Supplementary Information Target identification of Mycobacterium tuberculosis phenotypic hits using a concerted chemogenomic, biophysical and structural approach Grace Mugumbate1,2*#, Vitor Mendes2*#, Michal Blaszczyk2#, Mohamad Sabbah3, George Papadatos1§, Joel Lelievre4, Lluis Ballell4, David Barros4, Chris Abell3, Tom L. Blundell2 and John P. Overington1,5 1 - European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, CB10 1SD, United Kingdom 2 - Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB1 2GA, United Kingdom. 3 - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom. 4 - GSK, P.T.M Severo Ochoa, Madrid, 28760, Spain. 5 - Medicines Discovery Catapult Mereside, Alderley Park, Alderley Edge, SK10 4TG, United Kingdom. § - Current address: GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK. * Corresponding authors: grace@ebi.ac.uk vgm23@cam.ac.uk # These authors contributed equally to this work and are co-first authors. Supplementary Methods Table S1: Crystallographic and data collection statistics Compound 1 2 3 5 6 7 10 12 PDB ID 5MXV 5MYL 5MYM 5MYN 5MYR 5MYS 5MYT 5MYW Data collection* Space group P41212 P41212 P2 P41212 P41212 P41212 P41212 P41212 Cell parameters: a [Å] b [Å] c [Å] α=β=γ=90˚ (or β for P2) 121.23 121.23 33.84 120.66 120.66 33.66 86.62 56.48 98.19 90.01 121.81 121.81 33.70 121.36 121.36 33.70 121.55 121.55 33.79 121.17 121.17 33.72 121.53 121.53 33.76 Resolution range [Å] 121.23 – 1.62 (1.71 – 1.62) 42.66 – 1.72 (1.82 – 1.72) 98.19 – 2.28 (2.40 – 2.28) 43.07 – 1.56 (1.65 – 1.56) 121.36 – 1.83 (1.93 – 1.83) 121.55 – 1.59 (1.67 – 1.59) 38.32 – 1.61 (1.70 – 1.61) 60.76 – 1.77 (1.86 – 1.77) No. of observations total unique 713630 (54085) 32415 (4638) 331256 (44027) 26955 (3849) 146825 (21103) 43150 (6247) 396526 (26057) 36051 (4725) 290306 (42469) 22975 (3275) 431353 (51807) 34673 (4822) 375730 (30076) 32287 (3884) 316819 (46830) 25356 (3617) Rmerge 0.096(1.035) 0.077 (1.277) 0.058 (0.461) 0.054 (0.767) 0.107 (1.439) 0.061 (1.141) 0.089 (1.494) 0.077 (1.340) I/σ(I) 26.6 (2.3) 18.7 (1.9) 12.1 (2.2) 26.0 (1.9) 17.4 (2.3) 22.0 (2.1) 20.3 (1.9) 19.1 (2.3) Completeness [%] 100.0 (99.9) 100.0 (100.0) 98.7 (98.5) 98.8 (92.4) 100.0 (99.8) 99.2 (96.5) 97.5 (84.1) 99.7 (99.5) Multiplicity 22.0 (11.7) 12.3 (11.4) 3.4 (3.4) 11.0 (5.5) 12.6 (13.0) 12.4 (10.7) 11.6 (7.7) 12.5 (12.9) Refinement Refinement program PHENIX PHENIX PHENIX PHENIX PHENIX PHENIX PHENIX PHENIX Resolution [Å] 85.72 – 1.63 28.56 – 1.72 98.19 – 2.28 43.07 – 1.56 85.82 – 1.83 85.95 – 1.59 38.32 – 1.61 33.71 – 1.77 No. reflections 32356 26884 43140 35991 22915 34616 32185 25300 Rwork/Rfree [%] 17.1/19.2 18.4/21.5 17.8/22.7 17.1/19.5 17.7/20.3 17.5/20.8 16.9/17.8 17.7/19.8 RMS deviations Bonds [Å] 0.006 0.006 0.008 0.006 0.007 0.006 0.005 0.006 Angles [˚] 0.783 0.735 0.985 0.713 0.885 0.772 0.752 0.761 Ramachandran Favoured [%] 99 99 99 99 99 99 98 99 Outliers [%] 0 0 0 0 0 0 0 0 Mean ADP Protein [Å2] Ligand [Å2] 39.4 40.0 43.7 72.0 46.2 70.0 35.7 37.3 38.8 72.5 37.5 75.0 38.0 44.3 47.2 67.0 * Parameters shown in brackets are for the highest resolution shell Supplementary Results Table S2a: Number of compounds for each predicted M. tuberculosis target using MCNBC. Targets are ranked in descending order of number of assigned ligands M. tuberculosis Gene id Essentiality* M. tuberculosis UniProt Accession M. tuberculosis Protein Name Number of compounds 1 Rv2234 ptpA Y(1) P65716 Probable low molecular weight protein-tyrosine-phosphatase (PTPase) (EC 3.1.3.48) 102 2 Rv1527c pks5 N(2) O53901 Probable polyketide synthase pks5 100 3 Rv2933 ppsC N(2) P96202 Phthiocerol synthesis polyketide synthase type I PpsC (Beta-ketoacyl-acyl-carrier-protein synthase I) (EC 2.3.1.41) 100 4 Rv0363c fba Y(2) P67475 Fructose-bisphosphate aldolase (FBP aldolase) (FBPA) (EC 4.1.2.13) (Fructose-1,6-bisphosphate aldolase) 99 5 Rv2935 ppsE N(2) P96204 Phthiocerol synthesis polyketide synthase type I PpsE (Beta-ketoacyl-acyl-carrier-protein synthase I) (EC 2.3.1.41) 98 6 Rv1181 pks4 N(2) O50437 Probale polyketide beta-ketoacyl synthase Pks4 EC 2.3.1.-) 97 7 Rv2934 ppsD N(2) P96203 Phthiocerol synthesis polyketide synthase type I PpsD (Beta-ketoacyl-acyl-carrier-protein synthase I) (EC 2.3.1.41) 95 8 Rv2121c hisG Y(2) P60759 ATP phosphoribosyltransferase (ATP-PRT) (ATP-PRTase) (EC 2.4.2.17) 94 9 Rv1661 pks7 N(2) P94996 Polyketide synthase (Probable polyketide synthase pks7) 94 10 Rv0734 mapA Y(3) Q7D9D5 Methionine aminopeptidase 1 (MAP) (EC 3.4.11.18) (Peptidase M) 94 11 Rv2048c pks12 Y(4) O53490 Probable polyketide synthase pks12 92 12 Rv3800c pks13 Y(2) O53579 Polyketide synthase Pks13 (Polyketide synthase) 92 13 Rv1662 pks8 N(2) O65933 Probable polyketide synthase pks8 91 14 Rv2931 ppsA N(2) Q10977 Phthiocerol synthesis polyketide synthase type I PpsA (Beta-ketoacyl-acyl-carrier-protein synthase I) (EC 2.3.1.41) 91 15 Rv1664 pks9 N(2) O06586 Polyketide synthase (Probable polyketide synthase pks9) 91 16 Rv0405 pks6 N(5) O86335 Probable membrane bound polyketide synthase polyketide synthase Pks6 90 17 Rv3855 ethR N(2) P96222 HTH-type transcriptional regulator EthR 89 18 Rv1180 pks3 N(2) O50436 Probable polyketide beta-ketoacyl synthase Pks3 (EC 2.3.1.