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MONASH PHARMACY & PHARMACEUTICAL SCIENCES Metabolomics helps to unravel the mode of action of novel anti-malarial compounds Anubhav Srivastava Research Fellow Pharmaceutical Metabolomics Laboratory @RUNubhav @CreekLab Malaria: we need

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Metabolomics helps to unravel the mode of action of novel anti-malarial compounds

Anubhav Srivastava Research Fellow Pharmaceutical Metabolomics Laboratory

MONASH PHARMACY & PHARMACEUTICAL SCIENCES

@RUNubhav @CreekLab

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Malaria: we need new drugs

P. falciparum

Anopheles Mosquito

India Myanmar China (Yunnan) Thailand Lao PDR Vietnam Cambodia

High ACT failure rate (>10%)

1 ACT 2 ACTs 5 ACTs

P. falciparum has developed resistance to the first line of malaria

treatment, Artemisinin-based Combination Therapies (ACTs) in many countries in South-East Asia1,2

Resistance has also been reported from Africa3
We need new anti-malarials to combat the on-going threat of anti-

malarial drug-resistance

1. Dondorp AM, et al. 2009 N Engl J Med; 361:455-467
2. Ashley EA, et al. 2014 N Engl J Med; 371:411-423
3. Feng Lu, et al. 2017 N Engl J Med; 376:991-993

>200 million cases of malaria and an estimated 429000 deaths in 2015 (WHO 2016)

WHO Status report on artemisinin and ACT resistance (April 2017)

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Target-based screening/design Poor Known

Approach Success Rate Mode of Action

Phenotypic Screening Better Unknown Small molecules METABOLOMICS Known

Anti-parasitic drug discovery approaches

Efficacy
Combinations
Resistance

?

Parasite death

Screening library

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Drug Discovery Systems Pharmacology

Genomics

Transcriptomics

Proteomics

Metabolomics

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Drug Discovery Systems Pharmacology Metabolomics

Proteomics

Transcriptomics

Genomics

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Metabolomics Methodology

Incubation/ Extraction

80°C

LC-MS: HILIC-Orbitrap (Untargeted)

HILIC chromatography (Zwitterionic) High resolution Accurate mass MS

Data Analysis: IDEOM workflow

Drug

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Using Metabolomics to study mode of drug action

Drug

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Compounds used in this study

Inactive

Novel anti-malarials

Potent activity
No known target

http://opensourcemalaria.org

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MMV000073 MMV397264 MMV390482

PfATP4 inhibitors

Known anti-malarials

Atovaquone Chloroquine Dihydroartemisinin

? ? Compounds used in this study

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P_397264:P_397264_4 P_S313:P_S313_2 P_000073:P_000073_2 P_000073:P_000073_4 P_000073:P_000073_1 P_000073:P_000073_3 P_S5:P_S5_2 P_S5:P_S5_4 P_397264:P_397264_2 P_S313:P_S313_4 P_DHA:P_DHA_3 P_DHA:P_DHA_4 P_DHA:P_DHA_1 P_DHA:P_DHA_2 P_S5:P_S5_1 P_S5:P_S5_3 P_390482:P_390482_2 P_390482:P_390482_4 P_390482:P_390482_1 P_390482:P_390482_3 P_S313:P_S313_1 P_397264:P_397264_3 P_397264:P_397264_1 P_S313:P_S313_3 P_CQ:P_CQ_3 P_CQ:P_CQ_4 P_CQ:P_CQ_1 P_CQ:P_CQ_2 P_S4:P_S4_3 P_S291:P_S291_2 P_S4:P_S4_4 P_S106:P_S106_1 P_S106:P_S106_3 P_S106:P_S106_2 P_S106:P_S106_4 P_S291:P_S291_1 P_DMSO:P_DMSO_4 P_S133:P_S133_4 P_S133:P_S133_2 P_DMSO:P_DMSO_2 P_DMSO:P_DMSO_3 P_DMSO:P_DMSO_1 P_S133:P_S133_1 P_S133:P_S133_3 P_S4:P_S4_1 P_S4:P_S4_2 P_S291:P_S291_3 P_S291:P_S291_4 P_S112:P_S112_2 P_S112:P_S112_4 P_S112:P_S112_1 P_S112:P_S112_3 P_S37:P_S37_1 P_S37:P_S37_3 P_ATV:P_ATV_3 P_ATV:P_ATV_4 P_S37:P_S37_2 P_S37:P_S37_4 P_ATV:P_ATV_1 P_ATV:P_ATV_2

NDF: No Discriminating Features

Results: Multivariate analysis Unsupervised Hierarchical Clustering

SLIDE 11 s b d s r i

Peptides Nucleotides Cofactors Lipids Carbohydrates and energy metabolism Amino acid metabolism Unclassified

0.2 1 5

Fold change

Results: Multivariate analysis Heat map

471 putative metabolites Classification Pathway mapping

MMV PfATP4 inhibitors Known antimalarials OSM compounds

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Compounds used in this study

Inactive

Novel anti-malarials

Potent activity
No known target

http://opensourcemalaria.org

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DHODH

DMSO control Dihydroorotate dehydrogenase Pyrimidine biosynthesis is affected in parasites treated with OSM-S-112 and OSM-S-37 DHODH Inhibition OSM-S-37 >> OSM-S-112

MoA of OSM-S-112 and OSM-S-37 Similar to Atovaquone

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Compounds used in this study

Inactive

Novel anti-malarials

Potent activity
No known target

http://opensourcemalaria.org

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OSM-S-106 OSM-S-5

Multivariate

analysis (PCA)

No unique

changes

requires further investigation

e.g. enriched

(>90%) parasite cultures

MoA of OSM-S-106 and OSM-S-5 Unknown

PfATP4 inhibitors DHA OSM-S-106 ATV DMSO CQ PfATP4 inhibitors DHA OSM-S-5 ATV DMSO CQ

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Compounds used in this study

Inactive

Novel anti-malarials

Potent activity
No known target

http://opensourcemalaria.org

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Multivariate analysis (PCA) showed that parasites treated with OSM-S-313 cluster together with parasites treated with the MMV PfATP4 inhibitors

MoA of OSM-S-313 Similar to MMVPfATP4 inhibitors

PfATP4 inhibitors DHA OSM-S-313 ATV DMSO CQ

OSM-S-313

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MoA of OSM-S-313 Similar to MMVPfATP4 inhibitors

Cell pellet Spent media

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RBC Plasma Parasite PVM

PfATP4 maintains Na+ homeostasis in the malaria parasite

Na+ NPPs PfATP4

Adapted from: Spillman NJ, Kirk K. Int J Parasitol Drugs Drug Resist. 2015;5: 149–162

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RBC Parasite PVM High Na+ Inhibition of metabolic enzymes Inhibition of physiological processes dependent on Na+ electrochemical gradient Cell lysis due to

smotic imbalance

Disruption of Na+ regulation: Potential mechanisms of parasite killing

Adapted from: Spillman NJ, Kirk K. Int J Parasitol Drugs Drug Resist. 2015;5: 149–162

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High Na+ concentration can inhibit glycolysis

P P P P

Glucose

P

Fructose-1-6-bi-P Glyceraldehyde-3-P Glycerone-P Phosphoenolpyruvate Pyruvate Can be inhibited by high Na+

Utter M.F., J Biol Chem. 1950 Aug;185(2):499-517

This can prevent re-phosphorylation of adenylates AMP

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Summary

Pyrimidine synthesis Glycolysis

MMV397264 MMV390482

Unknown Unknown

Phenotypic Screens Metabolomics

Higher success Known MoA

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