Chemically Induced Haploinsufficiency Screens to Identify Drug Mechanism of Action in Aspergillus Fumigatus

Ref ID: 19021

Author:

D. A. Macdonald, A. E. Johns, M. Eberle, P. Bowyer, D. Denning, M. J. Bromley

Author address:

Institute of Inflammation and Repair, Respiratory & Allergy Centre, University of Manchester,
Manchester, United Kingdom

Full conference title:

27th Fungal Genetics Conference

Date: 12 March 2014

Abstract:

Current drugs used to treat Aspergillus infections are limited and suffer from a variety of shortcomings including low efficacy, toxicity and increasing
resistance. Despite the discovery of numerous promising drug targets, few lead compounds have been discovered by target based approaches. This can be
explained, in part, by the ’druggability’ of a target as some compounds which demonstrate promising activity against an enzyme are not active against the
whole cell or are toxic to humans. Consequently most of the antimicrobials presently on the market were originally discovered by random screening of
compounds against whole cell screens. A solution to this problem is to identify gene targets utilizing compounds that already show antifungal activity and
have clean toxicity profiles.
Chemical genetic profiling aids identification of drug mechanism of action as a diploid strain lacking a single copy of a drug’s target is hypersensitive to
that drug. Heterozygote S. cerevisiae and C. albicans libraries have been used to identify the mechanism of action of several promising compounds;
however, this has been hindered in A. fumigatus by the complexity in generating an adequate set of heterozygous strains. A high-throughput targeted
gene KO method for A. fumigatus has been established by employing fusion-PCR to generate targeted gene disruption cassettes, optimizing the common
transformation protocol for A. fumigatus high-throughput gene disruption, and utilising a diploid Ku80-
/Ku80-mutant to facilitate more reliable
homologous recombination. Preliminary efforts have produced 46 heterozygous KO strains and subsequently, the feasibility of chemical genetic
haploinsufficiency studies in filamentous fungi has been demonstrated with several compounds. High-throughput methods of chemical genetic profiling by
pooling multiple heterozygous KO strains into a single culture is currently being validated and preliminary data is promising. This will enable highthroughput methods for surveying the genome of A. fumigatus for new drug targets and supports unveiling the mechanisms of action of antifungal drug

Abstract Number: N/A

Conference Year: 2013

Link to conference website: http://www.fungalgenetics.org/2013/documents/FungalProgramBook2013.pdf

New link: NULL

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