#METABOLOMICS WORKBENCH nlpm_20190524_101216 DATATRACK_ID:1728 STUDY_ID:ST001186 ANALYSIS_ID:AN001978 PROJECT_ID:PR000798 VERSION 1 CREATED_ON June 2, 2019, 11:31 am #PROJECT PR:PROJECT_TITLE A Multi-Omics Interpretable Machine Learning Model Reveals Modes of Action of PR:PROJECT_TITLE Small Molecules PR:PROJECT_SUMMARY High-throughput screening and gene signature analyses frequently identify lead PR:PROJECT_SUMMARY therapeutic compounds with unknown modes of action (MoAs), and the resulting PR:PROJECT_SUMMARY uncertainties can lead to the failure of clinical trials. We developed an PR:PROJECT_SUMMARY approach for uncovering MoAs through an interpretable machine learning model of PR:PROJECT_SUMMARY transcriptomics, epigenomics, metabolomics, and proteomics. Examining compounds PR:PROJECT_SUMMARY with beneficial effects in models of Huntington’s Disease, we found common PR:PROJECT_SUMMARY MoAs for compounds with unrelated structures, connectivity scores, and binding PR:PROJECT_SUMMARY targets. The approach also predicted highly divergent MoAs for two FDA-approved PR:PROJECT_SUMMARY antihistamines. We experimentally validated these effects, demonstrating that PR:PROJECT_SUMMARY one antihistamine activates autophagy, while the other targets bioenergetics. PR:PROJECT_SUMMARY The use of multiple omics was essential, as some MoAs were virtually PR:PROJECT_SUMMARY undetectable in specific assays. Our approach does not require reference PR:PROJECT_SUMMARY compounds or large databases of experimental data in related systems and thus PR:PROJECT_SUMMARY can be applied to the study of agents with uncharacterized MoAs and to rare or PR:PROJECT_SUMMARY understudied diseases. PR:INSTITUTE Massachusetts Institute of Technology PR:LABORATORY Fraenkel Lab PR:LAST_NAME Patel-Murray PR:FIRST_NAME Natasha PR:ADDRESS 77 Massachusetts Avenue PR:EMAIL nlpm@mit.edu PR:PHONE 6179490941 #STUDY ST:STUDY_TITLE Untargeted metabolomics on control and compound-treated STHdhQ111 cells and ST:STUDY_TITLE control STHdhQ7 cells ST:STUDY_SUMMARY Cells expressing mutant huntingtin were treated in triplicate with serum-free ST:STUDY_SUMMARY DMEM with vehicle (Q111SST) or serum-free DMEM with one of 14 protective ST:STUDY_SUMMARY compounds for 24 hours. Wild type cells were also treated with serum-free DMEM ST:STUDY_SUMMARY with vehicle (Q7SST) as an additional control for 24 hours. We examined the ST:STUDY_SUMMARY compounds' metabolomic effects on the cells using untargeted mass spectrometry, ST:STUDY_SUMMARY which measured lipids and polar metabolites. ST:INSTITUTE Massachusetts Institute of Technology ST:LABORATORY Fraenkel Lab ST:LAST_NAME Patel-Murray ST:FIRST_NAME Natasha ST:ADDRESS 77 Massachusetts Avenue, Building 16 Room 244 ST:EMAIL nlpm@mit.edu ST:PHONE 6179490941 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Q7SST_1 Time:1 | Treatment:Q7SST_Control Batch=1 SUBJECT_SAMPLE_FACTORS - Q7SST_2 Time:2 | Treatment:Q7SST_Control Batch=2 SUBJECT_SAMPLE_FACTORS - Q7SST_3 Time:3 | Treatment:Q7SST_Control Batch=3 SUBJECT_SAMPLE_FACTORS - Q111SST_1 Time:1 | Treatment:Q111SST_Control Batch=1 SUBJECT_SAMPLE_FACTORS - Q111SST_2 