© 2009 Nature America, Inc. All rights reserved. nature CH nature Received 19 December 2008; accepted 22 April 2009; published online 28 June 2009; Novartis AG, Novartis Institutes for BioMedical Research, Basel, Switzerland. Correspondence should be addressed to H.W. ([email protected]). Medicine, Albuquerque, New Mexico, USA. Frankfurt am Main, Germany. Planck-Society, Dortmund, Germany. Germany. 1 affinity higher with molecules larger form to linked are affinity low with molecules small in which approaches, design and screening properties. A similar reasoning also underlies current fragment share likely they which with molecules larger into incorporated folds may allow for the identification of smaller, structurally simplified scaf similar yet still ­simpler to complex from trees) botanical in branches along primates of tion target biological same the on activity share also may but related, structurally be only may not tree scaffold the of branch a given within classes compound were built by means of exclusively chemistry rules could meet this demand. Previous investigations using trees that bioactivity both by guided trees scaffold of construction the In principle, desirable. be highly would activity and archically system classification structured that encodes both structure hier similar a activities, biological and biochemical investigated the relationships structural complex of recognition intuitive the facilitate to method efficient and valid a is trees’ ‘scaffold of means by scaffolds library the of representation bioactivity. investigated the integrate taneously the of analysis simul that and library and by a compound given defined space chemical navigation visualization, mapping, allow that needed are approaches applicable readily and accessible intuitively knowledge, into data these of conversion and data screening bio­ and logical biochemical primary of sets large of analysis efficient For identifying new active scaffolds for 5-lipoxygenase and the estrogen receptor ER target classes and allows for the of identification new inhibitor types for a given target. We provide proof of principle by simple scaffolds with retained yet varying bioactivity is feasible at high frequency for the five major pharmaceutically relevant investigated bioactivity and further compound design. Brachiation along the branches of such trees from structurally complex to and navigation of the chemical space defined by a given library, which in turn allows correlation of this chemical space with the selection criterion for structural during simplification tree allowconstruction efficient and intuitive mapping, visualization intuitive means to map and navigate chemical space. We demonstrate that scaffold trees built using bioactivity as the key The structure- and chemistry-based hierarchical of organization library scaffolds in tree-like arrangements provides a valid, Luc Brunsveld Steffen Renner space chemical Bioactivity Bettina Hofmann Bettina Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Dortmund, Germany. The chemistry The 3 Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa. E MIC 1–7 A - 4 based hierarchical classification, organization and and organization classification, hierarchical based 1 L BIOLOGY L , , 8 . Such ‘brachiation’ (analogous to the locomo the to (analogous ‘brachiation’ Such . 2 , Daniel Rauh , 9 5 , Willem A L van Otterlo , Stefan Wetzel 8 structures along lines of biological relevance relevance biological of lines along structures 6 Novartis Institutes for BioMedical Research, Basel, Switzerland. - guided mapping and navigation of of navigation and mapping guided

advance online publication online advance 5 Institute of Pharmaceutical Chemistry, Center for Drug Research, Development and Safety, Johann Wolfgang Goethe-University, 1–3 . For the simultaneous analysis of of .analysis For the simultaneous 8 4 Technische Universiteit Eindhoven, Department of Biomedical Engineering, Eindhoven, the Netherlands. & Herbert Waldmann 1 , 2 , Ansgar Schuffenhauer - - based based rules indicate that and chemistry and 1 , 3 , Marta Dominguez Seoane - derived derived - based based

9,10 1 . , 2 ­ ­ ­ ­ the WOMBAT database ( database WOMBAT the linear all derived we from target on the same biological analysis, activity with scaffolds of branches the for dataset basic a generate To building tree scaffold Bioactivity-guided RESULTS for scaffolds active 5 new identifying by approach this of utility the We demonstrate target. given a for types inhibitor new of tification among all major pharmaceutical target classes and phenomenon allows for the prevalent iden widely a is targets protein given on activity from complex to simpler scaffolds structural with retained yet varying relevance biological proven of lines along brachiation that revealed design. Our compound analysis and further bioactivities investigated the with space chemical this of correlation both allows library, which to means accessible map, visualize, navigate and analyze the chemical intuitively space defined by the efficient, an in provide library tree compound scaffold a given a of organization and analysis guided scaffolds product natural of characteristic are that libraries prevalidation biological of elements product–inspired retaining natural tractable more synthetically of active in it may because the guide design products importance the natural complex case of known particular of of is analogs simplification new Structural simpler, ­molecules. of synthesis the direct to 6 , Peter Ertl - lipoxygenase and the estrogen receptor ER receptor estrogen the and lipoxygenase In addition to library analysis, brachiation could also be used used be also could brachiation analysis, library to addition In Here we provide empirical proof that the hierarchical bi hierarchical the that proof empirical provide we Here doi:10.1038/nchembio.18 7 Division of Biocomputing, University of New Mexico School of 2 Technische Universität Dortmund, Fakultät Chemie, Dortmund, 6  , Tudor I Oprea . 4 , Sabine Möcklinghoff Figs. 1 Figs. 8 4 . 4 Chemical Genomics Centre of the Max- and and 7 , Dieter Steinhilber 2 ) 11,12