-) 87 19 Rv2940c mas N(2) P96291 Mycocerosic acid synthase (probable multifunctional mycocerosic acid synthase membrane-associated Mas) 87 20 Rv2932 ppsB N(2) Q10978 Phthiocerol synthesis polyketide synthase type I PpsB (Beta-ketoacyl-acyl-carrier-protein synthase I) (EC 2.3.1.41) 87 21 Rv2068c blaC N(2) P0C5C1 Beta-lactamase (EC 3.5.2.6) (Penicillinase) 87 22 Rv2946c pks1 N(2) P96285 Putative inactive phenolphthiocerol synthesis polyketide synthase type I Pks1 81 23 Rv2781c N(4) O33323 2-nitropropane dioxygenase, putative (possible alanine rich oxireductase) (EC 1.-.-.-) 79 24 Rv2947c pks15 N(2) P96284 Putative inactive phenolphthiocerol synthesis polyketide synthase type I Pks15 75 25 Rv2861c mapB N(3) P0A5J2 Methionine aminopeptidase 2 (MAP) (EC 3.4.11.18) (Peptidase M) 74 26 Rv0435c N(2) P96281 Cell division control protein, putative (putative conserved ATPase) (EC 3.6.1.-) 71 27 Rv3553 N(2) P71847 Possible oxireductase (EC 1.-.-.-) 69 28 Rv0533c fabH N(2) P0A574 3-oxoacyl-[acyl-carrier-protein] synthase 3 (EC 2.3.1.180) (3-oxoacyl-[acyl-carrier-protein] synthase III) (Beta-ketoacyl-ACP synthase III) (KAS III) (MtFabH) 67 29 Rv3197 N(2) O53343 Probable conserved ATP-Binding Protein ABC transporter 65 30 Rv2045c lipT N(2) O53488 Probable carboxylesterase LipT (EC 3.1.1.-) 63 31 Rv3588c canB Y(4) O53573 Carbonic anhydrase (EC 4.2.1.1) (Carbonate dehydratase) 60 32 Rv2210c ilvE Y(2) Q10399 Branched-chain-amino-acid aminotransferase (BCAT) (EC 2.6.1.42) 57 33 Rv1484 inhA Y(2) P0A5Y6 Enoyl-[acyl-carrier-protein] reductase [NADH] (EC 1.3.1.9) (NADH-dependent enoyl-ACP reductase) 57 34 Rv3825c pks2 N(2) O07798 Phthioceranic/hydroxyphthioceranic acid synthase (EC 2.3.1.-) (Polyketide synthase pks2) 55 35 Rv1640c lysX Y(6) P94974 Lysylphosphatidylglycerol biosynthesis bifunctional protein LysX [Includes: Lysine--tRNA ligase (EC 6.1.1.6) (Lysyl-tRNA synthetase) (LysRS); Phosphatidylglycerol lysyltransferase (EC 2.3.2.3) (Lysylphosphatidylglycerol synthetase) (LPG synthetase)] 54 36 Rv3598c lysS Y(2) P67607 Lysine--tRNA ligase 1 (EC 6.1.1.6) (Lysyl-tRNA synthetase 1) (LysRS 1) 52 37 Rv0902c prrB Y(7) P0A5Z8 Sensor-type histidine kinase prrB (EC 2.7.13.3) 51 38 Rv2737c recA N(8) P0A5U4 Protein RecA (Recombinase A) [Cleaved into: Endonuclease PI-MtuI (EC 3.1.-.-) (Mtu RecA intein)] 51 39 Rv0427c xthA Y(4) P96273 Exodeoxyribonuclease III (EC 3.1.11.2) 43 40 Rv1938 ephB N(2) P95276 Epoxide hydrolase (Probable Epoxide Hydrolase EphB (Epoxide Hydratase)) (EC 3.3.2.3) 42 41 Rv2361c uppS Y(2) P60479 Undecaprenyl pyrophosphate synthase (UPP synthase) (EC 2.5.1.31) (Di-trans,poly-cis-decaprenylcistransferase) (Ditrans,polycis-undecaprenyl-diphosphate synthase) (Undecaprenyl diphosphate synthase) (UDS) 40 42 Rv0429c Def Y(2) P96275 Peptide deformylase (PDF) (EC 3.5.1.88) (Polypeptide deformylase) 40 43 Rv3608c folP1 Y(2) P0A578 Dihydropteroate synthase 1 (DHPS 1) (EC 2.5.1.15) (Dihydropteroate pyrophosphorylase 1) 37 44 Rv2523c acpS N(2) P0A4W8 Holo-[acyl-carrier-protein] synthase (Holo-ACP synthase) (EC 2.7.8.7) (4'-phosphopantetheinyl transferase AcpS) 37 45 Rv2443 dctA N(2) P71906 C4-dicarboxylate transport protein (probable C4-dicarboxylate-transport transmembrane protein Dcta) 37 46 Rv1730c N(2) P71988 Possible penicillin-binding protein 36 47 Rv2870c Dxr Y(9) P64012 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXP reductoisomerase) (EC 1.1.1.267) 36 48 Rv1436 gap Y(2) P64178 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) 34 49 Rv0482 murB Y(2) P65460 UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) (UDP-N-acetylmuramate dehydrogenase) 33 50 Rv3670 ephE N(2) O69638 Hydrolase, alpha/beta hydrolase fold family (possible epoxide hydrolase EphE (epoxide hydratase) (arene-oxide hydratase)) (EC 3.3.2.3) 31 51 Rv3273 N(2) P96878 Probable transmembrane carbonic anhydrase (carbonate dehydratase) (carbonic dehydratase) (EC 4.2.1.1) (Sulfate transporter/carbonic anhydrase, putative) 30 52 Rv2537c aroD Y(2) P0A4Z6 3-dehydroquinate dehydratase (3-dehydroquinase) (EC 4.2.1.10) (Type II DHQase) 30 53 Rv1284 canA Y(4) P64797 Putative carbonate dehydratase-like protein Rv1284 (EC 4.2.1.-) 30 54 Rv1599 hisD Y(2) P63950 Histidinol dehydrogenase (HDH) (EC 1.1.1.23) 29 55 Rv0764c cyp51 N(2) P0A512 Lanosterol 14-alpha demethylase (EC 1.14.13.70) (CYPLI) (Cytochrome P450 51) (Cytochrome P450-14DM) (Cytochrome P450-LIA1) (Sterol 14-alpha demethylase) 29 56 Rv3627c Y(2) O06380 Putative uncharacterized protein 29 57 Rv2971 Y(2) P95124 Uncharacterized oxidoreductase Rv2971/MT3049 (EC 1.-.-.-) 29 58 Rv1707 N(2) O33206 Probable conserved transmembrane protein (Sulfate transporter) 28 59 Rv1086 N(2) O53434 Short-chain Z-isoprenyl diphosphate synthase (EC 2.5.1.68) ((2Z,6E)-farnesyl diphosphate synthase) (Z-FPP synthase) (Z-FPPS) (Z-isoprenyl diphosphate synthase) 28 60 Rv3170 aofH N(2) P63533 Putative flavin-containing monoamine oxidase AofH (EC 1.4.3.