Antifungal Target Discovery and Validation

Michael Bromley, Paul Carr, Sandra Howsley, Sarah Kaye, Danny Tuckwell and Jason Oliver*
F2G Ltd., Lankro Way, Eccles, Manchester M30 0BH, UK. www.f2g.com

Introduction Target Validation II

New antifungal drugs are required to treat the increasing numbers of patients suffering For the further validation of promising targets a variety of techniques are available to us:
from serious fungal infections. Existing antifungal drugs are active on only a limited number
of molecular targets, so new agents acting on novel targets are needed. To address this 1. Confirm essentiality of a gene by:
need F2G carries out target-based screening as well as more traditional whole cell • directed knockout or disruption
screening approaches. The steps from gene to screen are as follows: • rescue – can Mycobank KO strain be rescued by an intact copy of the gene?
Target discovery - Mycobank® identifies genes that are essential for the growth of
Aspergillus fumigatus. 2. Conditional expression – Does down-regulation of a gene affect growth?
Target validation - determines whether these genes will make good antifungal drug Cellobiohydrolase B & AlcA promoters can be used to vary gene expression. They can
targets. replace native promoters to control gene expression directly or regulate gene silencing
Screen development - selected genes are then chosen for assay and screen by RNAi [2].
development.
3. Is a gene expressed during infection? RNA is extracted from infected tissue followed by
real time PCR analysis of gene expression (see figure 4).
Target Discovery - Mycobank®
Genes essential for the growth of Aspergillus fumigatus are identified by the Mycobank Quantification of expressed target
process. Genes are disrupted by a tagged transposon in a modified diploid strain of A infected mouse tissue
fumigatus. The diploid is then haploidised, a process that yields colour mutant haploids
(brown and white). The haploids are inoculated on media selective for the tag. If no growth is
observed then the transposon has disrupted a gene essential for growth. That gene can be RNA
identified by investigation of the diploid Mycobank strain.
cDNA

Brown haploid White haploid Green diploid cDNA from static culture (16h)
real time PCR cDNA from infected mouse kidney (48h)
Protoplast fusion
A
B
cDNA from non-infected mouse kidney (48h)
-ve control (no cDNA)
Viable - growth Tagged transposon
C (pyrG cassette) Figure 4. Real-time PCR analysis of a Mycobank hit. Expression of the gene is compared to housekeeping
controls in infected & non-infected tissue and in A fumigatus culture.

Forced breakdown
Selection
D
Screen Development
Gene knock-out
Mycobank®
-
®
How a target is assayed is obviously dependent on the function of the target. The assay
must be amenable to high-throughput screening. Suitable endpoints include
Lethal – no growth
E spectrophotometric, fluorimetric and luminescent detection. The process is exemplified
Figure 1. The Mycobank process below by a typical Mycobank target – oxidoreductase 2031. Oxidoreductase 2031 belongs to
a family of enzymes related to Old Yellow Enzyme of yeast [3]. It is a novel broad spectrum
target with no human homologue.
Mycobank output

h h
eluates

w oug
73 genes have been identified by Mycobank as being essential for growth.

2
wa 1
IPTG

r
flo e

sh
th
E1 E2 E3 E4

at

as
Following recent improvements, this number will increase rapidly (current rate of discovery - +

w
s
ly
is 150 genes per year).
A diverse range of cellular processes are involved:
RNA metabolic process, 17
Recombinant protein expression
ribosome biogenesis
and assembly, 14 For most assays reliable supplies of target
process unknown, 20
protein are required. Most commonly this is
transport, 13
produced by heterologous expression in E
organelle organization
response to stress, 9
coli.
and biogenesis, 25

translation, 8

transcription, 7
sporulation, 1
DNA metabolic process, 6
conjugation, 1
cytoskeleton organization
membrane organization
and biogenesis, 6
and biogenesis, 1
protein modification, 5
Figure 5. Expression in E coli and purification of A
carbohydrate metabolic process, 1
amino acid and derivative
fumigatus oxidoreductase 2031. 2031 protein is indicated
cell budding, 1

cell wall organization and biogenesis, 1

metabolic process, 5
by arrow.
cell cycle, 5
signal transduction, 2

cellular respiration, 2 lipid metabolic process, 4

Assay Development
nuclear organization and biogenesis, 2 vesicle-mediated transport, 4 An assay has been described for this family of enzymes [4]. The substrate 2-cyclohexenone
protein catabolic process, 2 generation of precursor metabolites and energy, 3 is reduced and NADPH is oxidised. NADPH oxidation is accompanied by decrease in
anatomical structure morphogenesis, 2
cytokinesis, 2
absorbance at 340nm.
nmoles NADPH converted

100
90
NADPH NADP
Figure 2. Classification of Mycobank hits according to GO-slim annotation scheme [1]. 80
70

Genes can be classified in more than one category 60

50
40
30
20
10

2-cyclohexenone cyclohexanone 0
0 200 400 600 800 1000

Target Validation I uM cyclohexenone

Figure 6. 2-cyclohexenone reduction assay for oxidoreductase 2031. Graph shows the amount of
Bioinformatics and literature analysis provides information for each gene. This work NADPH oxidised by 2031 for increasing amount of substrate.
provides an initial assessment of the suitability of a gene as an antifungal target.
The 2031 assay was developed into a 384-well high-throughput screen. The output of the
screen is currently being evaluated.
Putative Gene Targets

Summary
Novelty Absence of close IP position Presence in other
human homologue fungal species • Mycobank provides choice and variety of novel antifungal targets
• Target validation combines in silico and experimental approaches to identify the
best targets
• Screens have been developed for oxidoreductase 2031 and 3 other targets
Orphan Known Characterized Filamentous Yeast and Mixed
sequences function homologue only filamentous

References
Structural Other Enzyme/receptor [1] Ashburner, M. et al. (2000) Nat Genet. 25:25-9.
[2] Bromley, M, Gordon, C, Rovira-Graells, N & Oliver, J. (2006) FEMS Microbiol Lett. 264:246-54.
[3] Williams, R.E. and Bruce, N.C. (2002) Microbiology 148, 1607-1614.
Figure 3. Bioinformatic analysis of putative gene targets.
[4] Abramovitz and Massey (1976) J. Biol. Chem. 251, 5321-5326.

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