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© 2009 Nature America, Inc. All rights reserved. peptides and linear molecules of low relevance to medicinal chem medicinal to relevance low of molecules linear and ­peptides ent scaffolds found, parent found, scaffolds ent istry,  the is one in scaffold child the which for pairs scaffold two from 2a Fig. Supplementary par the For WOMBATscaffolds. unique of set the in for searched from the child scaffolds child the from derived scaffolds parent meaningful chemically All parent). the fold formed scaf were smaller the and scaffold, ring child the is termed one scaffold larger (the by size in differing pairs scaffold struction, con sequence For sequence. larger a within already contained not were that generated were to sequences possible All subjected generation. then branch were which identified, were scaffolds molecular unique 46,036 molecules, 170,980 remaining WOMBAT. in the For molecules 186,114 the from removed were bonds peptide successive in given is analysis the of t r a b a e d c

Paclitaxel O O O

13 molecules without a ring and molecules with at least three three least at with molecules and ring a without molecules 3 NH NH O NH H N L C I 7 8-ring scaffold OH OH N S scaffold Epothilone O H O O O O 1 N N O O 5 O O O O H 10 O N O O H O O O O H s e H N O O O O OH OH OH C O O O O O O H H HO HO H H O O O O pair child parent- Not O O O H H ). Next, linear branches were constructed constructed were branches linear Next, ). O 2

O (step 1 in in 1 (step child scaffold Addition oflarger HN O NH - 14 Supplementary Figure 1 Figure Supplementary child pairs were constructed (step 2 in in 2 (step constructed were pairs child NH H N Terminal sidechains Linker Ring systems 7-ring scaffold O OH 11 N 2 1 (child scaffold Paclitaxel O O H O O H N N O O ) O NH NH O OH Supplementary Fig. 2a Fig. Supplementary O O O O O OH O O O 2 1 O O child pair Initial parent- H HO H O O O O O O OH O O Br H O O O O 15 H HO H H N O O O 12 N O O O H N NH H 6-ring scaffold NH O OH 8 pair chil Parent- . To filter out out filter To . parent scaffold Addition ofsmaller 4 O O d O

O HO O scaffold Molecular O (parent) scaffold Docetaxel HO O OH ) were were ) O O O H HO H NH O 16 O O ­ ­ ­ ­ H N O O O

H 2 O O 4 6 NH O ber of targets modulated by ber modulated targets of each at compound a half closely related receptors or enzymes investigated for selectivity of the of for selectivity investigated or receptors enzymes related closely are frequently targets Such compounds. the of for 86% recorded was only one target is described in WOMBAT. A maximum of three targets 3 Fig. (IC concentration itory promiscuous compounds recorded in of WOMBAT, activities to multiple linked be may we proportion analyzedhigh this thewhether num determine to WOMBAT. by order In covered targets protein the of 1,649) of is, 517 (that third one about for identified was brachiation ( short highest relatively are the targets with annotated of numbers populated branches active Most 9. to 4 length of Figure in alkaloids indole and derivates paclitaxel for detail more in trated in given is analysis cheminfor matic the of overview general A 9. to 3 from ranging lengths 2 O O O O NH O O O OH 9 5-ring scaffold O O O advance online publication online advance O ). The analysis revealed that for about 50% of the compounds, compounds, the of 50% about for that revealed analysis ). The O F 1 F F . O O O HO Figure Figure O OH O O O O 17 H HO H O O NH O 2 H 2 displays five selected examples for sequences sequences for examples selected five displays 50 the branches with activity on the same target target same the on activity with branches the ( scaffolds parent ( scaffolds child different that demonstrates example The 5HT-2A.on active alkaloids indole of branch ( retained. were target same the on active scaffolds contiguous three least at with those only structure, chemical the by defined branches scaffold ( branches. longer toward 2) length of (branches pairs initial the extend ( other. each from rings other disconnect not would that a ring of removal by is, ring—that peripheral one exactly of removal by scaffold child the from constructed be can scaffolds parent Valid scaffolds. of pool the from identified were pairs scaffold scaffold–child parent branches, scaffold longer of generation the for points ( a linker. or a ring either to bonded double not were that and systems, ring linking not were that atoms nonring all is, chains—that side terminal all of removal the ( 5HT-2A. receptor 5-hydroxytryptamine the on active alkaloids indole and stabilizers tubulin paclitaxel-derived for exemplified tree scaffold 1 Figure one third of all unique linear sequences with with sequences linear unique all of third one about is, branches—that 18,655 to led step ( scaffolds consecutive three least at to assigned target same the on sequences were retained that featured activity and activity, biological and biochemical for ( 9 length to scaffolds) 3 including is, (that 3 length of branches linear led unique 57,490 procedure to this to according branches 2b (see Fig. other the of parent (see branch the in scaffolds neighboring the as target same the on active molecules of design the for points 1 ( annotation bioactivity the in Gaps not. was 1 sequence the example, for retained; were scaffolds consecutive three least at to assigned a Table 5 0 ) ) or Supplementary Figures 1 Figures Supplementary ) Molecules were converted to scaffolds by scaffolds to converted were ) Molecules ) can be identified and may serve as starting starting as serve may and identified be ) can  1 K 7 c i was retained but the sequence sequence the but retained was of 30 of 1 ) Parent-child pairs were combined to combined were pairs ) Parent-child ). Iterative growth of these linear linear these of growth Iterative ).

