Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila.
The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).
Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich , 80336 Munich, Germany.
Christopher F
CF
Harrison
School of Pharmaceutical Sciences, Department of Pharmaceutical Biochemistry, University of Geneva and University of Lausanne , 1211 Geneva, Switzerland.
Gianpaolo
G
Chiriano
Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich , 80336 Munich, Germany.
Ivo
I
Finsel
Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich , 80336 Munich, Germany.
Christian
C
Manske
Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich , 80336 Munich, Germany.
Christine
C
Hoffmann
Institute of Medical Microbiology, Department of Medicine, University of Zurich , Gloriastrasse 30/32, 8006 Zurich, Switzerland.
Bernhard
B
Steiner
School of Pharmaceutical Sciences, Department of Pharmaceutical Biochemistry, University of Geneva and University of Lausanne , 1211 Geneva, Switzerland.
Agata
A
Kranjc
School of Pharmaceutical Sciences, Department of Pharmaceutical Biochemistry, University of Geneva and University of Lausanne , 1211 Geneva, Switzerland.
Ophelie
O
Patthey-Vuadens
Sébastien
S
Kicka
Valentin
V
Trofimov
Hajer
H
Ouertatani-Sakouhi
Thierry
T
Soldati
School of Pharmaceutical Sciences, Department of Pharmaceutical Biochemistry, University of Geneva and University of Lausanne , 1211 Geneva, Switzerland.
Leonardo
L
Scapozza
Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich , 80336 Munich, Germany.|Institute of Medical Microbiology, Department of Medicine, University of Zurich , Gloriastrasse 30/32, 8006 Zurich, Switzerland.
Hubert
H
Hilbi
Legionella; amoeba; antibiotics; antivirulence; calcineurin; intracellular replication; macrophage; pathogen vacuole; screen; structure−activity relationship (SAR); type IV secretion