HMT Drug and Biomarker Discovery by HMT’s Next-gen Metabolomics

Human Metabolome Technologies (HMT) has been providing metabolome analysis service to researchers worldwide since 2003 and contributed to over 6,000 projects with more than 500 publications. Continuously innovating CE-MS-based metabolomics technology, HMT dramatically improved analytical sensitivity and coverage of target metabolites recently and developed a sophisticated chemoinformatics approach for predicting the structure of unknown compounds, with which dozens of new metabolites were successfully identified. Taken together, HMT introduces a novel solution for elucidating drug efficacy and toxicity mechanisms and discovering patient stratification biomarkers using the cutting-edge metabolomics technologies.

Human Metabolome Technologies, Inc. Company Profile

Basic Information l Business ü Metabolome analysis service ü Biomarker development l Foundation : July 1st, 2003 from Keio University l IPO : TSE Mothers in 2013 l Employees : 78 (As of April 2021) l President : Katsuhito Hashizume l Headquarter: Tsuruoka, Yamagata, Japan l Sales office : Tokyo, Japan l Subsidiary : HMT America (Boston, USA) HMT Europe (Leiden, Netherlands) l Major clients: Academic institutions, pharma, food and cosmetics companies worldwide

2 Metabolomics Market Growth

10000 CAGR (2010-2020): 20.0%!! 9121 9000 “Genome”: 4.2% 8000 “Proteome”: 6.2%

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1000 Search key in PubMed “metabolomic OR metabolome OR 0 metabolomics OR metabonomic OR metabonomics OR 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 metabonome” Year of Publication

3 CE-MS-based Metabolomics

Best platform for comprehensive analysis of hydrophilic metabolites

Capillary Electrophoresis (CE)

Mass Spectrometry (MS) Capillary Compounds are measured separately based on their mass and time.

m/z m/z Compounds are separated based Buffer on their charge and ionic radius. solution μ = ionic mobility q = ion charge η = solution viscosity r = ion radius Migration Time

4 CE-FTMS: World’s Only Platform Patented Technology Agilent’s CE & Thermo’s Orbitrap MS connected by a patented ESI adaptor World’s only platform combining + + CE with high separation power and + + Orbitrap with high resolution & sensitivity + + + + → 10-fold inc. in sensitivity + + → 2-fold inc. in # metabolites detected + The potential of nebulizer is 0V Potential discovery of novel metabolites that have never been detected by the A glass capillary for isolating from a high voltage other platforms based on LC- or GC-MS

Best-suited to • Comprehensive Metabolomics High voltage plate Thermo ion transfer tube • Biomarker Discovery Positive Ion mode : -4000V modified to connect a glass Negative Ion mode : +3500V capillary

Sasaki, K., et al. Anal Chem. 2019, 15;91(2):1295-1301. 5 UDP-Glc Gal1P ATP ATP Metabolomic Profile of UDP Glycogen dATP Glycogen synthase MCF-10A Cells Debranching Pentose Purine Phosphotransferase enzyme ADP Hexose-1-phosphate NDP ADP ATP Pi PPi uridylyltransferase UDP-glucose In collaboration with Prof. Brugge & Dr. Coloff, Phosphate Metabolism Nucleoside-diphosphate pyrophosphorylase kinase UTP PPi 2+ Inactivation of de novo Mg Adenylate Harvard Medical School, MA, USA ATP G1P UDP-Gal Pathway cAMP cyclase ADP NTP Thioredoxin edoxin NADPH Glycolysis disulfide Glucose ADP-Rib H O Ribonucleotide 2 Phosphotransferase (from left to right) + reductase NADP NADPH AMP dADP dAMP Glucokinase/ purine synthesis? Pi ATP DMSO vs Mek Inhibitor (n=3) Glucose Hexokinase ADP R1P 6-phosphatase Phosphodiesterase + NADP H O 10A 2 H O 2 1-Oxidoreductase H O 2 G6P Phosphogluco 6-PG Ru5P ADP-ribose Fumaric acid Adenylate mutase 2+ CO diphosphatase kinase ADP Mg 2 R1P Pi Metabolic Parameters ATP 6-Phosphoglucono Succinyl AMP AMP Adenosine Adenine Sorbitol Adenylate Energy Charge Total Adenylate Guanylate Energy Charge Total Guanylate GSH/GSSG + delta-lactone Purine nucleoside NAD G6P 6-PG Gln Glu Gly Gln Glu Asp Fumaric acid H O Lactonase phosphorylase dehydrogenase 2 dehydrogenase