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© 2009 Nature America, Inc. All rights reserved. nature CH nature scaffold. guiding the than scaffolds simpler much structurally at arrive still but respectively, four and rings, rings five at stopped I inhibitors topoisomerase camptothecin-derived and stabilizers tubulin paclitaxel-derived of branches The scaffold. ring one- simpler a much with end both and respectively, scaffold, a six-ring and scaffold a seven-ring with start trypsin and (AChE) esterase acetylcholine enzymes the of branches The scaffold. one-ring a simpler with ends and morphine, product natural the from derived scaffold nine-ring a complex with 2 Figure size Scaffold

1 ring 2 ring 3 ring 4 ring 5 ring 6 ring 7 ring 8 ring 9 ring Selected examples of identified scaffold branches. The longest branch (length of 9) was found for the the for found was 9) of (length branch longest The branches. scaffold identified of examples Selected E MIC A HO HO HO HO HO HO HO HO Cl Cl L BIOLOGY L receptor κ 19 25 24 21 20 23 22 O O O O 18 opioi 26 N N N N N N N H H OH OH OH OH O N d O O O Br N

N advance online publication online advance Cl HN HN HN N N N esterase Acetylcholine Neostigmine HN N F N 33 O 32 O NH N Cl 29 27 31 30 NH N 2 O Cl 28 2 O N + NH NH NH N N N NH N

Benzamidine HN HN HN HN HN HN Trypsin HN HN HN NH S NH S NH O S O O HN HN O 2 36 2 O 2 O NH NH NH S S 35 O O 34 O O O 39 2 38 37 2 O O 2 N N N O O O H N N N N O O O O O O O O O O Paclitaxel Docetaxel NH NH NH NH 1 OH OH OH OH 7 9 8 O O O O O Tubulin O O O O O O HO HO O O O  O O O O OH OH OH OH opioid receptor. This branch starts starts branch This receptor. opioid O O O O O O O O HO HO HO HO H H H H H H H H O O O O O O O O O O O O H H H H O O O O HO HO Topoisomerase HO O O HO O O O N HO HO t r a Camptothecin N N O O Cl N N N N N 43 42 41 40 N N L C I O O O O I s e  © 2009 Nature America, Inc. All rights reserved. nels, including voltage including nels,  5 4 Branch length Table 1 from Protein Data Bank entries 2CKM (acetylcholine esterase) and 1K1M (trypsin). 1K1M and esterase) (acetylcholine 2CKM entries Bank Data Protein from obtained were structures cocrystal the of coordinates The Asp189. at recognition substrate for pocket specificity the in is hotspot binding the trypsin, In site. active the in triad catalytic the is hotspot binding this esterase, acetylcholine For scaffold. child larger the into incorporated substructure analogous the by targeted is that site same the to bind scaffolds smallest the cases both In ( esterase acetylcholine of branches scaffold the from ligands and proteins ( target. particular a to WOMBATrespect in with reported were that molecules inactive and active all includes molecules reported of number The target. respective the for reported molecules of number the and a target of branch longest the of length the between observed was (0.65) correlation WOMBAT.in reported ( molecules 100 than more with targets considered only we results, relevant more statistically obtain To proteins’). (‘other families target the of any to assigned not proteins and kinases for found were Exceptions more. 4 or length of branches had targets the of half than more classes target the of most for that reveals classes target the over target per branches longest the ( classes. 3 Figure 3 (length branches short with targets substantially for was lower target per scaffolds) (and molecules reported of the longest branch of the target (see of unique WOMBAT molecules reported for a target and the length of the with receptors nuclear for found were (see trypsin (thrombin, proteases and receptor, EGFR) factor growth for The identified. (epidermal branches longest kinases (see esterase acetylcholine types (see (GPCR) receptor coupled the for found was 9 of length observed interest, pharmaceutical current ( 5 were identified length at least of of branches classes protein major all For classes target major within Brachiation dataset. analyzed the in promiscuity abundant of is no indication there Thus compounds. b a Total 9 8 7 6 3 t r a and HIV and scaffolds were considered instead of molecules. The median The molecules. of instead considered were scaffolds (peroxisome proliferator Number ofuniquebranchesthatwereannotated;multipleannotationsarenotconsidered. Each targetisonlycountedonceforitslongestbranch.

A A weak Pearson 0.65 of was correlation the found between number  and

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2 ) A weak ) A weak 18,655 12,561 ); for the opioid receptor sub receptor opioid the for ); d 1,023 4,756 219 ). ).

