Discovering Modes of Discovering Modes of Action for Therapeutic Action for Therapeutic Compounds Using a Compounds Using a

Genome-Wide Screen of Genome-Wide Screen of Yeast HeterozygotesYeast HeterozygotesPek Yee Lum, Christopher D. Armour, Pek Yee Lum, Christopher D. Armour,

Daniel D. Shoemaker, Daniel D. Shoemaker, et al.et al.

Cell, Vol. 116, 121-137, January 9, 2004Cell, Vol. 116, 121-137, January 9, 2004

Purpose of this paper:Purpose of this paper:

Knowledge of the underlying molecular Knowledge of the underlying molecular mechanisms of drugs + their targetsmechanisms of drugs + their targets

Use of “Fitness Profiling” for Use of “Fitness Profiling” for understanding drug activitiesunderstanding drug activities

INTRODUCTIONINTRODUCTION

Need for new tools that can rapidly Need for new tools that can rapidly identify protein targets of small identify protein targets of small molecules.molecules.

i.e. Protein arrays, reverse transfection, and i.e. Protein arrays, reverse transfection, and DNA microarraysDNA microarrays

Saccharomyces cerevisiaeSaccharomyces cerevisiae

APPROACHAPPROACH A study by Giaever A study by Giaever et al. et al. (1999) demonstrated that parallel (1999) demonstrated that parallel

analysis of yeast strains with heterozygous deletions of analysis of yeast strains with heterozygous deletions of drug target genes can be used to monitor compound drug target genes can be used to monitor compound activities activities in vivoin vivo..

reducing the gene copy number of drug targets in a diploid cell can reducing the gene copy number of drug targets in a diploid cell can result in sensitization to the drug of interest.result in sensitization to the drug of interest.

NOW……in this paper:NOW……in this paper: They extended this approach to analyze the activities of 78 They extended this approach to analyze the activities of 78

chemical entities, most of which are medically relevant.chemical entities, most of which are medically relevant. Increased the number of mutant strains…why??Increased the number of mutant strains…why?? Used high-density oligonucleotide arrays with a two-color Used high-density oligonucleotide arrays with a two-color

labelling strategy…..to do what ??labelling strategy…..to do what ?? Finally, a strain-specific error model was used….to do what ??Finally, a strain-specific error model was used….to do what ?? In this study, they correctly identified the reported targets for In this study, they correctly identified the reported targets for

many well-characterized compounds in addition to discovering many well-characterized compounds in addition to discovering many potentially novel drug targets.many potentially novel drug targets.

Result Result #1:#1:

Study Design Study Design RationaleRationale

Figure 1.Figure 1. Schematic Schematic

Representation Representation of the Fitness of the Fitness

Profiling Profiling Experimental Experimental

Strategy Strategy

RESULT #2:RESULT #2: Identifying Drug-Specific Growth Identifying Drug-Specific Growth DefectsDefects

Result Result #3:#3:

Large-Scale Large-Scale Analysis of Analysis of

78 78 CompoundsCompounds

Figure 3.Figure 3. Comprehensive Comprehensive View of Fitness View of Fitness Profiles for 78 Profiles for 78 CompoundsCompounds

Result #4: Result #4: Inhibition of Lanosterol Synthase Inhibition of Lanosterol Synthase (Erg7p) by Molsidomine(Erg7p) by Molsidomine

Result #5: Result #5: Disruption of Exosome-Specific rRNA Disruption of Exosome-Specific rRNA Processing by 5-FluorouracilProcessing by 5-Fluorouracil

Result #5….continuedResult #5….continued

DiscussionDiscussion Use of Fitness Profiling for Understanding Drug Use of Fitness Profiling for Understanding Drug

ActivitiesActivities Advantages:Advantages:

Requires no prior knowledge of compound mode of action, which allows Requires no prior knowledge of compound mode of action, which allows truly novel drug activities to be uncovered in a systematic and truly novel drug activities to be uncovered in a systematic and unbiased fashionunbiased fashion

Biological processes that are affected by a given compound are Biological processes that are affected by a given compound are identified in addition to the precise protein target(s)identified in addition to the precise protein target(s)

Limitations and Technical Considerations:Limitations and Technical Considerations: The compound of interest must be able to affect the growth rate of the The compound of interest must be able to affect the growth rate of the

cell. cell. However, the ability of a compound to affect the growth rate of yeast However, the ability of a compound to affect the growth rate of yeast

does not guarantee that a target will be identified by this approach.does not guarantee that a target will be identified by this approach. The activity level of the targeted protein must be influenced by the The activity level of the targeted protein must be influenced by the

dosage level of the corresponding gene under the conditions profiled.dosage level of the corresponding gene under the conditions profiled. Finally, compounds that exert their effects through direct interaction Finally, compounds that exert their effects through direct interaction

with nonprotein elements in the cell, such as DNA or ergosterol, do not with nonprotein elements in the cell, such as DNA or ergosterol, do not appear suitable for this approach. appear suitable for this approach.