+ NADPH Phosphogluco NADP ADP R5P mutase Succinyl AMP Adenosine Phosphoglucose isomerase lyase kinase 2-Oxidoreductase GDP NADH isomerase Mg2+ Ribulose phosphate X5P R5P PRPP 2+ Pi H O 3-epimerase Mg H O 2 Fructose 2 Pi ADP ATP R1P F6P GAR N10-formyl FGAM AIR Pi SCAAIR N10-formyl H O PPi THF CO THF Total Glutathione NADPH/NADP+ NADH/NAD + Lactate/Pyruvate Glycerol 3-phosphate/DHAP 2 synthetase THF synthetase synthetase 2 synthetase THF Pi Adenosine Hexokinase ASP beta 5-Phospho deaminase GAR FGAR FGAM AIR CAIR SAICAR AICAR FAICAR Succinyl AMP 2+ ribosylamine Mg GTP synthetase NH Ribose phosphate PRPP GAR AIR AICAR H O 3 ATP Adenylosuccinase 2 ATP pyrophosphokinase amidotransferase Pi transformylase carboxylase transformylase Pi AMP IMP Inosine Hypoxanthine deaminase H O ATP ADP ATP ADP ATP ADP ATP ADP 2 PPi Purine nucleoside ATP AMP 5-Nucleotidase Pi S7P Glyceraldehyde 3-phosphate phosphorylase NH + 2+ H O 3 Total Amino Acids Total Essential Total Non-essential Total Glucogenic Total Ketogenic Fructokinase K ,Mg Phosphofructo Fructose Transketolase 2 ADP kinase-1 1,6-bisphosphatase Amino Acids Amino Acids Amino Acids Amino Acids ADP IMP 2+ H O TDP Mg 2 cyclohydrolase D-F1P F1,6P Transaldolase + GMP TDP NAD + reductase NADP O H O H O NADPH 2 2 2 ATP AMP Hypoxanthine PPi PRPP Xanthine E4P F6P phosphoribosyltransferase oxidase Total BCAA Total Aromatic Fischer's Ratio Total Glu-related Total Pyr-related IMP Transaldolase NADH dehydrogenase H O Amino Acids Amino Acids Amino Acids 2 2 H O H O + + 2 2 2 SAM SAH Met Homocysteine NADH NAD NADH NAD AcCoA CoA H O Xanthine Uric acid PPi GMP 2 Pi Guanosine O Aldolase A Aldolase XMP 2 Glyceraldehyde Xanthine 5-Nucleotidase oxidase DHAP Glyceraldehyde 3-phosphate FADH FAD Triose phosphate 2 O H O GMP Total Acetyl CoA-related Total Fumarate-related Total Succinyl CoA-related Total Oxaloacetate-related Malate/Asp isomerase Glycolic acid 2 2 2 Gly Sarcosine DMG Betaine BTL Choline Pi Amino Acids Amino Acids Amino Acids Amino Acids O-acetyltransferase synthase + NADH + H ATP Purine-nucleoside NAD phosphorylase R1P NH Glyoxylic 3 Acetylcholine Gln Glu acid PPi Glycolate-oxygen Alanine-glycine Glycine N-methyl Dimethylglycine S-methyl Betaine-aldehyde Betaine-aldehyde FADH Acetylcholinesterase Adenine phosphoribosyl- 2 H O Pi 2-oxidoreductase transaminase transferase dehydrogenase transferase dehydrogenase dehydrogenase 2 transferase Guanine Glycerol-NAD+ Glycerol 3-phosphate Glyceraldehyde 3-phosphate deaminase Guanylate kinase oxidoreductase + dehydrogenase dehydrogenase NADH ADP NAD Threonine PRPP PLP H O + H O H O Citrulline/Ornithine Glu/2-Oxoglutarate G6P/R5P SAM/SAH Putrescine/Spermidine FAD Ala Pyruvic acid