Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen
The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a ne...
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doaj-8d1728843dd04b85a73fc6e17f622b9d2021-03-14T00:01:48ZengMDPI AGMicroorganisms2076-26072021-03-01959259210.3390/microorganisms9030592Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion ScreenMohamed Belal Hamed0Ewa Burchacka1Liselotte Angus2Arnaud Marchand3Jozefien De Geyter4Maria S. Loos5Jozef Anné6Hugo Klaassen7Patrick Chaltin8Spyridoula Karamanou9Anastassios Economou10Laboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumCistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, BelgiumCistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumCistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, BelgiumCistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumLaboratory of Molecular Bacteriology, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, BelgiumThe increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC<sub>50</sub> < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC<sub>50</sub> of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC<sub>50</sub> ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of <i>E. coli</i> strains with IC<sub>50</sub> < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an <i>E. coli</i> strain missing TolC to improve permeability with IC<sub>50</sub> 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.https://www.mdpi.com/2076-2607/9/3/592<i>E. coli</i>alkaline phosphatasesmall molecule inhibitorsantibacterialsprotein secretion |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mohamed Belal Hamed Ewa Burchacka Liselotte Angus Arnaud Marchand Jozefien De Geyter Maria S. Loos Jozef Anné Hugo Klaassen Patrick Chaltin Spyridoula Karamanou Anastassios Economou |
spellingShingle |
Mohamed Belal Hamed Ewa Burchacka Liselotte Angus Arnaud Marchand Jozefien De Geyter Maria S. Loos Jozef Anné Hugo Klaassen Patrick Chaltin Spyridoula Karamanou Anastassios Economou Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen Microorganisms <i>E. coli</i> alkaline phosphatase small molecule inhibitors antibacterials protein secretion |
author_facet |
Mohamed Belal Hamed Ewa Burchacka Liselotte Angus Arnaud Marchand Jozefien De Geyter Maria S. Loos Jozef Anné Hugo Klaassen Patrick Chaltin Spyridoula Karamanou Anastassios Economou |
author_sort |
Mohamed Belal Hamed |
title |
Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen |
title_short |
Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen |
title_full |
Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen |
title_fullStr |
Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen |
title_full_unstemmed |
Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen |
title_sort |
effective small molecule antibacterials from a novel anti-protein secretion screen |
publisher |
MDPI AG |
series |
Microorganisms |
issn |
2076-2607 |
publishDate |
2021-03-01 |
description |
The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC<sub>50</sub> < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC<sub>50</sub> of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC<sub>50</sub> ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of <i>E. coli</i> strains with IC<sub>50</sub> < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an <i>E. coli</i> strain missing TolC to improve permeability with IC<sub>50</sub> 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics. |
topic |
<i>E. coli</i> alkaline phosphatase small molecule inhibitors antibacterials protein secretion |
url |
https://www.mdpi.com/2076-2607/9/3/592 |
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