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% 7000 6000 5000 4000 Publication 3000 2000 1472 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 biopterin 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