Deciphering Drug Modes of Action by MetabolomicsIsabel Vincent1,2, David Ehmann3, Manos Perros3, Scott Mills3, Deborah Woods4, Srinivasa Rao5, Karl Burgess2, Michael Barrett1,2

1Wellcome Trust Centre for Molecular Parasitology and 2Glasgow Polyomics, University of Glasgow, Glasgow G12 8TA3Astrazeneca, Waltham, MA, USA, 4Zoetis LLC (formerly Pfizer Animal Health), Kalamazoo, MI, USA, 5Novartis Tropical Diseases, Singapore.

Knowing the mode of action of a drug is useful. Antiparasitic drugs are often old and their modes of action (MOAs) are unknown. Even in the pharmaceutical industry, the MOA of the lead compounds can be elusive. Knowing the MOA of a drug allows drugs to be more efficiently targeted, reducing toxicity, predicting resistance mechanisms and helps licencing approval. Current tests for the modes of action are inadequate. Current methods to determine the MOA of a drug can be time consuming. At Astrazeneca, there is a distinct need to add a higher resolution to the current radioactive metabolite incorporation assay. Metabolomics provides a high level of detail, is non-radioactive and is cost effective. The data presented here show that an untargeted metabolomics approach can isolate specific drug targets and can be performed in a relatively high throughput manner.

Proof of principleThe ornithine

decarboxylase inhibitor eflornithine is used in human African trypanosomiasis. Our metabolomics technique detected an increase in the substrate (ornithine) and a decrease in the product (putrescine) of this enzyme after eflornithine treatment of bloodstream form trypanosomes [1].

References [1] Vincent, IM et al., Untargeted metabolomics reveals a lack of synergy between nifurtimox and eflornithine against Trypanosoma brucei. PLoS NTDs. 2012; 6(5) [2] Vincent, IM et al., A molecular mechanism for eflornithine resistance in African trypanosomes. PLoS Pathogens 2010; 6(11)..

ConclusionsMetabolomics analysis is a powerful tool for the study of the MOA of a drug. It is hoped that the techniques developed here will add more in-depth data to the current radioactivity-based MOA studies. Eventually, heavy atom labelled precursor metabolites may be incorporated into the cell growth medium to test the incorporation of these metabolites after drug addition in the same way as radioactive compounds are used, but producing a much higher resolution and eliminating the need for ionising radiation.

Drug resistance

Eflornithine transport was found to be reduced in a one hour metabolomics assay of eflornithine resistant trypanosomes [2].

High throughput method

DNA biosynthesis inhibitors

The Astrazeneca test compound, AZ1 was tested on E. coli at 4 x minimum inhibitory concentration (MIC).

Cell wall biosynthesis inhibitors

A known bacterial cell wall biosynthesis inhibitor, ceftazidime was added to TolC- E. coli. Changes to cell wall-related metabolites were seen.

Ceftazidime (CAZ) treatment of E. coli at 2 and 4 hours compared to a non-drugged control (NDR)

The compound caused a significant increase in dTMP and metabolites upstream of dTMP kinase and a significant decrease in dTDP and metabolites downstream of dTMP kinase. dTDP-sugars were also decreased and gamma-glutamylputrescine was increased. Otherwise, the metabolome was stable.

AZ1 treatment of E. coli at 2 and 4 hours (red) compared to a non-drugged control (blue). Metabolites marked “ND” were not detected. Standards were run for thymidine, deoxyuridine, dCMP, and dUMP. Other metabolites were annotated based on mass and predicted retention time.

Eflornithine uptake over one hour was measured by metabolomics in a wildtype T. brucei line and one resistant to eflornithine.

Bacteria

Protozoa

4 x Drug MIC

IC90

Chloroform:methanol:water and 4 freeze-thaw cycles

Chloroform:methanol:water and 1 hour shaking at 4°C

Zic-pHILIC column

Orbitrap QExactive in switching mode