-) 27 61 Rv3062 ligB N(2) P95096 Probable DNA ligase (EC 6.5.1.1) (Polydeoxyribonucleotide synthase [ATP]) 27 62 Rv2764c thyA N(10) P67044 Thymidylate synthase (TS) (TSase) (EC 2.1.1.45) 26 63 Rv0153c ptbB N(2) P96830 Phosphotyrosine protein phosphatase PtpB (protein-tyrosine-phosphatase) (PTPase) (EC 3.1.3.48) 26 64 Rv0373c N(2) O53708 Carbon monoxide dehydrogenase, large subunit, putative (probable carbon monoxide dehydrogenase (large chain)) (EC 1.2.99.2) 26 65 Rv1323 fadA4 Y(4) P66926 Probable acetyl-CoA acetyltransferase (EC 2.3.1.9) (Acetoacetyl-CoA thiolase) 25 66 Rv2763c dfrA Y(2) P0A546 Dihydrofolate reductase (EC 1.5.1.3) 25 67 Rv0091 Mtn N(2) P67656 MTA/SAH nucleosidase (EC 3.2.2.9) (5'-methylthioadenosine nucleosidase) (S-adenosylhomocysteine nucleosidase) 23 68 Rv1338 murI Y(11) P63635 Glutamate racemase (EC 5.1.1.3) 22 69 Rv1747 Y(12) O65934 Probable conserved transmembrane ATP-binding protein ABC transporter 22 70 Rv2110c prcB Y(13) O33245 Proteasome subunit beta (EC 3.4.25.1) (20S proteasome beta subunit) (Proteasome core protein PrcB) 19 71 Rv1536 ileS Y(2) Q10765 Isoleucine--tRNA ligase (EC 6.1.1.5) (Isoleucyl-tRNA synthetase) (IleRS) 18 72 Rv2299c htpG N(2) P64411 Chaperone protein htpG (Heat shock protein htpG) (High temperature protein G) 18 73 Rv2182c Y(2) O53516 1-acylglycerol-3-phosphate O-acyltransferase (EC 2.3.1.51) (Acyltransferase family protein) 18 74 Rv2139 pyrD Y(2) P65908 Dihydroorotate dehydrogenase (quinone) (EC 1.3.5.2) (DHOdehase) (DHOD) (DHODase) (Dihydroorotate oxidase) 18 75 Rv3617 ephA N(5) O06266 Epoxide hydrolase (EC 3.3.2.9) (probable epoxide hydrolase EphA (epoxide hydratase) (arene-oxide hydratase)) (EC 3.3.2.3) 17 76 Rv1981c nrdF1 N(2) Q10840 Ribonucleoside-diphosphate reductase subunit beta nrdF1 (EC 1.17.4.1) (Ribonucleotide reductase R2-1 small subunit) (Ribonucleotide reductase small subunit 1) 16 77 Rv2855 Mtr N(2) O07927 Mycothione reductase (EC 1.8.1.15) (Mycothiol-disulfide reductase) (NADPH-dependent mycothione reductase) 16 78 Rv1649 pheS Y(2) P94984 Phenylalanine--tRNA ligase alpha subunit (EC 6.1.1.20) (Phenylalanyl-tRNA synthetase alpha subunit) (PheRS) 16 79 Rv2483c plsC Y(4) O53208 Acyltransferase family protein 16 80 Rv3218 N(2) O05848 Diacylglycerol kinase catalytic domain-containing protein 15 81 Rv3602c panC N(14) P0A5R0 Pantothenate synthetase (PS) (EC 6.3.2.1) (Pantoate--beta-alanine ligase) (Pantoate-activating enzyme) 15 82 Rv2677c hemY Y(2) P0A5A7 Protoporphyrinogen oxidase (PPO) (EC 1.3.3.4) 15 83 Rv0183 N(2) O07427 Possible lysophospholipase (EC 3.1.-.-) 15 84 Rv2384 mbtA N(2) P71716 2,3-dihydroxybenzoate-AMP ligase (bifunctional enzyme Mbta: salicyl-AMP ligase (SAL-AMP ligase) + salicyl-S-ArCP synthetase) (EC 6.-.-.-) 15 85 Rv1007c metS Y(2) O05593 Methionine--tRNA ligase (EC 6.1.1.10) (Methionyl-tRNA synthetase) (MetRS) 14 86 Rv3397c phyA N(4) P65860 Probable phytoene synthase (EC 2.5.1.32) 14 87 Rv0005 gyrB Y(2) P0C5C5 DNA gyrase subunit B (EC 5.99.1.3) 14 88 Rv0233 nrdB N(2) P96416 R2-like ligand binding oxidase (EC 1.-.-.-) (Ribonucleotide reductase R2 subunit homolog) (Ribonucleotide reductase small subunit homolog) 13 89 Rv0211 pckA N(2) P65686 Phosphoenolpyruvate carboxykinase [GTP] (PEP carboxykinase) (PEPCK) (EC 4.1.1.32) (Phosphoenolpyruvate carboxylase) 13 90 Rv3255c manA Y(2) O05898 Mannose-6-phosphate isomerase (EC 5.3.1.8) 13 91 Rv1916 aceAb N(2) O07717 Probable isocitrate lyase aceAb [second part] (isocitrase) (isocitratase) (ICL) (EC 4.1.3.1) 13 92 Rv1629 polA Y(2) P0A550 DNA polymerase I (POL I) (EC 2.7.7.7) 12 93 Rv3314c deoA N(2) O53366 Thymidine phosphorylase (EC 2.4.2.4) (TdRPase) 11 94 Rv1617 pykA Y(2) O06134 Pyruvate kinase (PK) (EC 2.7.1.40) 11 95 Rv2614c thrS Y(2) P67582 Threonine--tRNA ligase (EC 6.1.1.3) (Threonyl-tRNA synthetase) (ThrRS) 10 96 Rv1915 aceAa N(5) O07718 Probable isocitrate lyase aceAa [first part] (isocitrase) (isocitratase) (ICL) (EC 4.1.3.1) 10 97 Rv0129c fbpC N(2) P0A4V4 Antigen 85-C (Antigen 85 complex C) (85C) (Ag85C) (Fibronectin-binding protein C) (Mycolyl transferase 85C) (EC 2.3.1.-) 9 98 Rv2157c murF Y(2) P0A5L4 UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase (EC 6.3.2.10) (D-alanyl-D-alanine-adding enzyme) (UDP-MurNAc-pentapeptide synthetase) 9 99 Rv3411c guaB2 Y(15) P65167 Inosine-5'-monophosphate dehydrogenase (IMP dehydrogenase) (IMPD) (IMPDH) (EC 1.1.1.205) 9 100 Rv3313c Add N(2) P63907 Adenosine deaminase (EC 3.5.4.4) (Adenosine aminohydrolase) 9 101 Rv0467 icl1 Y(2) P0A5H3 Isocitrate lyase (ICL) (Isocitrase) (Isocitratase) (EC 4.1.3.1) 9 102 Rv0957 purH Y(2) P67541 Bifunctional purine biosynthesis protein PurH [Includes: Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (ATIC) (IMP synthase) (Inosinicase)] 8 103 Rv2129c N(2) O33263 Probable oxidoreductase (EC 1.-.-.-) 8 104 Rv3410c guaB3 Y(2) P65170 Uncharacterized oxidoreductase Rv3410c/MT3518 (EC 1.-.-.-) 8 105 Rv1293 lysA Y(2) P0A5M4 Diaminopimelate decarboxylase (DAP decarboxylase) (DAPDC) (EC 4.1.1.20) 8 106 Rv1703c N(2) O33202 Probable catechol-o-methyltransferase (EC 2.1.1.6) 7 107 Rv1905c aao N(2) O07727 Probable D-amino-acid oxidase (DAAO) (DAMOX) (DAO) (EC 1.4.3.3) 7 108 Rv1547 dnaE1 Y(2) P63977 DNA polymerase III subunit alpha (EC 2.7.7.7) 6 109 Rv2903c lepB Y(2) Q10789 Probable signal peptidase I (SPase I) (EC 3.4.21.89) (Leader peptidase I) 6 110 Rv0050 ponA1 N(2) P71707 Probable bifunctional penicillin-binding prtein 1A/1B PonA1 (Murein polymerase) (PBP1): penicillin-insensitive transglycosylase (peptidoglycan tgase + penicillin-sensitive transpeptidase (DD- transpeptidase) (EC 2.4.2.-) (EC 3.4.-.