aldolase Formaldehyde 2 2H 2 Acetate 2 Glycerol 3-phosphate H O Serotonin 2 Thr Acetaldehyde ATP CO Choline Metabolism 2 + NAD Choline Ser Aromatic amino Phosphodiesterase Guanine CO NH GDP 1,3-Bisphospho 2 3 acid decarboxylase cGMP 10A Glycerol Pi glycerate Thioredoxin Glycerol 2+ ADP Glycine Dihydro 5-Hydroxy PLP Thioredoxin Ribonucleotide H O ADP Mg disulfide 2 kinase PLP cleavage Choline Choline Trp reductase system H O dGDP kinase kinase ADP 2 Bisphospho + NADH Glu 2-OG Phosphatidylserine PPi CTP Trp 5-mono ATP ATP NAD NADH CMP CO glycerate mutase Folic acid Ethanolamine 2 decarboxylase Ph sphatidyl Cholinephospho DP-choline H O oxygenase ATP ADP 2 O Diphosphoglycerate Phosphoglycerate phosphotransferase serine synthase I transferase synthetase 2 + Ethanolamine CDP-ethanol Phosphatidyl Phosphatidyl Phosphatidyl Tetrahydro NAD kinase ATP Serine hydroxy CDP-choline Phosphocholine phosphate amine ethanolamine serine choline (Lecithin) biopterin methyl transferase ATP Ethanolamine phosphate Guanylate PLP Ser Phosphatidyl Lys Trp Nucleoside-diphosphate 2,3-biosphospho cytidylyltransferase CMP cyclase Phosphotransferase serine synthase II kinase ADP ADP Phosphotransferase glycerate mutase 3-PG Ser CTP PPi Ethanolamine PPi Glyceraldehyde-NAD+ H O Pi Glycerate 2 GTP NADH oxidoreductase 2+ Glycolysis inhibition? kinase Mg 3-Phosphohydroxy 3-Phospho Glycerate NADPH pyruvate serine Triglycerol Phosphoglycerate Phosphoserine Phosphoserine CoA 2-OG dGTP dGMP dehydrogenase aminotransferase phosphohydrolase H O 2 Ala dGTP + Triacylglycerol Acyl-CoA-1,2-diacylgly erol H O diphosphohydrolase ADP ATP + NADH 2 Tryptophan NAD acylhydrolase O-acyltransferase Lysine-2-OG Ala 2 3-Dioxygenase reductase + H O Phosphoglycerate Palmitoyl-CoA NADP N-Formyl- 2 PPi H O H O PLP mutase LDH 2 2 kynurenine H O 2+ Reductant Oxidant Mg Serine 1,2-Diacylglycerol 2 Hydroxyproline Glu Asn dehydratase Pi Formamidase Kinureninase + 2-PG NADP NADPH Phosphatic acid Formate + H O H 2 phosphatase Kynurenine Anthranilate Glu Glycerate H O + 2-Oxidoreductase 2 NH CoA + 2-kinase Serine-pyruvate 4 H O Saccharopine O H NADPH 2-OG Glu 3-Hydroxy 1,2-Diacylglycerol 2 + 2 Succinic acid AcCoA Glycerate aminotransferase inhibition? Phosphatidic acid NAD Kynurenine acylhydrolase H O pyruvate 2 3-Monooxygenase ADP 2-OG PLP H O AMP Monoacyl 2 GTP GDP Cys glycerol Lysophosphatidic 3-Hydroxy- Cysteine O acid-acyltransferase H O kynurenine Gln Glu CO PPi Gln 2 H O Palmitoyl-CoA 2 2 dioxygenase 2 Glu Saccharopine PLP N-acetylglutamate PLP Cysteine H+ Ala Kynurenine + Oxidoreductase ATP ADP dehydrogenase NADH NADP + + synthase sulfinic acid 3-Monooxygenase NAD NADPH-NAD O 2-OG CoA Asparagine Aspartate H