Time:2 | Treatment:Q111SST_Control Batch=2 SUBJECT_SAMPLE_FACTORS - Q111SST_3 Time:3 | Treatment:Q111SST_Control Batch=3 SUBJECT_SAMPLE_FACTORS - Clio_1 Time:1 | Treatment:Clioquinol Batch=3 SUBJECT_SAMPLE_FACTORS - Clio_2 Time:2 | Treatment:Clioquinol Batch=2 SUBJECT_SAMPLE_FACTORS - Clio_3 Time:3 | Treatment:Clioquinol Batch=3 SUBJECT_SAMPLE_FACTORS - Cypro_1 Time:1 | Treatment:Cyproheptadine Batch=3 SUBJECT_SAMPLE_FACTORS - Cypro_2 Time:2 | Treatment:Cyproheptadine Batch=2 SUBJECT_SAMPLE_FACTORS - Cypro_3 Time:3 | Treatment:Cyproheptadine Batch=3 SUBJECT_SAMPLE_FACTORS - Cyst_1 Time:3 | Treatment:Cysteamine Batch=1 SUBJECT_SAMPLE_FACTORS - Cyst_2 Time:1 | Treatment:Cysteamine Batch=2 SUBJECT_SAMPLE_FACTORS - Cyst_3 Time:2 | Treatment:Cysteamine Batch=3 SUBJECT_SAMPLE_FACTORS - Dime_1 Time:2 | Treatment:Dimethyl fumarate Batch=1 SUBJECT_SAMPLE_FACTORS - Dime_2 Time:3 | Treatment:Dimethyl fumarate Batch=2 SUBJECT_SAMPLE_FACTORS - Dime_3 Time:1 | Treatment:Dimethyl fumarate Batch=3 SUBJECT_SAMPLE_FACTORS - DKI_1 Time:1 | Treatment:Diacylglycerol kinase inhibitor II Batch=3 SUBJECT_SAMPLE_FACTORS - DKI_2 Time:2 | Treatment:Diacylglycerol kinase inhibitor II Batch=2 SUBJECT_SAMPLE_FACTORS - DKI_3 Time:3 | Treatment:Diacylglycerol kinase inhibitor II Batch=3 SUBJECT_SAMPLE_FACTORS - DOP_1 Time:3 | Treatment:4-Deoxypyridoxine Batch=1 SUBJECT_SAMPLE_FACTORS - DOP_2 Time:1 | Treatment:4-Deoxypyridoxine Batch=2 SUBJECT_SAMPLE_FACTORS - DOP_3 Time:2 | Treatment:4-Deoxypyridoxine Batch=3 SUBJECT_SAMPLE_FACTORS - FTYP_1 Time:2 | Treatment:Fingolimod phosphate Batch=1 SUBJECT_SAMPLE_FACTORS - FTYP_2 Time:3 | Treatment:Fingolimod phosphate Batch=2 SUBJECT_SAMPLE_FACTORS - FTYP_3 Time:1 | Treatment:Fingolimod phosphate Batch=3 SUBJECT_SAMPLE_FACTORS - Halo_1 Time:2 | Treatment:Haloperidol Batch=1 SUBJECT_SAMPLE_FACTORS - Halo_2 Time:3 | Treatment:Haloperidol Batch=2 SUBJECT_SAMPLE_FACTORS - Halo_3 Time:1 | Treatment:Haloperidol Batch=3 SUBJECT_SAMPLE_FACTORS - Lox_1 Time:3 | Treatment:Loxapine Batch=1 SUBJECT_SAMPLE_FACTORS - Lox_2 Time:1 | Treatment:Loxapine Batch=2 SUBJECT_SAMPLE_FACTORS - Lox_3 Time:2 | Treatment:Loxapine Batch=3 SUBJECT_SAMPLE_FACTORS - Mec_1 Time:2 | Treatment:Meclizine Batch=1 SUBJECT_SAMPLE_FACTORS - Mec_2 Time:3 | Treatment:Meclizine Batch=2 SUBJECT_SAMPLE_FACTORS - Mec_3 Time:1 | Treatment:Meclizine Batch=3 SUBJECT_SAMPLE_FACTORS - NaB_1 Time:3 | Treatment:Sodium Butyrate Batch=1 SUBJECT_SAMPLE_FACTORS - NaB_2 Time:1 | Treatment:Sodium Butyrate Batch=2 SUBJECT_SAMPLE_FACTORS - NaB_3 Time:2 | Treatment:Sodium Butyrate Batch=3 SUBJECT_SAMPLE_FACTORS - Nico_1 Time:3 | Treatment:Nicotinamide Batch=1 SUBJECT_SAMPLE_FACTORS - Nico_2 Time:1 | Treatment:Nicotinamide Batch=2 SUBJECT_SAMPLE_FACTORS - Nico_3 Time:2 | Treatment:Nicotinamide Batch=3 SUBJECT_SAMPLE_FACTORS - Nort_1 Time:3 | Treatment:Nortriptyline Batch=1 SUBJECT_SAMPLE_FACTORS - Nort_2 Time:1 | Treatment:Nortriptyline Batch=2 SUBJECT_SAMPLE_FACTORS - Nort_3 Time:2 | Treatment:Nortriptyline Batch=3 SUBJECT_SAMPLE_FACTORS - Pizo_1 Time:2 | Treatment:Pizotifen Batch=1 SUBJECT_SAMPLE_FACTORS - Pizo_2 Time:3 | Treatment:Pizotifen