-) 6 111 Rv2447c folC Y(2) O53174 Folylpolyglutamate synthase (EC 6.3.2.17) (Probable folylpolyglutamate synthase protein FolC (folylpoly-gamma-glutamate synthetase) (FPGS)) (EC 6.3.2.17) 6 112 Rv0781 ptrBa N(2) P71835 Probable protease II PtaBa [first part] (Oligopeptidase B) (EC 3.4.21.83) 5 113 Rv3566c nhoA N(2) P0A5L8 Arylamine N-acetyltransferase (EC 2.3.1.5) 5 114 Rv0162c adhE1 N(2) Q7DAC8 Alcohol dehydrogenase, zinc-containing (Probable zinc-type alcohol dehydrogenase (E subunit) AdhE) (EC 1.1.1.1) 5 115 Rv1449c tkt Y(16) O06811 Transketolase (TK) (EC 2.2.1.1) 5 116 Rv1206 fadD6 N(2) O05307 Probable fatty-acid-CoA ligase FadD6 (fatty-acid-CoA synthetase) (fatty-acid-CoA synthase) (EC 6.2.1.-) (Very-long-chain acyl-CoA synthetase, putative) 5 117 Rv3247c tmk Y(2) O05891 Thymidylate kinase (EC 2.7.4.9) (Thymidine monophosphate kinase) (dTMP kinase) (TMPK) 5 118 Rv2173 idsA2 N(2) O53507 Probable geranylgeranyl pyrophosphate synthetase IdsA2 (GGPPSASE) (GGPP synthetase) (geranylgeranyl diphosphate synthase) (EC 2.5.1.-) (Polyprenyl synthetase) 4 119 Rv0548c menB Y(2) O06414 1,4-Dihydroxy-2-naphthoyl-CoA synthase (DHNA-CoA synthase) (EC 4.1.3.36) 3 120 Rv0194 N(5) O53645 Probable drug-transport transmembrane ATP-binding protein ABC transporter 3 121 Rv0761c adhB N(2) P71818 Alcohol dehydrogenase B (EC 1.1.1.1) 3 122 Rv1316c adaB N(2) P0A696 Methylated-DNA--protein-cysteine methyltransferase (EC 2.1.1.63) (6-O-methylguanine-DNA methyltransferase) (MGMT) (O-6-methylguanine-DNA-alkyltransferase) 3 123 Rv0198c zmp1 N(2) O53649 Endopeptidase, peptidase family M13 (Probable zinc metalloprotease) (EC 3.4.24.-) 3 124 Rv3086 adhD N(2) O53303 Probable zinc-type alcohol dehydrogenase AdhD (aldehyde reductase) (EC 1.1.1.-) (Zinc-binding dehydrogenase) 3 125 Rv1987 N(2) P64905 Uncharacterized protein Rv1987/MT2041 3 126 Rv2540c aroF Y(2) P63611 Chorismate synthase (EC 4.2.3.5) (5-enolpyruvylshikimate-3-phosphate phospholyase) 3 127 Rv3315c cdd N(2) O53367 Cytidine deaminase (EC 3.5.4.5) (probable cytidine deaminase CDD (cytidine aminohydrolase) (cytidine nucleoside deaminase)) (EC 3.5.4.5) 2 128 Rv0809 purM Y(2) O53823 Phosphoribosylformylglycinamidine cyclo-ligase (EC 6.3.3.1) 1 129 Rv0772 purD Y(2) P65893 Phosphoribosylamine--glycine ligase (EC 6.3.4.13) (GARS) (Glycinamide ribonucleotide synthetase) (Phosphoribosylglycinamide synthetase) 1 130 Rv2089c pepE N(2) P65810 Probable dipeptidase pepE (EC 3.4.13.-) 1 131 Rv1844c gnd1 N(2) Q79FJ2 6-phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44) 1 132 Rv1122 gnd2 Y(2) O06574 6-phosphogluconate dehydrogenase, decarboxylating, putative (EC 1.1.1.44)) 1 133 Rv2754c thyX Y(10) P66930 Thymidylate synthase thyX (TS) (TSase) (EC 2.1.1.148) 1 134 Rv0458 N(2) P63937 Probable aldehyde dehydrogenase (EC 1.2.1.3) 1 135 Rv3856c N(2) P96221 Putative uncharacterized protein 1 136 Rv2201 asnB Y(2) P64247 Putative asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) 1 137 Rv3307 deoD N(2) P0A538 Purine nucleoside phosphorylase (PNP) (EC 2.4.2.1) (Inosine phosphorylase) 1 * - Essentiality was attributed by first consulting any in vivo data when available, then by looking at individual knock outs in vitro and finally using information from in vitro transposon mutagenesis studies. Table S2b: Number of compounds for each predicted M. tuberculosis target using SEA. Targets are ranked in descending order of number of assigned ligands M. tuberculosis Gene id Essentiality* M. tuberculosis UniProt Accession M. tuberculosis Protein Name Number of compounds 1 Rv0404 fadD30 N(2) P95213 Long-chain-fatty-acid-AMP ligase FadD30 (EC 6.2.1.-) (Acyl-AMP synthetase) 77 2 Rv3170 aofH N(2) P63533 Putative flavin-containing monoamine oxidase AofH (EC 1.4.3.-) 69 3 Rv2505c fadD35 N(2) O06168 Probable Fatty-Acid-CoA Ligase FADD35 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 68 4 Rv0435c N(2) P96281 Cell division control protein, putative (putative conserved ATPase) (EC 3.6.1.-) 55 5 Rv3617 ephA N(6) O06266 Epoxide hydrolase (EC 3.3.2.9) (probable epoxide hydrolase EphA (epoxide hydratase) (arene-oxide hydratase)) (EC 3.3.2.3) 54 6 Rv3089 fadD13 N(2) O53306 Probable Fatty-Acid-CoA Ligase FADD13 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 48 7 Rv0533c fabH N(2) P0A574 3-oxoacyl-[acyl-carrier-protein] synthase 3 (EC 2.3.1.180) (3-oxoacyl-[acyl-carrier-protein] synthase III) (Beta-ketoacyl-ACP synthase III) (KAS III) (MtFabH) 45 8 Rv3411c guaB2 Y(15) P65167 Inosine-5'-monophosphate dehydrogenase (IMP dehydrogenase) (IMPD) (IMPDH) (EC 1.1.1.205) 38 9 Rv0270 fadD2 N(2) P95227 Probable Fatty-Acid-CoA Ligase FADD2 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 37 10 Rv2045c lipT N(2) O53488 Probable carboxylesterase LipT (EC 3.1.1.-) 35 11 Rv0183 N(2) O07427 POSSIBLE LYSOPHOSPHOLIPASE (EC 3.1.-.-) 29 12 Rv0119 fadD7 N(2) O07169 Probable Fatty-Acid-CoA Ligase FADD7 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 28 13 Rv3588c canB Y(4) O53573 Carbonic anhydrase (EC 4.2.1.1) (Carbonate dehydratase) 27 14 Rv2139 pyrD Y(2) P65908 Dihydroorotate dehydrogenase (quinone) (EC 1.3.5.2) (DHOdehase) (DHOD) (DHODase) (Dihydroorotate oxidase) 27 15 Rv3410c guaB3 Y(2) P65170 Uncharacterized oxidoreductase Rv3410c/MT3518 (EC 1.-.-.