New Multidrug Efflux Inhibitors for Gram-Negative Bacteria
ABSTRACT Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiot...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-08-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01340-20 |
| _version_ | 1818927445549514752 |
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| author | Robert L. Marshall Georgina S. Lloyd Amelia J. Lawler Sarah J. Element Jaswant Kaur Maria Laura Ciusa Vito Ricci Andreas Tschumi Holger Kühne Luke J. Alderwick Laura J. V. Piddock |
| author_facet | Robert L. Marshall Georgina S. Lloyd Amelia J. Lawler Sarah J. Element Jaswant Kaur Maria Laura Ciusa Vito Ricci Andreas Tschumi Holger Kühne Luke J. Alderwick Laura J. V. Piddock |
| author_sort | Robert L. Marshall |
| collection | DOAJ |
| description | ABSTRACT Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study. |
| first_indexed | 2024-12-20T03:13:08Z |
| format | Article |
| id | doaj.art-2629c888b070459bbab02735e914fc43 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-20T03:13:08Z |
| publishDate | 2020-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-2629c888b070459bbab02735e914fc432022-12-21T19:55:25ZengAmerican Society for MicrobiologymBio2150-75112020-08-0111410.1128/mBio.01340-20New Multidrug Efflux Inhibitors for Gram-Negative BacteriaRobert L. Marshall0Georgina S. Lloyd1Amelia J. Lawler2Sarah J. Element3Jaswant Kaur4Maria Laura Ciusa5Vito Ricci6Andreas Tschumi7Holger Kühne8Luke J. Alderwick9Laura J. V. Piddock10Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomRoche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, SwitzerlandRoche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, SwitzerlandSchool of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, United KingdomInstitute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United KingdomABSTRACT Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.https://journals.asm.org/doi/10.1128/mBio.01340-20efflux inhibitorsGFPhigh throughputRamA |
| spellingShingle | Robert L. Marshall Georgina S. Lloyd Amelia J. Lawler Sarah J. Element Jaswant Kaur Maria Laura Ciusa Vito Ricci Andreas Tschumi Holger Kühne Luke J. Alderwick Laura J. V. Piddock New Multidrug Efflux Inhibitors for Gram-Negative Bacteria mBio efflux inhibitors GFP high throughput RamA |
| title | New Multidrug Efflux Inhibitors for Gram-Negative Bacteria |
| title_full | New Multidrug Efflux Inhibitors for Gram-Negative Bacteria |
| title_fullStr | New Multidrug Efflux Inhibitors for Gram-Negative Bacteria |
| title_full_unstemmed | New Multidrug Efflux Inhibitors for Gram-Negative Bacteria |
| title_short | New Multidrug Efflux Inhibitors for Gram-Negative Bacteria |
| title_sort | new multidrug efflux inhibitors for gram negative bacteria |
| topic | efflux inhibitors GFP high throughput RamA |
| url | https://journals.asm.org/doi/10.1128/mBio.01340-20 |
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