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                <term xml:lang="en">bacterial transcription inhibitors</term>
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                <term xml:lang="en">bacterial RNA polymerase (RNAP)</term>
                <term xml:lang="en">protein-protein interaction inhibitor</term>
                <term xml:lang="en">protein-protein interaction (PPI)</term>
                <term xml:lang="en">antibiotics</term>
                <term xml:lang="en">drug-discovery workflow</term>
                <term xml:lang="en">yeast Bioluminescence Resonance Energy Transfer (yBRET)</term>
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              <p>Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein–protein interaction between the β’ subunit and the σ70 initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the β’–σ70 interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87–1.56 μM) thus far reported for β’–σ PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors</p>
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