-) 26 16 Rv1747 Y(12) O65934 Probable conserved transmembrane ATP-binding protein ABC transporter 25 17 Rv0194 N(5) O53645 Probable drug-transport transmembrane ATP-binding protein ABC transporter 23 18 Rv2763c dfrA Y(2) P0A546 Dihydrofolate reductase (EC 1.5.1.3) 21 19 Rv3512 N(2) Q6MWW7 PE-PGRS Family protein 21 20 Rv0781 ptrBa N(2) P71835 Probable protease II PtaBa [first part] (Oligopeptidase B) (EC 3.4.21.83) 18 21 Rv1484 inhA Y(2) P0A5Y6 Enoyl-[acyl-carrier-protein] reductase [NADH] (EC 1.3.1.9) (NADH-dependent enoyl-ACP reductase) 17 22 Rv0166 fadD5 N(2) O07411 Probable Fatty-Acid-CoA Ligase FADD5 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 17 23 Rv0429c Def Y(2) P96275 Peptide deformylase (PDF) (EC 3.5.1.88) (Polypeptide deformylase) 15 24 Rv1640c lysX Y(6) P94974 Lysylphosphatidylglycerol biosynthesis bifunctional protein LysX [Includes: Lysine--tRNA ligase (EC 6.1.1.6) (Lysyl-tRNA synthetase) (LysRS); Phosphatidylglycerol lysyltransferase (EC 2.3.2.3) (Lysylphosphatidylglycerol synthetase) (LPG synthetase)] 15 25 Rv2443 dctA N(2) P71906 C4-dicarboxylate transport protein (probable C4-dicarboxylate-transport transmembrane protein Dcta) 15 26 Rv2764c thyA N(10) P67044 Thymidylate synthase (TS) (TSase) (EC 2.1.1.45) 15 27 Rv2971 Y(2) P95124 Uncharacterized oxidoreductase Rv2971/MT3049 (EC 1.-.-.-) 15 28 Rv2483c plsC Y(4) O53208 Acyltransferase family protein 13 29 Rv3273 N(2) P96878 Probable transmembrane carbonic anhydrase (carbonate dehydratase) (carbonic dehydratase) (EC 4.2.1.1) (Sulfate transporter/carbonic anhydrase, putative) 13 30 Rv3569 hsaD Y(6) P96851 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oate hydrolase (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) (HOPDA hydrolase) (EC 3.7.1.8) (Meta-cleavage product hydrolase) (MCP hydrolase) 13 31 Rv3561 fadD3 N(2) P96843 Probable Fatty-Acid-CoA Ligase FADD3 (Fatty-Acid-CoA synthase) (EC 6.2.1.-) (Substrate-CoA ligase) 12 32 Rv2129c N(2) O33263 Probable oxidoreductase (EC 1.-.-.-) 12 33 Rv2855 Mtr N(2) O07927 Mycothione reductase (EC 1.8.1.15) (Mycothiol-disulfide reductase) (NADPH-dependent mycothione reductase) 12 34 Rv1599 hisD Y(2) P63950 Histidinol dehydrogenase (HDH) (EC 1.1.1.23) 11 35 Rv2182c Y(2) O53516 1-acylglycerol-3-phosphate O-acyltransferase (EC 2.3.1.51) (Acyltransferase family protein) 11 36 Rv3598c lysS Y(2) P67607 Lysine--tRNA ligase 1 (EC 6.1.1.6) (Lysyl-tRNA synthetase 1) (LysRS 1) 11 37 Rv2361c uppS Y(2) P60479 Undecaprenyl pyrophosphate synthase (UPP synthase) (EC 2.5.1.31) (Di-trans,poly-cis-decaprenylcistransferase) (Ditrans,polycis-undecaprenyl-diphosphate synthase) (Undecaprenyl diphosphate synthase) (UDS) 10 38 Rv2234 ptpA Y(1) P65716 Probable low molecular weight protein-tyrosine-phosphatase (PTPase) (EC 3.1.3.48) 10 39 Rv0764c cyp51 N(2) P0A512 Lanosterol 14-alpha demethylase (EC 1.14.13.70) (CYPLI) (Cytochrome P450 51) (Cytochrome P450-14DM) (Cytochrome P450-LIA1) (Sterol 14-alpha demethylase) 10 40 Rv1086 N(2) O53434 Short-chain Z-isoprenyl diphosphate synthase (EC 2.5.1.68) ((2Z,6E)-farnesyl diphosphate synthase) (Z-FPP synthase) (Z-FPPS) (Z-isoprenyl diphosphate synthase) 9 41 Rv1284 canA Y(4) P64797 Putative carbonate dehydratase-like protein Rv1284 (EC 4.2.1.-) 9 42 Rv1730c N(2) P71988 Possible penicillin-binding protein 9 43 Rv1707 N(2) O33206 Probable conserved transmembrane protein (Sulfate transporter) 9 44 Rv0373c N(2) O53708 Carbon monoxide dehydrogenase, large subunit, putative (probable carbon monoxide dehydrogenase (large chain)) (EC 1.2.99.2) 9 45 Rv2781c N(4) O33323 2-nitropropane dioxygenase, putative (possible alanine rich oxireductase) (EC 1.-.-.-) 8 46 Rv3218 N(2) O05848 Diacylglycerol kinase catalytic domain-containing protein 8 47 Rv1899c lppD N(2) O07733 Possible lipoprotein LppD 8 48 Rv3553 N(4) P71847 Possible oxireductase (EC 1.-.-.-) 7 49 Rv3397c phyA N(4) P65860 Probable phytoene synthase (EC 2.5.1.32) 7 50 Rv0058 dnaB Y(2) P71715 Replicative DNA helicase DnaB (EC 3.6.4.12) 7 51 Rv3825c pks2 N(2) O07798 Phthioceranic/hydroxyphthioceranic acid synthase (EC 2.3.1.-) (Polyketide synthase pks2) 6 52 Rv0482 murB Y(2) P65460 UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) (UDP-N-acetylmuramate dehydrogenase) 6 53 Rv1987 N(2) P64905 Uncharacterized protein Rv1987/MT2041 6 54 Rv0198c zmp1 N(2) O53649 Endopeptidase, peptidase family M13 (Probable zinc metalloprotease) (EC 3.4.24.-) 6 55 Rv2766c fabG5 N(2) Q7D6M3 Oxidoreductase, short-chain dehydrogenase/reductase family (probable short-chain type dehydrogenase/reductase) (EC 1.-.-.