover an entirely novel therapeutic class of anti-virulence agents to improve human health.