A new anti-Aspergillosis inhibitor targeting N- myristoyltransferase in Aspergillus fumigatus

Ref ID: 19430

Author:

W. Fang,1 D. A. Robinson,2 D. E. Blair,1 O. G. Raimi,1
L. S. Torrie,2 I. H. Gilbert1 and D. M. F. Van Aalten1

Author address:

1University of Dundee, United Kingdom and 2Drug Discovery
Unit, University of Dundee, United Kingdom

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives MyristoylCoA:protein N-myristoyltransferase (NMT, EC:
2.1.3.97) is a highly conserved protein that catalyzes the covalent
attachment of a 14-carbon saturated fatty acid myristate onto the N-
terminal glycine residue of a variety of eukaryotic proteins. Many of
the target proteins are crucial components of signaling pathways,
and myristoylation typically promotes membrane binding that is
essential for proper protein localization or biological function. NMT
has been genetically validated as a therapeutic target in several
human pathogens, including parasitic protozoa Leishmania major1
and Trypanosoma brucei2 as well as fungal species Candida albicans3
and Cryptococcus neoformans4. A number of NMT inhibitors have
been reported in those species. However, little in known about NMT
in human opportunistic pathogen A. fumigatus. Our work focuses on
1) does NMT exist in A. fumigatus? 2) is it essential and 3) is it drug-
gable in A. fumigatus?
Methods Single nmt gene was cloned from A. fumigatus. Recombi-
nant AfNMT from Escherichia coli was characterized. No knockout
strain of AfNMT was obtained after several trials. Therefore, a con-
ditional mutant was constructed by replacing the native promoter
of the A.fumigatus nmt gene with the Aspergillus nidulans alcA pro-
moter. Phenotypic analyses, including growth rate, temperature sensitivity, germination and cell wall structure, were performed for
conditional mutant. A small library of previously described CaNMT
and TbNMT inhibitors was tested on both AfNMT enzyme and A.
fumigatus. Furthermore, crystal structure of AfNMT was deter-
mined. Promising inhibitors from the screening were co-crystallized
with AfNMT.
Results A. fumigatus possesses an active NMT, which shows low Km
for both myristyl-CoA and peptide substrates. Under strict repression
conditions conditional NMT mutant cannot grow, demonstrating that
nmt is essential for A. fumigatus survival. Under partial repression
condition the mutant exhibits retarded hyphal growth, cell wall
defect, delayed germination and abnormal polarity establishment and
maintenance. Different from the NMT mutants in other fungal spe-
cies, the condition mutant is not susceptible to high temperature and
myristic supplementation doesn’t restore its normal hyphal growth,
suggesting NMT targets in A. fumigatus are different from other fungi.
Inhibitors screening revealed that although several inhibitors are
very potent with IC50 in the nM range against AfNMT, only a deriva-
tive of TbNMT inhibitors showed inhibition on A. fumigatus in a
AfNMT-dependent manner. Co-crystallized AfNMT-inhibitors com-
plexes provided structure guides for more potent anti-aspergillosis
inhibitors.
Conclusion A. fumigatus has an essential and active NMT that tar-
gets proteins different from other fungi. A promising AfNMT inhibitor
showed potent inhibition both on this enzyme and the organism, rep-
resenting a new anti-Aspergillosis inhibitor that would be exploitable
in the future.
Reference
1. C. Panethymitaki et al., Biochem J 396, 277 (Jun 1, 2006).
2. J. A. Frearson et al., Nature 464, 728 (Apr 1,2010).
3. R. A. Weinberg et al., Mol Microbiol 16, 241 (Apr, 1995).
4. J. K. Lodge, E. Jackson-Machelski, D. L. Toffaletti, J. R. Perfect, J. I.
Gordon, Proc Natl Acad Sci U S A 91, 12008 (Dec 6, 1994).

Abstract Number: p254

Conference Year: 2013

Link to conference website: NULL

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