-) 5 56 Rv1601 hisB Y(2) P64368 Imidazoleglycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19) 5 57 Rv3627c Y(2) O06380 Putative uncharacterized protein 5 58 Rv3855 ethR N(2) P96222 HTH-type transcriptional regulator EthR 5 59 Rv2947c pks15 N(2) P96284 Putative inactive phenolphthiocerol synthesis polyketide synthase type I Pks15 5 60 Rv2299c htpG N(2) P64411 Chaperone protein htpG (Heat shock protein htpG) (High temperature protein G) 5 61 Rv1703c N(2) O33202 Probable catechol-o-methyltransferase (EC 2.1.1.6) 4 62 Rv2157c murF Y(2) P0A5L4 UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase (EC 6.3.2.10) (D-alanyl-D-alanine-adding enzyme) (UDP-MurNAc-pentapeptide synthetase) 4 63 Rv2110c prcB Y(13) O33245 Proteasome subunit beta (EC 3.4.25.1) (20S proteasome beta subunit) (Proteasome core protein PrcB) 4 64 Rv0957 purH Y(2) P67541 Bifunctional purine biosynthesis protein PurH [Includes: Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (ATIC) (IMP synthase) (Inosinicase)] 4 65 Rv3602c panC N(14) P0A5R0 Pantothenate synthetase (PS) (EC 6.3.2.1) (Pantoate--beta-alanine ligase) (Pantoate-activating enzyme) 4 66 Rv1315 murA Y(2) P0A5L2 UDP-N-acetylglucosamine 1-carboxyvinyltransferase (EC 2.5.1.7) (Enoylpyruvate transferase) (UDP-N-acetylglucosamine enolpyruvyl transferase) (EPT) 3 67 Rv3525c N(2) P71876 Possible siderophore-binding protein 3 68 Rv2068c blaC N(2) P0C5C1 Beta-lactamase (EC 3.5.2.6) (Penicillinase) 3 69 Rv2523c acpS N(2) P0A4W8 Holo-[acyl-carrier-protein] synthase (Holo-ACP synthase) (EC 2.7.8.7) (4'-phosphopantetheinyl transferase AcpS) 3 70 Rv0091 Mtn N(2) P67656 MTA/SAH nucleosidase (EC 3.2.2.9) (5'-methylthioadenosine nucleosidase) (S-adenosylhomocysteine nucleosidase) 3 71 Rv2200c ctaC Y(2) P63854 Cytochrome c oxidase subunit 2 (EC 1.9.3.1) 3 72 Rv2210c ilvE Y(2) Q10399 Branched-chain-amino-acid aminotransferase (BCAT) (EC 2.6.1.42) 3 73 Rv0458 N(2) P63937 Probable aldehyde dehydrogenase (EC 1.2.1.3) 2 74 Rv2121c hisG Y(2) P60759 ATP phosphoribosyltransferase (ATP-PRT) (ATP-PRTase) (EC 2.4.2.17) 2 75 Rv1323 fadA4 Y(4) P66926 Probable acetyl-CoA acetyltransferase (EC 2.3.1.9) (Acetoacetyl-CoA thiolase) 2 76 Rv0427c xthA Y(4) P96273 Exodeoxyribonuclease III (EC 3.1.11.2) 2 77 Rv3608c folP1 Y(2) P0A578 Dihydropteroate synthase 1 (DHPS 1) (EC 2.5.1.15) (Dihydropteroate pyrophosphorylase 1) 2 78 Rv1629 polA Y(2) P0A550 DNA polymerase I (POL I) (EC 2.7.7.7) 2 79 Rv2946c pks1 N(2) P96285 synthase type I Pks1 2 80 Rv3197 N(2) O53343 Probable conserved ATP-Binding Protein ABC transporter 2 81 Rv3255c manA Y(2) O05898 Mannose-6-phosphate isomerase (EC 5.3.1.8) 2 82 Rv1338 murI Y(11) P63635 Glutamate racemase (EC 5.1.1.3) 2 83 Rv1436 gap Y(2) P64178 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) 2 84 Rv2845c proS Y(2) O05814 Proline-tRNA ligase (EC 6.1.1.15) Prolyl-tRNA synthetase 2 85 Rv1649 pheS Y(2) P94984 Phenylalanine--tRNA ligase alpha subunit (EC 6.1.1.20) (Phenylalanyl-tRNA synthetase alpha subunit) (PheRS) 2 85 Rv0005 gyrB Y(2) P0C5C5 DNA gyrase subunit B (EC 5.99.1.3) 2 86 Rv3566c nhoA N(2) P0A5L8 Arylamine N-acetyltransferase (EC 2.3.1.5) 1 88 Rv3313c Add N(2) P63907 Adenosine deaminase (EC 3.5.4.4) (Adenosine aminohydrolase) 1 89 Rv0800 pepC N(2) O06634 Probable M18 family aminopeptidase 2 (EC 3.4.11.-) 1 90 Rv2701c suhB N(2) P65165 Inositol-1-monophosphatase SuhB (I-1-Pase) (IMPase) (Inositol-1-phosphatase) (EC 3.1.3.25) 1 91 Rv1547 dnaE1 Y(2) P63977 DNA polymerase III subunit alpha (EC 2.7.7.7) 1 92 Rv1316c adaB N(2) P0A696 Methylated-DNA--protein-cysteine methyltransferase (EC 2.1.1.63) (6-O-methylguanine-DNA methyltransferase) (MGMT) (O-6-methylguanine-DNA-alkyltransferase) 1 93 Rv0809 purM Y(2) O53823 Phosphoribosylformylglycinamidine cyclo-ligase (EC 6.3.3.1) 1 94 Rv3307 deoD N(2) P0A538 Purine nucleoside phosphorylase (PNP) (EC 2.4.2.1) (Inosine phosphorylase) 1 95 Rv2540c aroF Y(2) P63611 Chorismate synthase (EC 4.2.3.5) (5-enolpyruvylshikimate-3-phosphate phospholyase) 1 96 Rv0772 purD Y(2) P65893 Phosphoribosylamine--glycine ligase (EC 6.3.4.13) (GARS) (Glycinamide ribonucleotide synthetase) (Phosphoribosylglycinamide synthetase) 1 97 Rv2384 mbtA N(2) P71716 2,3-dihydroxybenzoate-AMP ligase (bifunctional enzyme Mbta: salicyl-AMP ligase (SAL-AMP ligase) + salicyl-S-ArCP synthetase) (EC 6.-.-.-) 1 98 Rv1850 ureC N(2) P0A660 Urease subunit alpha (EC 3.5.1.5) (Urea amidohydrolase subunit alpha) 1 99 Rv1872c lldD2 N(2) P95143 Putative L-lactate dehydrogenase [cytochrome] (EC 1.1.2.3) 1 100 Rv3086 adhD N(2) O53303 Probable zinc-type alcohol dehydrogenase AdhD (aldehyde reductase) (EC 1.1.1.-) (Zinc-binding dehydrogenase) 1 101 Rv0162c adhE1 N(2) Q7DAC8 Alcohol dehydrogenase, zinc-containing (Probable zinc-type alcohol dehydrogenase (E subunit) AdhE) (EC 1.1.1.1) 1 102 Rv0761c adhB N(2) P71818 Alcohol dehydrogenase B (EC 1.1.1.1) 1 103 Rv1007c metS Y(2) O05593 Methionine--tRNA ligase (EC 6.1.1.10) (Methionyl-tRNA synthetase) (MetRS) 1 104 Rv2447c folC Y(2) O53174 Folylpolyglutamate synthase (EC 6.3.2.17) (Probable folylpolyglutamate synthase protein FolC (folylpoly-gamma-glutamate synthetase) (FPGS)) (EC 6.3.2.17) 1 105 Rv1905c aao N(2) O07727 Probable D-amino-acid oxidase (DAAO) (DAMOX) (DAO) (EC 1.4.3.3) 1 106 Rv2467 pepD N(2) Q7D736 Probable serine protease PepD (serine proteinase) (EC 3.4.21.-) 1 107 Rv2332 mez N(2) P71880 Putative malate oxidoreductase [NAD] (EC 1.1.1.38) (Malic enzyme) 1 108 Rv0211 pckA N(2) P65686 Phosphoenolpyruvate carboxykinase [GTP] (PEP carboxykinase) (PEPCK) (EC 4.1.1.32) (Phosphoenolpyruvate carboxylase) 1 109 Rv2173 idsA2 N(2) O53507 Probable geranylgeranyl pyrophosphate synthetase IdsA2 (GGPPSASE) (GGPP synthetase) (geranylgeranyl diphosphate synthase) (EC 2.5.1.