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6-ring scaffold tal structure of the enzyme and a molecule with the six the with a molecule and enzyme the of structure tal possible and successful for proteins with different sizes and types types and sizes different with proteins for successful and ­possible mal lengths per target ( target per lengths mal 6. length than may molecules limit the branch identified length for branches shorter that in the examined dataset, the lower number of documented tested of natural ligands—that is, brachiation is not restricted to target target to restricted not is brachiation is, ligands—that natural of morphine. to relative scaffolds, smaller and larger both of direction the into extended was branch The scaffold. derivatives of the natural product morphine with five rings in the core the for by activity. the of result and may This means remaining be illustrated or non natural to smaller products natural of complexity structural the reduce to possibility a up open more. or 4 of lengths sequence displayed targets the of 50% not to belonging proteins the major target classes (‘other the proteins’), more and than kinases for Except more. or 3 of brachiation lengths displayed sequence molecules reported 100 least at with targets a binding hotspot at the S1 pocket. This pocket is responsible for for responsible is pocket This pocket. S1 the at hotspot binding a peptide The triad. catalytic the to close acetylcholine substrate natural the of is, hotspot) (that binding the site binding the at directly interacts that inhibitor petitive ( neostigmine into product incorporated natural the scaffold ring single the identifies molecule ring compound in present is that an is esterase substrate natural a small with enzyme Acetylcholine trypsin. and esterase acetylcholine by provided are examples Illustrative ligands. natural large with classes 6 6 per target, length 5 4 length target, per scaffolds) (35 ligand comes close to the catalytic triad. Brachiation from the six the from Brachiation triad. catalytic the to close comes ligand

= 50% The analysis also provides evidence that brachiation appears appears brachiation that evidence provides also analysis The for branch The this identified receptor longest synthetic of consists the of maxi frequencies different displayed classes target Different Our Our analysis also indicates that bioactivity

1,265 molecules (424 scaffolds) per target). This finding suggests per suggests target). This finding (424 scaffolds) molecules 1,265 advance online publication online advance  75% opioid receptor ( receptor opioid triad Catalyti 100% length branch Max. c

9 8 7 6 5 4 3 <3 =

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Number of molecules 3 ). In all target classes at least 75% of the the of 75% least at classes ). target In all 2 Trypsi

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biology ­ ­ ­ - © 2009 Nature America, Inc. All rights reserved. C activity nature CH nature scaffold largest the in rings 4 or 5 6, with 3 length branch of tions combina The respectively. rings, 2 to 5 from and rings 3 to 6 from simplification scaffold a substantial represents scaffold largest the in 6 4 or 5 with rings length branch of Thus, combination the scaffolds. ( scaffold in the largest rings 6 to 3 and 6, to 3 lengths for found were 93%) or targets, 517 the of ring. one than more have must scaffolds smallest the diagonal, main the of left branches all For ring. a single in terminate must member largest the in 4 rings a one contain all and size. They for length values equal with branches all represents the length/size combinations is shown in of each of The population were the target, considered. of irrespective branches, active all comparison, for analysis, second the In chosen. was scaffold largest the in rings of number highest the with example the length, maximal with sequences multiple of case the In targets. for each target to sequence avoid the bias potential by imposed longest heavily investigated the to restricted was analysis This targets. the case we focused on the distribution of brachiation length and size over In one were performed. two analyses branches, in the size and length two tractable at synthetically thereby arriving simplification, to structural lead to frequently for brachiation it desirable efforts, would be highly synthesis subsequent for guidance provide to order In sequence. the by covered scaffold smallest and largest the in rings of number the is by our approach simplification for structural criterion An important scaffolds active of Size pocket. S1 the of bottom the at Asp189 for partner tor ( 3 Fig. 3 for natural product inhibitors and for most synthetic inhibitors substrates peptide large of cleavage selective and recognition the ( 1T8I) code Bank Data I (Protein topoisomerase to bound camptothecin ( datasets. both for line), black (dashed 3–6 size and 3–6 length between found are size and length branch of combinations frequent most The targets. all from branches all ( ( examples campthotecin and paclitaxel The scaffold). one-ring the to down brachiate not do that branches all is, (that found are size than length a smaller have that branches all diagonal, the above and Left diagonal. ( example trypsin the and esterase acetylcholine receptor, The scaffold). parent a one-ring to down brachiate that branches is, (that found are size and length same the with branches scaffold all length, branch the and scaffold largest the in rings of number the by defined line) gray (dotted diagonal the On target. each for considered was scaffolds largest the with branch longest the only targets, ( scaffolds. the of scaffold) largest the in rings of (number size 4 Figure c b Fig. 6 - , ) Distribution of size and length over over length and size of ) Distribution The highest populated combinations identified for (499 the targets identified combinations populated The highest d ) terminal to lysine or to arginine lysine terminal 1 ) Cocrystal structures of proteins and ligands from the scaffold branches of paclitaxel and tubulin (Protein Data Bank code 1JFF) ( 1JFF) code Bank Data (Protein tubulin and paclitaxel of branches scaffold the from ligands and proteins of structures ) Cocrystal - 6 3 ring ring to four 2 . Brachiation .from a Brachiation six d 9 ) are found in this category. category. this in found ) are for the cocrystal structure with a with four structure for the cocrystal , -