O Enolase 2 2 Transaminase ATP kinase oxidoreductase synthase transaminase 2+ Glu Lipid 3-Hydroxy- ATP Mg Pro N-AcGlu PEP + anthranilate + NADH NAD 2-Aminoadipate O NAD H O CO 2 3-Hydroxyanthranilate Quinolinate H O PPi 2 2 semialdehyde + 2 3-Sulfino Metabolism 3 4-dioxygenase phosphoribosyl Deamido-NAD + Cys NAD synthase Asp b-Ala pyruvate transferase pyrophosphorylase Oxaloacetate Cysteic acid 1-Acylglycerol Glycerol Lysophosphatidic + 2-Amino-3-carboxy- Nicotinate + PLP Cystine NADPH + NADPH + NAD Quinolinate Deamino-NAD + Aspartate CO2 decarboxylase NADP NADP acylhydrolase kinase acid muconic semialdehyde ribonucleotide NAD PEP reductase 1-carboxy-lyase ADP CoA carboxykinase CO NADH Cystine 2-OG + CO Picolinate PPi 2 Glycerol Glycerol 3-phosphate NAD 2 H O Carbamoyl-Asp SO carboxylase 2 PRPP CO ATP ATP ADP 2 Hypotaurine 2 PPi H O H O IMP 2 2 2-Aminomuconate Pyrroline 5-carboxylate Glycerophosphate 2 Aminoadipate semi + NADH H O ACP semialdehyde H O Reductant Oxidant reductase + NAD Hypotaurine 2 transacylase aldehyde dehydrogenaseNADH 2 NADH Pyruvate + beta-Ketoacyl-ACP beta-Hydroxyacyl Enoyl ACP Acyl-ACP H+ H O dehydrogenase H Acetoacetyl ACP reductase ACP dehydratase reductase hydrolase 2 kinase ATP Aminomuconate-semi + (C ) or Acyl ACP 3-Hydroxy Palmitoyl 2-Aminoadipate NAD Pyrroline + Carbamoyl phosphate 4 Glu NADH aldehyde dehydrogenase H 2+ + Taurine Enoyl ACP Acyl ACP Palmitic acid Palmitoyl-CoA glycohydrolase Mg , K acyl ACP ACP (C16) 5-carboxylate synthetase - (C /C /C /C /C /C ) 2-Aminoadipate HCO 6 8 10 12 14 16 Long-chain acyl + H O 2H O 3 CO PPi aminotransferase 2H ADP-Rib Nicotinamide 2 2 2 H O CoA synthetase CoA NH + GTP 2 4 Lactic acid Pyruvic acid beta-Ketoacyl CoA ATP AMP 1-pyrroline-5 carboxylate Glu Acetyl-CoA ACP synthase dehydrogenase Nicotinamides Aspartate transacylase (outer)Carnitine 2-Oxoadipate 2-Aminomuconate Carbamoyl-P CoA transcarbamoylase AcCoA CoA palmitoyltransferase + Glutamate Succinyl AMP CoA NAD H+ NH Oxidoreductase 5-semialdehyde Pi synthetase Deaminase 3 H O GDP Lactate Carnitine 2 Ornithine dehydrogenase + NADH Pi Palmitoyl NAD aminotransferase 2-Oxoadipate 2-OG AcCoA CoA NADH + carnitine Succinyl AMP NAD CO dehydrogenase 2 + Ornithine Citrulline Malonyl ACP H NADH Glyoxylate Gly + 2+ NAD Mg ,TDP (inner)Carnitine Glutaryl-CoA Pi ATP ADP lipoic acid palmitoyltransferase CoA FAD BCAA Metabolism AMP FADH FAD Pyruvate Malonyl CoA-ACP 2 Glutaryl-CoA transacylase 3-Ketoacyl- L-3-Hydroxyacyl- Enoyl-CoA Acyl-CoA Ornithine dehydrogenase dehydrogenase Leu Ile Val Urea CoA thiolase CoA dehydrogenase hydratase dehydrogenase Palmitoyl-CoA transcarbamoylase PLP 2 NADH Acyl carrier 3-Ketoacyl L-3-Hydroxy trans-Δ - (C ) or Acyl-CoA Palmitoyl-CoA FADH CO Acyl-CoA 16 2 Alanine-glyoxylate 2 protein (ACP) -CoA acyl-CoA (C ) Enoyl-CoA (C /C /C /C /C /C ) 16 Lys & Trp aminotransferase 2+ H O 14 12 10 8 6 4 Glutaconyl-CoA Succinyl AMP AcCoA - biotin,Mg Malonyl-CoA 3-Hydroxy 2 HCO Acetone ATP lyase 3 Pi butyric acid CO 2-OG 2-OG 2-OG Acetyl CoA 2 carboxylase CO Metabolism 2 NADPH + CoA ATP ADP ATP ADP Ala CoA Acetoacetate NADP Crotonyl-CoA + H O Synthetase AcCoA NAD 2 Crotonyl CO 2+ AcCoA CoA hydrase 2 Mg BCAA amino BCAA amino BCAA amino Arginase Mn2+ HMG-CoA 3-Hydroxy AMP lyase transferase Glu transferase Glu transferase Glu H O PLP butyryl-CoA Ornithine 2 Alanine decarboxylase PPi aminotransferase Arg ArgSuccinate + CO Pi 3-Hydroxybutyryl-CoA AAcCoA HMG-CoA 2H Mevalonic acid 2 2-KIV PLP Adenylosuccinate NADH dehydrogenase HMG-CoA HMG-CoA Mevalonate 5-Phospho Diphosphomevalonate lyase Acetoacetyl-CoA H O + 2 synthase reductase kinase mevalonate kinase decarboxylase H Gly 2-OG Glu thiolase 2-Keto 2-Keto-beta- 5-Phospho 5-Diphospho Isopentenyl AAcCoA + ADP NAD isocaproate CoA methylvalerate mevalonate mevalonate diphosphate + ATP NAD CoA + CoA SAM SAH NAD ATP CoA + CoA NADPH NADP ATP ADP Pi Cholesterol lipoamide,TPP BC keto acid - Oxaloacetate NADH dehydrogenase lipoamide,TPP BC keto acid BC keto acid Guanidoacetate Urea HCO NADH dehydrogenase lipoamide,TPP dehydrogenase Pyruvate 3 NADH Glycine amidino ATP citrate Steroid CO Guanidinoacetate ADP carboxylase 2 CO CO transferase Pi lyase 2 2 Putrescine N-methyltransferase Creatine Citric acid Isovaleryl 2-methyl Cycle 2+ Isobutyryl CoA biotin,Mg CoA FAD buryryl CoA FAD FAD H O 2 H O b-Ala ATP ADP 2 Glutaminolysis Histamine ATP Acyl-CoA Acyl-CoA Acyl-CoA Oxaloacetate dehydrogenase dehydrogenase dehydrogenase Gln CO SAM ATP NADPH Citrate 2 beta-Methyl Phe CO 2 synthase PLP FADH2 Tiglyl CoA FADH2 Methylacrylyl CoA FADH2 His crotonyl CoA CO CoA Aconitase H O 2 decarboxylase 2 NADH 2+ Fe cis-Aconitic acid Methylcrotonyl-CoA inhibition? His Carnosine ADP + H O H O NH H 2 2 3 Pi carboxylase Spermidine 2+ Carnosine Pi biotin Creatine kinase Mg Malic enzyme Imidazolone Urocanate His synthase SAH ADP + ADP synthase NADP propionase hydratase ammonia-lyase beta-Methyl Malate N-Formimino- 4-Imidazolone- glutaconyl CoA Urocanate Spermidine Phosphocreatine dehydrogenase glutamate 5-propanoate + H O NAD Pi 2 Histidine H O Methylcrotonyl-CoA H O H O O Folic acid ATP ADP ATP ADP GSH + NADPH 2 2 2 2 NADP hydrolase carboxylase H O Methylcrotonyl-CoA Methylcrotonyl-CoA Malic acid 2 Phe 4-mono Glutamine Glutaminase carboxylase carboxylase oxygenase H O ATP synthetase 2 2-Methyl-beta-hydroxy beta-Hydroxy SAM H O b-Ala HMGCoA CO 2 Dihydrobiopterin