-) (Polyprenyl synthetase) 1 110 Rv2870c Dxr Y(9) P64012 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXP reductoisomerase) (EC 1.1.1.267) (1-deoxyxylulose-5-phosphate reductoisomerase) (2-C-methyl-D-erythritol 4-phosphate synthase) 1 * - Essentiality was attributed by first consulting any in vivo data when available, then by looking at individual knock outs in vitro and finally using information from in vitro transposon mutagenesis studies. Table S3: Target prediction scores, EthR inhibition values and structural information for the in vitro assayed 35 compounds Compound SPR Inhibition (%) SPR IC50 (µM) EthR Zscore E-value LEI InhA Zscore E-value LEI GSK1107112a (1) 100 12 nd nd 1.58 nd nd 1.11 GSK1570606a (2) 100 9.9 1.45 nd 1.17 nd nd 1.26 GSK2032710a (3) 100 3.9 1.4 0.78 nd nd 0.72 GSK735826a (4) 100 13 1.57 nd 1.24 1.13 GSK445886a (5) 99 30 nd nd 1.55 nd nd 1.24 GSK735816a (6) 97 22 1.95 nd 1.35 4.6 1.98E-03 1.28 GSK920684a (7) 93 50 2.71 nd 1.17 nd nd 1.15 3 GSK1742694a (8) 93 ND nd nd 1.29 nd nd 1.15 GSK1372568a (9) 80 ND 1.54 nd 1.42 nd nd 1.19 GSK921295a (10) 74 >100 nd nd 1.31 nd nd 1.09 GSK690382a (11) 72 ND 1.72 nd 1.39 3.71 128E-05 1.17 SB-435634 (12) 67 >100 nd nd 1.18 nd nd 1.41 GSK1365028a (13) 62 ND nd nd 1.35 1.67 nd 0.98 GSK1733953a (14) 62 ND nd nd 0.76 nd nd 0.84 GR135486x (15) 61 ND nd nd 1.52 nd nd 1.15 GSK888636a (16) 60 ND nd nd 1.05 nd nd 0.69 GSK381407a (17) 59 ND nd nd 0.83 nd nd Nd SB-204804-a (18) 59 ND nd nd 0.6 nd nd nd GSK2157753a (19) 58 45 2.25 nd 1.65 nd nd 1.5 GSK1731114a (20) 56 ND nd nd 0.73 nd nd Nd GSK270670a (21) 54 ND nd nd 0.66 nd nd 0.5 GW356807a (22) 54 ND nd nd 0.94 nd nd Nd GSK463114a (23) 54 ND nd nd 0.88 nd nd 0.94 GSK1402290a (24) 51 ND nd nd 1.15 nd nd Nd GSK1302651a (25) 50 ND nd nd 1.19 nd nd 1.12 GR135487x (26) 47 ND nd nd 1.36 nd nd 1.47 GSK1812410a (27) 45 ND nd nd 0.58 nd nd Nd GSK358607a (28) 42 ND 1.53 nd 0.78 nd nd 0.75 GV187303x (29) 42 ND nd nd 1.26 1.77 4.19E-07 1.46 BRL-51091am (30) 41 ND nd nd 0.72 2.50 5.67E-21 0.56 GSK353069a (31) 38 ND nd nd 1.08 nd nd 1.13 GSK353496a (32) 33 ND 1.42 nd 1.30 nd nd 1.72 GSK3011724a (33) 28 ND nd nd 1.35 nd nd 1.16 GSK957094a (34) 27 ND nd nd 1.07 nd nd 1.23 brl-10988sa (35) 24 ND nd nd 0.44 nd nd 2.74 · ND – IC50 not determined, nd – not detected Table S4: List of other predicted targets for the 8 EthR hits GSKnumber Z_score M. tuberculosis Protein Essentiality* SB-435634 (7 targets) Compound 12 2.18 Rv2443 DctA N(2) 1.93 Rv3588c CanB Y(4) 1.76 Rv1599 HisD Y(2) 1.60 Rv0363c Fba Y(2) 1.55 Rv1981c NrdF1 N(2) 1.55 Rv2737c RecA N(8) 1.53 Rv2384 MbtA N(2) GSK2157753A (24) compound 19 3.06 Rv2933 PpsC N(2) 3.06 Rv2121c HisG Y(2) 2.25 Rv2940c Mas N(2) 2.25 Rv3855 EthR N(2) 2.18 Rv3800 Pks13 Y(2) 2.18 Rv0429c Def Y(2) 2.05 Rv2934 PpsD N(2) 2.05 Rv0734 MapA Y(3) 1.96 Rv2045c LipT N(2) 1.96 Rv1661 Pks7 N(2) 1.86 Rv2234 PtpA Y(1) 1.81 Rv1180 Pks3 N(2) 1.81 Rv0482 MurB Y(2) 1.76 Rv1527c Pks5 N(2) 1.76 Rv2210c IlvE Y(2) 1.73 Rv1181 Pks4 N(2) 1.73 Rv1436 Gap Y(2) 1.69 Rv0427c xthA Y(4) 1.69 Rv2048c Pks12 Y(4) 1.66 Rv2737c RecA N(8) 1.62 Rv2068c BlaC N(2) 1.62 Rv1662 Pks8 N(2) 1.59 Rv3062 LigB N(2) 1.59 Rv0405 Pks6 N(5) GSK1570606A (8 targets) Compound 2 2.86 Rv2781c N(4) 2.48 Rv3553 N(4) 2.25 Rv1599 HisD Y(2) 1.79 Rv1323 FadA4 Y(4) 1.74 Rv2045c LipT N(2) 1.63 Rv0734 MapA Y(3) 1.63 Rv3855 EthR N(2) 1.54 Rv2121c HisG Y(2) GSK921295A (15 targets) Compound 10 3.35 Rv2781c N(4) 3.35 Rv2931 PpsA N(2) 3.35 Rv1662 Pks8 N(2) 2.93 Rv3553 N(4) 2.93 Rv0405 Pks6 N(5) 2.93 Rv2932 PpsB N(2) 2.55 Rv0435c N(2) 2.55 Rv2933 PpsC N(2) 2.10 Rv2934 PpsD N(2) 2.10 Rv2121c HisG Y(2) 1.92 Rv1181 Pks4 N(2) 1.92 Rv2947c Pks15 N(2) 1.92 Rv0734 MapA Y(3) 1.72 Rv2935 PpsE N(2) 1.72 Rv2045c LipT N(2) GSK920684A (12) Compound 7 2.79 Rv1181 Pks4 N(2) 2.79 Rv0734 MapA Y(3) 2.79 Rv2931 PpsA N(2) 2.71 Rv0435c N(2) 2.71 Rv1180 Pks3 N(2) 2.71 Rv3855 EthR N(2) 2.71 Rv2947c Pks15 N(2) 2.68 Rv2048c Pks12 Y(4) 2.68 Rv2861c MapB N(3) 2.68 Rv2932 PpsB N(2) 2.54 Rv2781c N(4) 2.54 Rv1662 Pks8 Y(2) 2.54 Rv2933 PpsC N(2) GSK735826A (6 targets) Compound 4 3.26 Rv0734 MapA Y(3) 2.70 Rv2861c MapB N(3) 1.98 Rv2121c HisG Y(2) 1.57 Rv3855 EthR N(2) 1.95 Rv2781c N(4) 1.57 Rv0435c N(2) GSK735816A (7 targets) Compound 6 4.60 Rv2121c HisG Y(2) 2.71 Rv0734 MapA Y(3) 2.45 Rv2781c N(4) 2.44 Rv2861c MapB N(3) 2.21 Rv2971 Y(2) 1.95 Rv3855 EthR N(2) 1.87 Rv3553 N(4) 1.68 Rv0435c N(2) GSK445886A (6 targets) Compound 5 3.29 Rv2045c LipT N(2) 3.29 Rv0435c N(2) 2.49 Rv3170 AofH N(2) 2.49 Rv2781c N(4) 2.45 Rv3553 N(4) 1.89 Rv0734 MapA Y(3) * - Essentiality was attributed by first consulting any in vivo data when available, then by looking at individual knock outs in vitro and finally using information from in vitro transposon mutagenesis studies. Figure S1: Omit maps” for each of ligands contoured at 1.5σ. These maps were generated using the phases from the final model. Figure S2: Superposition of X-ray crystal structures of compound X in green and two published EthR inhibitors (PDB codes: 4M3B in yellow and 5EYR in orange). The distance of the ketone groups of each compound to N179 in shown in (Å) References 1. H. Bach, K. G. Papavinasasundaram, D. Wong, Z. Hmama and Y. Av-Gay: Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe, 3(5), 316-22 (2008) doi:10.1016/j.chom.2008.03.008 2. M. A. DeJesus, E. R. Gerrick, W. Xu, S. W. Park, J. E. Long, C. C. Boutte, E. J. Rubin, D. Schnappinger, S. Ehrt, S. M. Fortune, C. M. Sassetti and T. R. Ioerger: Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis. MBio, 