Fig. Fig. guided brachiation very frequently identified much smaller smaller much identified frequently very brachiation guided Distribution of brachiation length and and length brachiation of Distribution E 2 MIC ) identifies the benzamidine that is a potent interaction is that a interaction potent the benzamidine ) identifies - ring scaffold; for example, a sequence of length 4 with 4 with length of a sequence example, for scaffold; ring - Fig. Fig. a ring ring scaffolds. To investigate the relationship between A ) For the distribution over over distribution the ) For L BIOLOGY L 2 ) are found on this this on found ) are Fig. Fig. 4 - ring inhibitor ( inhibitor ring

advance online publication online advance 1

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Tubulin in largest scaffold 10 11 12 2 3 4 5 6 7 8 9 - ring inhibi ring 3 2 Paclitaxel 1 4 5

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at least 6 to 4 known active scaffolds in the sequences, respectively. respectively. sequences, the in scaffolds active known 4 to 6 least at 8 to 6 length with of in branches annotation in activity the gaps with we for sequences searched bioactivity), desired with libraries focused of design the for is, (that manner prospective a in applicable also is approach data by tree/brachiation the scaffold bioactivity with tation In order to space chemical of mapping and investigate whether anno by brachiation design Scaffold here. described approach the of means by ligands smaller of cation identifi for opportunities fewer provide might sites binding flexible thus not be able to site, the that binding very stabilize which indicates the I topoisomerase enzyme ( the cleaved to site the bound of DNAwithin and intercalate substrate binding ligand upon formed be to known is camptothecin of site binding The molecules. Scifinder using Service Abstracts Chemical the in WOMBAT or in off pruned rings peripheral other activity biological for is essential structure in the paclitaxel embedded The oxetane ring ( compounds smaller accommodate well might that site ing tubulin the of site binding ( ( tothecin se per data the of or sites binding molecules small with by noncompatibility may approach by be for limited instance the available the brachiation 6. to 3 size 3 to 6,length of and range the within are found sequences) 17,450 of (16,725 96% even analysis, second the to According dataset. first the trend may reflect the of the selection longest and largest sequences for ( scaffolds smaller for bias described a moderate we found above, analysis the to compared and sequences all Considering effort. reasonable with synthesized be may that molecules to is, that investigated as tubulin binders due to the complex the to due binders tubulin as investigated 4 6 of simplification structural to equal are likewise Fig. Fig. 4c - ring scaffolds ring to scaffolds 4 Identification of small one small of Identification . The binding mode of paclitaxel ( paclitaxel of mode binding The . 1 9 , d ; see also ; also see 0 4 2 ) to topoisomerase I may serve to illustrate this notion notion this illustrate to serve may I topoisomerase to ) targets Number of 100 10 1 1 8 Fig. Fig. . However, we did not with find analogs paclitaxel - ring, ring, 3 2 0 . The rings of the inhibitor mimic a base pair pair base a mimic inhibitor the of rings .The 2 d b ). The ). cryo The Number of rings  - in largest scaffold ring ring and 2 10 11 12 13 14 15 - Topoisomerase 2 3 4 5 6 7 8 9 Fig. 4 subunit - ring or two or ring 3 2 1 9 d . Possibly they may not have been been have not may they . Possibly - Camptotheci ). Ligands with fewer might rings EM structure of paclitaxel in the paclitaxel of EM structure 1 4 - 7 Branch length ring ring molecules, respectively— shows a large and open bind open and a shows large 9 8 7 6 5 I 1 - ) to tubulin and of camp of and tubulin to ) ring scaffolds by means of of by means scaffolds ring n t r a

- synthesis of such such of synthesis c ring, 5 ring, ) and ) and 10 Fig. 4 Fig. L C I branches Number of - ring and and ring Fig. 4 Fig. b ). This ). This 5,000 1,000 100 10 1 s e c ).  ­ ­ ­ ­ © 2009 Nature America, Inc. All rights reserved. annotated four annotated even lead to ligands with better ligand efficiency. 5 ligand better with even to lead ligands fied 5 fied  IC revealed assay cell in enes a blood human genase–catalyzed conversion of arachidonic acid into several leukotri compounds ( corresponding four the of Investigation activity. with annotated not successfully identified a new scaffold most likely belonging to the the to class. belonging inhibitor same likely most scaffold new a identified successfully four seven guiding the for true also is This inhibitors. 3 class are likely most they rather, inhibitors; 2 class or 1 class as them qualify 3). (class sites identi The newly to or the binding active via allosteric function that inhibitors nonredox and polyphenols) as such groups ligands), iron as serve redox that groups functional contain typically 1; (class inhibitors ligand iron mechanisms: different by inhibited be for compound was obtained 4 IC 1.5 of for compound ( inhibitors genase activity. expected an with compounds active new of synthesis for the points starting may be promising scaffolds Such unannotated t r a 7