Aconitase NH butyryl CoA isobutyryl CoA 2 NH 3 3 Glutamate Tyr 2+ 2+ Fe Fe formyltransferase 3-Hydroxy-isobutyryl O H O Isocitric acid + + + CoA 2 2 Glu GSH H GSSG NAD NAD CoA hydrolase Tyr-dopa Glutamate oxidase gamma-glutamyl Glutathione Spermine Pi + dehydrogenase beta-hydroxy DOPA NH H O cysteine synthetase synthetase synthase SAH 4 2 isobutyrate 2-OG + Creatinine DOPA NAD g-Glu-Cys CO Spermine 2 decarboxylase H O Pi Pi beta-Hydroxyacyl beta-Hydroxyacyl PLP 2 Glutathione NADH dehydrogenase NADH dehydrogenase Glutamate H O + O Fumarase Cys Gly 2 2 peroxidase CoA NAD 2 vit C TCA dehydrogenase 2H O H O 2 Tyrosine Dopamine beta- 2 Methylmalonate Cu2+ α-Methylacetoacetyl CoA aminotransferase Glu monooxygenase NADH NADH + GSH semialdehyde CO NAD 2-Hydroxyglutaric Gamma-glutamyl- 2 Dehydro Cycle Isocitrate Met Cycle H O Fumaric acid dehydrogenase acid transpeptidase DNA 5-methyl 4-Hydroxy 2 ascorbate SAH DNA SAM Acetyl-CoA MMSA phenylpyruvate 2+ cytosine acyltransferase dehydrogenase O Noradrenaline SAM Mg Gamma-glutamyl AcCoA CoA NADH Tyrosine 2 Methyl Tyramine amino acid decarboxylase CO transferase 4-Hydroxyphenyl 2 H O vit C 2 Polyamines pyruvate oxidase 2-OG Cysteinylglycine PLP dipeptidase Glutamate CO Cysteinyl CO 2 Phenylethanolamine glycine 2 decarboxylase CO Homogentisate O 2 2 N-methyltransferase & Creatine SAH FADH2 FAD Folic acid Propionyl-CoA Thr PLP 2+ ATP Fe Homogentisate 2-Oxoglutarate Glutathione O Pi 2 FADH2 CoA GTP GDP NADH + CO + NADH 2-OG Glu 1,2-dioxygenase NAD reductase H O 2 H O NAD Ser 2 PPi 2 4-Maleyl acetoacetate Succinate Metabolism S-adenosyl S-adenosyl 2+ Amine dehydrogenase Mg Maleylacetoacetate H O homocysteinase transferase 2+ Propionyl-CoA oxidase 2 Acetoacetate CoA ADP ATP NADH + Adenosine ATP biotin, Mg isomerase NAD H O carboxylase ADP Fumarylacetoacetase FAD 2 H O NH 2+ 2 3 Fumaryl Mg ,TDP PLP H O + g-Aminobutyric acid Succinic acid Homoserine Cystathionine Homocysteine Met H O 2 H acetoacetate 2-OG lipoic acid H O D-Methylmalonyl CoA 2 2 CO Threonine 2 Pi 2 dehydrogenase NH Oxidative Stress? Aldehyde 4-Aminobutyrate Succinate-semialdehyde dehydratase 3 dehydrogenase aminotransferase dehydrogenase Succinate L-methyl D-methyl Methylmalonyl-CoA 4-Aminobutanal SucCoA Propionyl-CoA 2-Oxobutanoate semialdehyde malonyl-CoA malonyl-CoA racemase H O + Succinyl CoA Methylmalonyl-CoA Methylmalonyl-CoA Propionyl-CoA 2-Oxobutyrate Homoserine Serine S-methyl 2 2H CoA Cystathionase synthetase mutase racemase carboxylase dehydrogenase deaminase hydrolyase transferase L-Methylmalonyl CoA + NADH NAD cobamamide 2+ HCO - CO vit B Pi biotin,Mg 3 2 lipoamide,TDP PLP 12 Methylmalonyl-CoA mutase cobamamide SucCoA CARCINOSCOPE