8(1) (2017) doi:10.1128/mBio.02133-16 3. O. Olaleye, T. R. Raghunand, S. Bhat, J. He, S. Tyagi, G. Lamichhane, P. Gu, J. Zhou, Y. Zhang, J. Grosset, W. R. Bishai and J. O. Liu: Methionine aminopeptidases from Mycobacterium tuberculosis as novel antimycobacterial targets. Chem Biol, 17(1), 86-97 (2010) doi:10.1016/j.chembiol.2009.12.014 4. C. M. Sassetti and E. J. Rubin: Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A, 100(22), 12989-94 (2003) doi:10.1073/pnas.2134250100 2134250100 [pii] 5. A. G. Tsolaki, A. E. Hirsh, K. DeRiemer, J. A. Enciso, M. Z. Wong, M. Hannan, Y. O. Goguet de la Salmoniere, K. Aman, M. Kato-Maeda and P. M. Small: Functional and evolutionary genomics of Mycobacterium tuberculosis: insights from genomic deletions in 100 strains. Proc Natl Acad Sci U S A, 101(14), 4865-70 (2004) doi:10.1073/pnas.0305634101 6. J. Rengarajan, B. R. Bloom and E. J. Rubin: Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A, 102(23), 8327-32 (2005) doi:10.1073/pnas.0503272102 7. S. E. Haydel, V. Malhotra, G. L. Cornelison and J. E. Clark-Curtiss: The prrAB two-component system is essential for Mycobacterium tuberculosis viability and is induced under nitrogen-limiting conditions. J Bacteriol, 194(2), 354-61 (2012) doi:10.1128/JB.06258-11 8. P. Sander, K. G. Papavinasasundaram, T. Dick, E. Stavropoulos, K. Ellrott, B. Springer, M. J. Colston and E. C. Bottger: Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model. Infect Immun, 69(6), 3562-8 (2001) doi:10.1128/IAI.69.6.3562-3568.2001 9. A. C. Brown and T. Parish: Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake. Bmc Microbiology, 8 (2008) doi:Artn 78 10.1186/1471-2180-8-78 10. A. S. Fivian-Hughes, J. Houghton and E. O. Davis: Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid (vol 158, pg 308, 2012). Microbiology-Sgm, 158, 1388-1388 (2012) doi:10.1099/mic.0.X00002-0 11. S. Morayya, D. Awasthy, R. Yadav, A. Ambady and U. Sharma: Revisiting the essentiality of glutamate racemase in Mycobacterium tuberculosis. Gene, 555(2), 269-76 (2015) doi:10.1016/j.gene.2014.11.017 12. J. M. Curry, R. Whalan, D. M. Hunt, K. Gohil, M. Strom, L. Rickman, M. J. Colston, S. J. Smerdon and R. S. Buxton: An ABC transporter containing a forkhead-associated domain interacts with a serine-threonine protein kinase and is required for growth of Mycobacterium tuberculosis in mice. Infect Immun, 73(8), 4471-7 (2005) doi:10.1128/IAI.73.8.4471-4477.2005 13. S. Gandotra, M. B. Lebron and S. Ehrt: The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to nitric oxide. PLoS Pathog, 6(8), e1001040 (2010) doi:10.1371/journal.ppat.1001040 14. J. C. Evans, C. Trujillo, Z. Wang, H. Eoh, S. Ehrt, D. Schnappinger, H. I. Boshoff, K. Y. Rhee, C. E. Barry, 3rd and V. Mizrahi: Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis. ACS Infect Dis, 2(12), 958-968 (2016) doi:10.1021/acsinfecdis.6b00150 15. V. Singh, S. Donini, A. Pacitto, C. Sala, R. C. Hartkoorn, N. Dhar, G. Keri, D. B. Ascher, G. Mondesert, A. Vocat, A. Lupien, R. Sommer, H. Vermet, S. Lagrange, J. Buechler, D. F. Warner, J. D. McKinney, J. Pato, S. T. Cole, T. L. Blundell, M. Rizzi and V. Mizrahi: The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis. ACS Infect Dis, 3(1), 5-17 (2017) doi:10.1021/acsinfecdis.6b00102 16. G. S. Kolly, C. Sala, A. Vocat and S. T. Cole: Assessing essentiality of transketolase in Mycobacterium tuberculosis using an inducible protein degradation system. FEMS Microbiol Lett, 358(1), 30-5 (2014) doi:10.1111/1574-6968.12536 image2.emf F O NH NS N F ONH NS N image3.emf N NN N N S O N N N N N S O image4.emf N S N HN N S O O N S N HN N S O O image5.emf N S N NH N Cl N S N NH N Cl image6.emf N S N HN N S F N S N HN N S F image7.emf F O H N N NSO F O H N N N SO image8.emf CH3 H N NN S H3C O N S CH 3 H N N N S H 3 C O N S image9.emf N S N HN H3C S O N S N HN H 3 CS O image10.emf N S NH N S H3C O N S N H N S H 3 C O image11.emf N S N HN N S O H3C N S N HN N S O H 3 C image12.emf NH2 H NO O NH 2 H N O O image13.emf O O H2N O S O O H 2 N O S image14.emf O CH3 N H Cl F O O CH3 O CH 3 N H Cl F O O CH 3 image15.emf H NN NH2 Cl H NN NH 2 Cl image16.emf NH O H3C N N N N NH O H 3 C N N N N image17.emf N N N NH2 Cl N N N NH 2 Cl image18.emf N HN CH3 O N N CH3 N HN CH 3 O N N CH 3 image19.emf S H N N H S O O S H N N H S O O image20.emf N N S O O O NN S O O O image21.emf H N O H3C NH S CH3 NO H3C O O H N O H 3 C NH S CH 3 NO H 3 C OO image22.emf NH HN N NH O HN NH N HN O NH HN N NH O HN NH N HN O image23.emf H N O CH3 N O CH3 O H N O CH 3 N O CH 3 O image24.emf N H N N N N N H N N N N image25.emf Cl H N CH3N N Cl H N CH 3 N N image26.emf F F F NH N NH2 F F F NH N NH 2 image27.emf HO O N HO O N image28.emf H N O CH3 N O CH3 O O CH3 H N O CH 3 N O CH 3 O O CH 3 image29.emf O H2N HN O H 2 N HN image30.emf O O N HN N NH2 NH CH3H3C ClCl O O N HNNNH 2 NH CH 3 H 3 C ClCl image31.emf N N S CH3 HN CH3 N NS CH 3 HN CH 3 image32.emf N N S CH3 NH H3C S N NS CH 3 NH H 3 C S image33.emf N N H N S CH3 O O H3C N N H N S CH 3 O O H 3 C image34.emf HN H3C N N O HN H 3 C N N O image35.emf CH3 O CH3 O O N NH N NH2N HH3C H3C CH 3 O CH 3 O O N NH N NH 2 N H H 3 C H 3 C image36.emf 1235671012 image37.emf image1.emf NH N S O S F Cl NH N S O S F Cl