6-ring scaffolds 7-ring scaffolds a 5-ring scaffolds 4-ring scaffolds 1-ring scaffolds 2-ring scaffolds 3-ring scaffolds One example was found for a sequence identified for 5 for identified sequence a for found was example One is 0.22, and for compound compound for and 0.22, is 50 O O - ÷ number of heavy atoms) for the four the for atoms) heavy of number ÷ O O ring scaffolds scaffolds ring O O - -  lipoxygenase inhibitors do not contain functional groups that that groups functional contain do not inhibitors lipoxygenase No annotatedactivity type inhibitors (class 2; typically contain reducing functional functional reducing contain typically 2; (class inhibitors type 5-LOX branch OH O M in a comparable M assay in a comparable L C I OH OH 47 46 45 50 49 44 48 O O O O O O 4 s e 8 4 - , ring molecule molecule ring 8 5 and 3 ± 1 O 7 O O Fig. 5 Fig. 4 , OH OH N 5 4 O NH N O O N 8 , 2 O 4 and 5 a No annotatedactivity , ), for which the three the which for ), 4 HO HO HO HO  5 6 HO 9 M M for compound and 4 ER ; 8 4 Fig. 5 , which indicates that brachiation can brachiation that , indicates which 7 2 α 5 O OH O O 1 branch displayed only a slightly lower IC lower a slightly only displayed 4 7 . efficiency Thus, the ligand NH N N O N N O O 56 55 7 it is 0.21. The best value (0.28) (0.28) value best The 0.21. is it O O S S . Thus, the brachiation approach approach . Thus, the brachiation O O N F F b F F N N F F ) ) for of the inhibition 5 54 53 52 51 50 - ring scaffold molecule molecule scaffold ring values of 9.5 ± of 1.5 values 5 c b - 7 HO ring scaffold scaffold ring . The corresponding Polarization (mP) 5-LOX activity (% control) - 100 100 105 110 115 120 125 130 135 140 Designed compoundsfor5-LOX Designed compoundsforER Lipoxygenase can Lipoxygenase 20 40 60 80 90 95 63 55 1 0 × w/o 48 58

O NH NH 1 O 0 –9 1 × 1 HO 0 –8 - - - 2 lipoxy­

lipoxy 1 ring to to ring 4 2 64 Concentration × 60 (–log (–log 8 Concentration (M) 1 0 –7 was was 1 O  N N

1 O M O O 50 × 57 N ­ ­ ­ 1 0 –6

1 HO Estradiol H N × H 2 oee, l mlcls rm cfod wt fu t sx ig ( rings six to four 5 with scaffolds from molecules all However, ER toward selectivity pronounced more even an three the 338 ± 22 nM with respect to ER to respect with nM 22 ± 338 0.3 ER receptor estrogen ER the in molecules scaffold of mode a binding A for and ring activity. is is This important with finding in agreement phenolic hydroxy group of display binding affinities of 123 nM and 29 nM, respectively nM, 29 and nM 123 of affinities binding display estradiol than active less times to be 1,000 reported was of potency lower times 100 the with agreement In ligand. the of size the to respect with notable of affinity the particular, In hit. good a is compound this that labeled estradiol [ with assay reported the than tively) respec times, 900 about and 140 (about higher magnitude of orders EC estradiol determined the Due to the high protein concentration (35 (EC estradiol substrate endogenous 2.8 three N O 1 α 0 –5 61 2 65 ( 48 µ Compound Compound 3 ,

1 M)  5  × 65 57

10 N N advance online publication online advance 59 M M ( 1 M, whereas estradiol only showed a slightly decreased EC decreased slightly a showed only estradiol whereas M, - –4 O O ring and ring four ) were approximately fourfold more selective for ER for selective more fourfold approximately were ) O O CF