Copyright © 2012 Human Metabolome Technologies, Inc. All Rights Reserved. Duplication without permission is prohibited. ＊ TCA inactivation? HMT’s C-SCOPE contributed to this study. Data Analysis & Interpretation Support

Statistical Analysis Pathway Map

Principal Component Analysis Dec. 15 L18-2

L6-3 10 L12-2 Inc.

L0-3 Glucosamine L12-3 5 synthesis L6-2 L12-1 L6-5 L18-5 L0-5 L12-4 0 L6-4 L12-5 L0-2 L6-1 Cluster Analysis (13.2%) PC2 -5 L0-4 L18-1 L18-4 -10 L0-1 Volcano Plot L18-3 7 -15-15 -10 -5 0 5 10 15 PC1 (29.1%)

Glucose 6-phosphate 6 IMP Fructose 6-phosphate Carnitine

5 Unknown compound

3-Aminoisobutyric acid Potential biomarker! N-Acetylglutamic acid Trigonelline N5-Ethylglutamine Rhein Stachydrine 4 2-Aminobutyric acid value)

- Dihydroxyacetone phosphate SDMA p UDP-glucose ( UDP-galactose Phosphoenolpyruvic acid 3',5'-ADP 10 3-Phosphoglyceric acid 5-Aminovaleric acid 2-Hydroxybutyric acid Sedoheptulose 7-phosphate 1-Methyl-4-imidazole threo-β-Methylaspartic acid log Ketone body Spermidine Putrescine O-Succinylhomoserine − acetic acid p=0.001 6-Phosphogluconic acid 3 3'-Dephospho CoA 3-Aminobutyric acid Imidazolelactic acid Gly-Gly Uracil 3-Hydroxybutyric acid Uric acid Creatine synthesis 2-Hydroxyvaleric acid Fructose 1,6-diphosphate Ornithine Ophthalmic acid Lactic acid Gln 3-Methylhistidine ADMA Acetyl CoA 3-Hydroxy-3-methylglutaric acid Glucose 1-phosphate Isocitric acid CDP-choline CoA_divalent N-Acetyl-β-alanine Fumaric acid Glyceric acid Taurine 2-Aminoadipic acid Adenosine p=0.01 2 Thiaproline break down Malic acid 3-Ureidopropionic acid Propionyl CoA_divalent Saccharopine S-Nitrosoglutathione Cystathionine Suberic acid UTP p=0.05 Phenaceturic acid Butyric acid Kynurenine S-Lactoylglutathione 1 ATP Taurocholic acid

0 Adipic acid -3 -2 -1 0 1 2 3

log2(12h/6h) 7 Unexplored Metabolomic Space

Comprehensive Metabolome Compounds ???,???,??? unknown

PubChem DB 109,908,881 Compounds reported

Human Metabolome DB Detected & Predicted 115,398 Metabolome in human

Human Metabolome DB Detected 9,419 Source: https://hmdb.ca/ https://pubchem.ncbi.nlm.nih.gov/ as of 2021/5/24

8 ω Search – Structure Prediction

MS/MS × Chemoinformatics Precursor Candidate structure

= Structure Elucidation -NH

Score 0.96 1H-Imidazole- Histidine Almost identical 4-propionic acid “Michi-taro” except –NH2 group Ranking based on the structural Precursor similarity of candidates & targets MT estimation model search Model prepared by MT, m/z & theoretical pKa of metabolites in the HMT library

MT & m/z + MS/MS MT estimation model MT estimation Finger- printing Ranking based on the similarity of actual & estimated MTs Ranking based on the similarity of actual & simulated MS/MS spectra Combining the 3 results for extracting the most likely structure

Yamamoto, H. and Sasaki, K. Electrophoresis 2017, 38(7):1053-1059. 9 Total Solution for Drug Dev. & Biomarker Discovery

Informatics, Metabolite Clinical Validation & Interpretation & Biomarker Discovery Quantitation Identification

Comprehensive Data Analysis Support Quantitative Analysis Metabolomics • Maker search & MoA elucidation • Targeted analysis of marker • Early diagnosis marker discovery Structural Prediction candidates • CDx marker search • Prediction & identification of • Patent application of biomarkers • Drug target search metabolites • Statistical analysis and publication

10 Human Metabolome Technologies, Inc.

HQ & Labs: 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052, Japan +81-235-25-1447 Tokyo Office: Stern Chuo Bldg. 5F, 2-9-6 Shinkawa, Chuo, Tokyo 104-0033, Japan +81-3-3551-2180

Human Metabolome Technologies America, Inc. Boston Office: 24 Denby Road, Suite 217, Boston, MA 02134, USA +1-617-987-0554 Human Metabolome Technologies Europe, B.V. Leiden Office: J.H. Oortweg 21, 2333 CH Leiden, Netherlands +31 71 332 2040