Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant.
OBJECTIVE
Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. The aim of this study was to screen a library of small chemical compounds to identify cell wall inhibitors using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans. This mutant is specifically susceptible to cell wall damaging compounds under PKC-repressive growth conditions.


METHODS
The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment.


RESULTS
Three 'hit' compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole.


CONCLUSIONS
This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.
Mircus
Gabriel
G
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
Hagag
Shelly
S
Levdansky
Emma
E
Sharon
Haim
H
Shadkchan
Yona
Y
Shalit
Itamar
I
Osherov
Nir
N
D000935:Antifungal Agents / Q000494:pharmacology*; D001233:Aspergillus nidulans / Q000187:drug effects* / Q000201:enzymology* / Q000648:ultrastructure; D002473:Cell Wall / Q000187:drug effects* / Q000648:ultrastructure; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism
D000935:Antifungal Agents; D005656:Fungal Proteins; D011493:Protein Kinase C