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© 2009 Nature America, Inc. All rights reserved. to the establishment of a “structural tree of by space means chemical evant agonistic or antagonistic modes of action. Thus, chip Thus, action. of modes antagonistic or agonistic finding indicated that, in principle, a hierarchical bioactivity a hierarchical that, in principle, indicated finding nature CH nature all for lengths scaffold active four than more of branches scaffold space. chemical defined the with subspace this for recorded ity can be covered by the compound collection and correlates the bioactiv method only maps a subfraction of the biological space that potentially the compounds is not explored by a limited number of assays. Thus, the chemical space covered, the the define biological space potentially addressable precisely by members library the of structures chemical the with the biochemical and biological assays carried out. However, while bioactivity. with it correlates and space chemical maps ously simultane method generation tree scaffold this Thus, relevance. cal decreasing ring number hierarchically arranged along lines of with biologi scaffolds of sequences selects criteria guiding major as activity biological and biochemical of use the However, space. chemical of to leads mapping primarily arguments chemistry ture and medicinal chemistry of set a on exclusively based trees scaffold of construction the from design. compound further and bioactivities investigated the space with chemical this of the correlation by both allow defined would which space library, chemical the analyze and navigate visualize, map, to means accessible intuitively and efficient tree an scaffold provide a could in libraries compound of organization and analysis classes ligand protein new of development the for allowed branches ity on along the corresponding the same target. Brachiation the scaffold of tree may branch be structurally related given and a also share biological within activ classes compound that demonstrated functions. biological their exerting when and synthesis bio during proteins multiple with interaction in to due their evolution prevalidated and relevant biologically as regarded are general in products natural context, this In probability. enhanced with pounds bi new yield should which libraries, compound new of design the for space chemical in points starting as classes compound prevalidated biologically of scaffolds the use to is BIOS of principle (SCONP) and “biology oriented synthesis” (BIOS) Previous attempts to simultaneously map and navigate biologically rel DISCUSSION PPAR for found were for receptors nuclear branches the longest (previously receptors extended ER ( four the contrast, In agonist. an with obtained estradiol. This that finding supports this small ligand is file ( amounts of small pressor to peptides the estrogen receptor in the presence of saturating B.V.), which measures the binding affinities of 53 coactivator and core cofactor profiling with PamChip microarrays (PamGene International chemistry medicinal in objective desired and challenging often profiles—an selectivity tailored with molecules of toward brachiation smaller scaffolds, thus possibly guiding the design by identified be might patterns selectivity that demonstrates ­finding 5 3 Based on an analysis of the WOMBAT database, we identified identified we database, WOMBAT the of analysis an on Based for the library explored space In a sense, the biological it maps also differs branches scaffold valid select to data activity of use The tree” product “natural the from delineated examples Individual ER the in identified gap the filling by Finally, determine that unraveled be may parameters structural Similarly, , 5 Supplementary Fig. 5 Supplementary 2 , 5 1 - ) of the ER the ) of derived rules. Hierarchical classification based on struc on based classification Hierarchical rules. derived E  MIC branch of length 6 is now the longest branch for nuclear  - A and PPAR and molecule modulators, revealed that the binding pro L BIOLOGY L  branch have an antagonistic effect have antagonistic an branch ) of ) of 

, with length 5 each). 5 length , with 6 advance online publication online advance - 5 ring, five ring, for ER

classification classification of natural products” - like scaffold arrangements led arrangements like scaffold  is very to similar the profile - ring and six and ring 2 8 . 1,4,5  branch, the new new the branch, . The underlying - - based peptide peptide based ring scaffolds scaffolds ring o active com active 2 7 1,5,6 . - guided guided . This This .

­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ity ity that should be (that tractable synthetically is, two spective compound development, as demonstrated for the simpler active molecules. active simpler structurally of design the for guidance providing thereby scaffolds), of substantially smaller and less complex scaffolds with retained activ investigated cases, bioactivity the of proportion large a In types. ligand particular to restricted be not it should therefore and ligands, synthetic and natural of sizes ing vary widely with targets for possible appears approach The activity. or non to products natural complex of natural polycyclic smaller complexity structural reduce to possibility a provides brachiation with together notion. this for proof further provide will companies) pharmaceutical major inside example, for available, is (as classes compound additional covering dataset larger much a of analysis that likely very is It proteins. of range wider even for an true hold will and is widespread principle brachiation the that assumed be can it scope, limited this at even targets analyzed all of third one nearly for identified is brachiation successful that account the literature, its from scope is by no means complete.extracted However,data taking into bioactivity of set limited a of consists database WOMBAT the that Given frequency. high at possible is brachiation which along relevance pharmaceutical of classes protein major five sition and synthesis of new small ligand classes for both proteins that that proteins for both classes ligand small new of synthesis and sition receptor ER apply. not does argument this which for compounds of fraction substantial a of inclusion the by balanced is development historical analyzed dataset the possible influence of a more or less serendipitous is events, relevance. minor Thus, only of and serendipity in the whole structures musttheir reflect inherentenzymes, by requirements of molecularbiosynthesized recognition are and proteins target particular to binding by function biological a serve typically products natural products that have resulted from selection processes in nature. Because however, natural hand, other WOMBATnumerous the contains also On analysis. for available databases the to addition their and pounds com bioactive of development further with extend) particular, (in serendipity. It appears possible that the identified branches can change science represent logical, certainly and bioactivity scaffolds the between links feasibility. Thus, chemical (iv) or classes compound particular of ties drug likeness, physicochemical properties and pharmaceutical proper about knowledge and (ii) targets empirical their druggability, (iii) the intuition, chemical (i) on instance for based synthesis compound for compounds in medicinal chemistry research. This may include choices of development historical the reflects hand WOMBATin one the on products. natural by covered space is, chemical space chemical of in by evolution—that nature to explored have known been areas currently those into well extends bioactivity with it to and space correlate to trees chemical map scaffold of construction bioactivity the using of principle the Thus, frequently. very products natural beyond goes which dataset, expanded this in sible non numerous and products natural both contains here analyzed database WOMBAT The evolution. in selected were they because relevant biologically as which can scaffolds, be product exclusively contains regarded natural examples selected few a for demonstrated successfully been has tion of new of active for scaffolds the 5 enzyme The The previously developed “natural product tree” for which brachia Scaffold trees and brachiation may successfully be used to direct pro The composition of the scaffold tree obtained by analysis of the data Our Our analysis indicates that bioactivity - - natural molecules with fewer rings but with the same type of of type same the with but rings fewer with molecules natural derived reasoning, but in part they may also reflect elements of  . Identification of gaps in branch sequences guided acqui - natural molecules. However, brachiation is pos is However, brachiation molecules. natural - guided brachiation guided led brachiation to identification - - lipoxygenase lipoxygenase and the nuclear guided guided scaffold tree building t r a - ring ring to four

