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Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

Rasch J, Theuerkorn M, Ünal C, Heinsohn N, Tran S, Fischer G, Weiwad M, Steinert M - Front Bioeng Biotechnol (2015)

Bottom Line: Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria.Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity.The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays.

View Article: PubMed Central - PubMed

Affiliation: Institut für Mikrobiologie, Technische Universität Braunschweig , Braunschweig , Germany.

ABSTRACT
Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

No MeSH data available.


Related in: MedlinePlus

Cycloheximide derivatives differentially inhibit bacterial replication during infection and are not cytotoxic. (A) All inhibitors were tested in macrophage infection assays at concentrations ranging from 12.5 to 100 μM. The substances were added at the indicated concentrations 2 h post infection, and the bacterial replication was monitored by determining the colony forming units/milliliter (cfu/ml) 24 h post infection. The novel PPIase inhibitors MT_30.32 and MT_30.51 effectively suppressed bacterial replication during infection of differentiated THP-1 cells in a concentration dependent manner. The remaining seven derivatives had no effect at the highest concentration tested as demonstrated by the example MT_30.9, and were comparable to untreated infections containing only 1% (v/v) ethanol as the solvent at its final concentration. The graph depicts mean and SD of two independent experiments performed in duplicate as a representative of four biological replicates. (B) Differentiated THP-1 cells were incubated with 100 μM MT_30.32 or MT_30.51 for 24 h. After 20 h, alamar blue was added and cell viability was determined by measuring fluorescence at 590 nm. The medium of control cells was either free of additives (untreated) or contained 1% (v/v) EtOH as a solvent control. The graph shows the mean and SD of two independent experiments performed in triplicate.
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Figure 2: Cycloheximide derivatives differentially inhibit bacterial replication during infection and are not cytotoxic. (A) All inhibitors were tested in macrophage infection assays at concentrations ranging from 12.5 to 100 μM. The substances were added at the indicated concentrations 2 h post infection, and the bacterial replication was monitored by determining the colony forming units/milliliter (cfu/ml) 24 h post infection. The novel PPIase inhibitors MT_30.32 and MT_30.51 effectively suppressed bacterial replication during infection of differentiated THP-1 cells in a concentration dependent manner. The remaining seven derivatives had no effect at the highest concentration tested as demonstrated by the example MT_30.9, and were comparable to untreated infections containing only 1% (v/v) ethanol as the solvent at its final concentration. The graph depicts mean and SD of two independent experiments performed in duplicate as a representative of four biological replicates. (B) Differentiated THP-1 cells were incubated with 100 μM MT_30.32 or MT_30.51 for 24 h. After 20 h, alamar blue was added and cell viability was determined by measuring fluorescence at 590 nm. The medium of control cells was either free of additives (untreated) or contained 1% (v/v) EtOH as a solvent control. The graph shows the mean and SD of two independent experiments performed in triplicate.

Mentions: During lung infection, L. pneumophila parasitizes human alveolar macrophages for its replication. Therefore, we tested whether the strongest inhibitors MT_30.32 and 30.51 were also capable of inhibiting bacterial replication during infection in differentiated THP-1 macrophages. In accordance with the protease coupled PPIase and MIC assays, both substances suppressed bacterial replication in THP-1 cells in a concentration dependent manner starting at 50 μM. In contrast, MT_30.9 that also inhibited bacterial growth but was about 18-fold less effective than MT_30.32 in the protease coupled PPIase-assay, had no effect on bacterial replication in THP-1 cells at the highest tested concentration of 100 μM (Figure 2A). None of the substances were cytotoxic to the THP-1 cells (Figure 2B).


Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

Rasch J, Theuerkorn M, Ünal C, Heinsohn N, Tran S, Fischer G, Weiwad M, Steinert M - Front Bioeng Biotechnol (2015)

Cycloheximide derivatives differentially inhibit bacterial replication during infection and are not cytotoxic. (A) All inhibitors were tested in macrophage infection assays at concentrations ranging from 12.5 to 100 μM. The substances were added at the indicated concentrations 2 h post infection, and the bacterial replication was monitored by determining the colony forming units/milliliter (cfu/ml) 24 h post infection. The novel PPIase inhibitors MT_30.32 and MT_30.51 effectively suppressed bacterial replication during infection of differentiated THP-1 cells in a concentration dependent manner. The remaining seven derivatives had no effect at the highest concentration tested as demonstrated by the example MT_30.9, and were comparable to untreated infections containing only 1% (v/v) ethanol as the solvent at its final concentration. The graph depicts mean and SD of two independent experiments performed in duplicate as a representative of four biological replicates. (B) Differentiated THP-1 cells were incubated with 100 μM MT_30.32 or MT_30.51 for 24 h. After 20 h, alamar blue was added and cell viability was determined by measuring fluorescence at 590 nm. The medium of control cells was either free of additives (untreated) or contained 1% (v/v) EtOH as a solvent control. The graph shows the mean and SD of two independent experiments performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376002&req=5

Figure 2: Cycloheximide derivatives differentially inhibit bacterial replication during infection and are not cytotoxic. (A) All inhibitors were tested in macrophage infection assays at concentrations ranging from 12.5 to 100 μM. The substances were added at the indicated concentrations 2 h post infection, and the bacterial replication was monitored by determining the colony forming units/milliliter (cfu/ml) 24 h post infection. The novel PPIase inhibitors MT_30.32 and MT_30.51 effectively suppressed bacterial replication during infection of differentiated THP-1 cells in a concentration dependent manner. The remaining seven derivatives had no effect at the highest concentration tested as demonstrated by the example MT_30.9, and were comparable to untreated infections containing only 1% (v/v) ethanol as the solvent at its final concentration. The graph depicts mean and SD of two independent experiments performed in duplicate as a representative of four biological replicates. (B) Differentiated THP-1 cells were incubated with 100 μM MT_30.32 or MT_30.51 for 24 h. After 20 h, alamar blue was added and cell viability was determined by measuring fluorescence at 590 nm. The medium of control cells was either free of additives (untreated) or contained 1% (v/v) EtOH as a solvent control. The graph shows the mean and SD of two independent experiments performed in triplicate.
Mentions: During lung infection, L. pneumophila parasitizes human alveolar macrophages for its replication. Therefore, we tested whether the strongest inhibitors MT_30.32 and 30.51 were also capable of inhibiting bacterial replication during infection in differentiated THP-1 macrophages. In accordance with the protease coupled PPIase and MIC assays, both substances suppressed bacterial replication in THP-1 cells in a concentration dependent manner starting at 50 μM. In contrast, MT_30.9 that also inhibited bacterial growth but was about 18-fold less effective than MT_30.32 in the protease coupled PPIase-assay, had no effect on bacterial replication in THP-1 cells at the highest tested concentration of 100 μM (Figure 2A). None of the substances were cytotoxic to the THP-1 cells (Figure 2B).

Bottom Line: Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria.Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity.The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays.

View Article: PubMed Central - PubMed

Affiliation: Institut für Mikrobiologie, Technische Universität Braunschweig , Braunschweig , Germany.

ABSTRACT
Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

No MeSH data available.


Related in: MedlinePlus