“Multiplexed screen identifies a<i>Pseudomonas aeruginosa</i>-specific small molecule targeting the outer membrane protein OprH and its interaction with LPS”

Poulsen, Bradley E.; Warrier, Thulasi; Barkho, Sulyman; Bagnall, Josephine; Romano, Keith P.; White, Tiantian; Yu, Xiao; Kawate, Tomohiko; Nguyen, Phuong H.; Raines, Kyra; Ferrara, Kristina; Golas, Aaron; Fitzgerald, Michael; Boeszoermenyi, Andras; Kaushik, Virendar; Serrano-Wu, Michael; Shoresh, Noam; Hung, Deborah T.

doi:10.1101/2024.03.16.585348

Cited by 2 publications

References 89 publications

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Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics

Romano,

Bagnall,

Warrier

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the postgenomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called perturbation-specific transcriptional mapping (PerSpecTM), in which large-throughput expression profiling of wild-type or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small-molecule inhibition resemble those resulting from genetic depletion of essential targets by clustered regularly interspaced short palindromic repeats interference (CRISPRi) by PerSpecTM, demonstrating proof of concept that correlations between expression profiles of small-molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

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Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics

Romano,

Bagnall,

Warrier

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Perturbation-Specific Transcriptional Mapping for unbiased target elucidation of antibiotics

Romano,

Bagnall,

Warrier

et al. 2024

Preprint

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The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are beginning to combat the threat, target elucidation of new chemical entities remains challenging. In the post-genomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction based on the principle that molecules with similar MOAs elicit similar transcriptional responses. However, the broad application of transcriptomics-based MOA prediction remains limited by technical confounding that encumber experimental throughput. Here we report an unbiased strategy for MOA prediction, called Perturbation-Specific Transcriptional Mapping (PerSpecTM), in which large-throughput transcriptional sequencing enables the identification of perturbation-specific gene responses to either target inhibition or transcriptional repression. We applied this approach to build an antimicrobial reference set comprising compounds with known activity against the important bacterial pathogen P. aeruginosa. We rapidly elucidated the MOAs of three new molecules with anti-pseudomonal activity, and importantly, demonstrated the value of using hypersensitized strains, depleted for essential targets, to yield more robust transcriptional responses to perturbation. Using CRISPRi, we then constructed a transcriptionally repressed reference set and demonstrated proof-of-concept that gene responses to gene target depletion can mimic its chemical inhibition, affording MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM and mutant strains depleted in essential targets, this work enables an unbiased and readily scalable reference-based strategy for MOA elucidation to assist antibiotic development efforts in an era fraught with resistance.

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“Multiplexed screen identifies aPseudomonas aeruginosa-specific small molecule targeting the outer membrane protein OprH and its interaction with LPS”

Cited by 2 publications

References 89 publications

Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics

Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics

Perturbation-Specific Transcriptional Mapping for unbiased target elucidation of antibiotics

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