identification L C I - guided guided - ring ring s e  ­ ­ ­ ­ ­ ­ ­ ­ © 2009 Nature America, Inc. All rights reserved.  National Genome Research Network German for Federal Education Ministry and Research through the German Health and of 1R01CA127731 (to 1U54MH084690 grants T.I.O.) and by the Chemischen Industrie. Part this work of was bysupported US National Institutes on the Note: studies. of previous part as deposited were structures All respectively. 1T8I, and 1JFF entries ( I topoisomerase and to pro bound tubulin and of camptothecin paclitaxel from of structures branches scaffold cocrystal the from the ligands of and teins coordinates the (trypsin); 1K1M and in tures codes. Accession in WOMBAT. annotation tag drugs into developed been have successfully range this in activity with hits initial that fact the by validated further is choice The proteins. and assay compounds data between may be interactions by influenced nonspecific concentrations, higher At values. exact yield still assays most which for value the of tutions and authors others WOMBAT. In in accordance with to and select use as many for active which molecules exact data are in available aim the with chosen was analysis the in considered be to bioactivity for value an IC had scaffold that of molecule potent most the if target particular a on active to be annotated were then Scaffolds ring. one of by removal scaffold child the from generated be could scaffold parent the which is, for view—that of point the for scaffolds which the parent were from built branches First, possible all branches. active possible all obtain used the as annotation or biochemical a activity biological instead, criterion to Here scaffolds. valid we chemically the among set of one scaffold parent select structure of set a algorithm, tree scaffold original generate a well to applied be to needs criterion second a solution, possible one than more in results this If other. each from rings other disconnect not would that ring a of is, removal by ring—that peripheral one exactly of removal the by scaffold child the from scaffold parent the to generate possible is it that is relationship a such for requirement minimum The scaffolds. of pairs between defined be database. the in given as structures molecular not deglycosylated in a atom row. studies carbon one Unlike previous in our by separated each bonds, amide consecutive three least at had that removed To were avoid in sequences, all the molecules the peptides scaffold presence of atoms linking ring systems, and atoms double bonded to either rings or linkers. scaffolds by removing all terminal side chains—that is, retaining all ring atoms, Scaffold sequence generation. data. activity biological annotated with molecules 186,114 of source the was Data. METHODS the by tools. fostered analysis interactive be accessible, readily of would development application Its datasets. screening large the tree. of in classes, the are construction and which pruned substituents attached to the structural frameworks of active compound relationships, which often are dictated by the structure nature and properties of classical of delineation the for tool a be to meant not is approach tree/brachiation scaffold Wethe however, note, that action of the guiding of chemical modes structures inthe the to analyzed treeanalogy branches. by activity receptor and enzyme modulate t r a Ackn This This research was bysupported the Max

Targets were compared on the basis of the ‘ACT_LIST:TARGET_NAME’ the of basis the on compared were Targets to even very applicable We be widely will our approach that expect For the generation of scaffold branches, parent branches, scaffold of generation the For S 50 The WOMBAT (version 7.1, Sunset Molecular LCC)Discovery database N owled u , EC p a Figure Figure L C I p t u l e r 50 m e

C , ED e n h g - t defined defined tree with only one parent for each child scaffold. In the e 3 a me s e m were obtained from entries 2CKM (acetylcholine esterase) esterase) (acetylcholine 2CKM entries from obtained were r 50 Protein Data Bank: The coordinates of the cocrystal struc cocrystal the of coordinates The Bank: Data Protein y i c

in in silico (effective dose), n i a n l ts f

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w n All molecules in WOMBAT were converted into and e b 29,30 s - - i child principle child held principle true from the structural house experience in host insti the different experience house - t e Plus (grant Plus number (grant BMBF 01GS08102, to c . K h , 30 - i e , Planck m K i b  c or a M was selected M as was selected cutoff the highest l

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i 4 n M M or lower. This upper ) were obtained from from obtained were ) f o 1,2,7 r m a , molecules were , molecules t i o n is available 3 0 . - activity activity ­ ­ ­

6. 10. 4. 19. 28. 25. 18. 30. 29. 20. 7. 11. 17. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturechemicalbiology/. HTML version the of paper at http://www.nature.com/naturechemicalbiology/. C Georg Forster Research Fellowship for Experienced Researchers. D.R. and H.W.). W.A.L.v.O. thanks the Alexander von Humboldt Foundation for a 22. 3. 14. 26. 21. 15. 5. 24. 23. 12. 27. 9. 8. 2. 16. 13. The The authors declare competing financial interests: details accompany the full OMPETI

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