Batch=2 SUBJECT_SAMPLE_FACTORS - Pizo_3 Time:1 | Treatment:Pizotifen Batch=3 SUBJECT_SAMPLE_FACTORS - Seli_1 Time:1 | Treatment:Selisistat Batch=1 SUBJECT_SAMPLE_FACTORS - Seli_2 Time:2 | Treatment:Selisistat Batch=2 SUBJECT_SAMPLE_FACTORS - Seli_3 Time:3 | Treatment:Selisistat Batch=3 SUBJECT_SAMPLE_FACTORS - TSA_1 Time:2 | Treatment:Trichostatin A Batch=1 SUBJECT_SAMPLE_FACTORS - TSA_2 Time:3 | Treatment:Trichostatin A Batch=2 SUBJECT_SAMPLE_FACTORS - TSA_3 Time:1 | Treatment:Trichostatin A Batch=3 #COLLECTION CO:COLLECTION_SUMMARY STHdhQ111 cells were grown on 10cm dishes in triplicate at a seeding density of CO:COLLECTION_SUMMARY 1.06 million cells/well. Compound- or vehicle-treated cells were washed with CO:COLLECTION_SUMMARY cold 0.9% NaCl. To each 10cm dish of cells, 660uL LC/MS-grade methanol CO:COLLECTION_SUMMARY containing internal standards and 330uL LC/MS-grade water were added. Cells were CO:COLLECTION_SUMMARY scraped and transferred to Eppendorf tubes, where 450uL chloroform was added. CO:COLLECTION_SUMMARY Samples were vortexed at maximum speed (20,817 rcf) for 10 minutes at 4°C. Each CO:COLLECTION_SUMMARY layer was collected separately, avoiding the precipitate at the interface of the CO:COLLECTION_SUMMARY two layers, and dried by speedvac. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY STHdh cells were incubated in serum-free medium with a compound or vehicle TR:TREATMENT_SUMMARY control for 24 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipid profiling, cells were resuspended in 50uL 60/35/5 SP:SAMPLEPREP_SUMMARY acetonitrile/isopropanol/water (v/v/v) and 5uL was injected for LC/MS analysis. SP:SAMPLEPREP_SUMMARY For polar metabolite profiling, cells were resuspended in 100uL water and 2uL SP:SAMPLEPREP_SUMMARY was injected for LC/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME EMD Millipore ZIC-HILIC (100 x 2.1 mm, 3.5 um) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Whitehead Institute Metabolite Profiling Core Facility #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS For polar metabolite profiling, cells were resuspended in 100uL water and 2uL MS:MS_COMMENTS was injected for LC/MS analysis. Please see Birsoy et al. and Chen at al. for a MS:MS_COMMENTS detailed description of the LC/MS analysis (Chen et al, 2016; Birsoy et al, MS:MS_COMMENTS 2015). Untargeted analysis was performed using Progenesis CoMet (Nonlinear MS:MS_COMMENTS Dynamics) using the default settings. Features were filtered based on replicate MS:MS_COMMENTS injections and a dilution series of a pooled sample prepared by mixing equal MS:MS_COMMENTS aliquots of the biological samples. Specifically, the filtering criteria were CV MS:MS_COMMENTS < 0.4 across the four replicate injections and R > 0.9 across a four-point MS:MS_COMMENTS dilution series (comprising 0.1X, 0.3X and 1X concentrations, and a MS:MS_COMMENTS double-volume injection). Features that were not lowest according to the MS:MS_COMMENTS Progenesis quantification in the blank water injection samples were discarded. MS:MS_RESULTS_FILE ST001186_AN001978_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END