a structure-guided fragment-based approach for the discovery of allosteric inhibitors targeting the...

Biochem. J. (2014) 458, 387–394 (Printed in Great Britain) doi:10.1042/BJ20131127 387

A structure-guided fragment-based approach for the discovery of allostericinhibitors targeting the lipophilic binding site of transcription factor EthRSachin SURADE*1, Nancy TY†, Narin HENGRUNG*, Benoit LECHARTIER‡, Stewart T. COLE‡, Chris ABELL† andTom L. BLUNDELL*1

*Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.‡Global Health Institute, Ecole Polytechnique Federale de Lausanne-EPFL, Lausanne, Switzerland

A structure-guided fragment-based approach was used totarget the lipophilic allosteric binding site of Mycobacteriumtuberculosis EthR. This elongated channel has many hydrophobicresidues lining the binding site, with few opportunities forhydrogen bonding. We demonstrate that a fragment-basedapproach involving the inclusion of flexible fragments in thelibrary leads to an efficient exploration of chemical space, thatfragment binding can lead to an extension of the cavity, and

that fragments are able to identify hydrogen-bonding oppor-tunities in this hydrophobic environment that are not exploitedin Nature. In the present paper, we report the identification ofa 1 μM affinity ligand obtained by structure-guided fragmentlinking.

Key words: EthR, fragment linking, fragment screening,tuberculosis.

INTRODUCTION

Structure-guided fragment-based approaches have providedefficient routes to the design of selective chemical tools and noveltherapeutics, not only for conventional ligandable targets [1], butalso for more challenging protein–protein interaction sites [2]. Inthe present study we explore the use of this approach to target thelipophilic allosteric binding site of EthR.

EthR has been implicated in ethionamide drug action [3–5]and is regulated allosterically by hexadecyl octanoate, a highlylipophilic molecule [6]. EthR, a TetR transcription family member[7], has an elongated ligand-binding site; many hydrophobicresidues line the binding site, leaving very few opportunities forhydrogen bonding [8]. Nevertheless, previous work on EthR hasreported the discovery of drug-like high-affinity EthR inhibitors[9–11]. In the present study we demonstrate that a fragment-based approach can also be used to identify novel chemotypes,forming hydrogen bonds within the largely lipophilic bindingsite. We describe the identification of new chemical scaffolds thatbind EthR and trigger dissociation from its cognate DNA-bindingsite. We demonstrate successful fragment linking to yield a 1 μMcompound as characterized by a functional SPR (surface plasmonresonance) assay. Early evaluation suggests that this compoundcan pass through the Mycobacterium tuberculosis cell membraneand shows a functional effect by decreasing the MIC (minimum in-hibitory concentration) of ethionamide when used in combination.

The campaign described in the present paper emphasizesseveral conclusions likely to be of wider application. First theinclusion of flexible fragments in the library can lead to amore efficient exploration of chemical space; secondly, thatfragments can lead to conformational changes in the proteinthat create an extension of the binding cavity, thus providinga new opportunity to discover novel ligands; and thirdly, thateven in a very hydrophobic environment, fragments are able toidentify hydrogen-bonding opportunities which are not exploitedin Nature.

EXPERIMENTAL

EthR: cloning, expression and purification

The EthR gene was cloned in a pHAT5 vector [12] with BamHIand EcoRI restriction sites. For expression, the Escherichia coliBL21 (DE3) (Novagen) strain was used. For protein expression,LB media was inoculated with fresh overnight liquid culture(25 ml per 1 litre) and grown to exponential phase (37 ◦C,230 rev./min). The cultures were then induced with IPTG (0.5–1 mM). After 3 h, the cells were harvested by centrifugation(4200 g for 15 min at 4 ◦C). Cell pellets from 1 litre of culture werere-suspended in 30 ml of lysis buffer [50 mM Hepes (pH 7.5) and150 mM NaCl] supplemented with EDTA-free complete proteaseinhibitor cocktail (Roche). The cells were lysed by sonication(10 pulses of 30 s each). Debris was removed by centrifugation(35000 g for 1 h at 4 ◦C) and the supernatant was passed througha 5 ml HiTrap IMAC Fast Flow column (GE Healthcare) chargedwith Ni2 + . After washing with 50 ml of wash buffer [50 mMHepes (pH 7.5), 150 mM NaCl and 20 mM imidazole], theprotein was eluted with 50 mM Hepes (pH 7.5), 150 mM NaCland 250 mM imidazole. The protein was further purified bysize-exclusion chromatography (Superdex 200) and concentrated(4500 g at 4 ◦C) using 10 kDa Amicon® Ultra concentrators.

Screening with a thermal-shift assay

For identification of hits, a thermal-shift assay [13] was used.Each 100 μl reaction contained 20 μM EthR, 150 mM NaCl,20 mM Tris/HCl (pH 8.0), 2.5×Sypro Orange dye (Invitrogen)and 10 mM test fragment with 10% DMSO (fragment stocksolutions were prepared in DMSO). The temperature of thesample was raised from 25 ◦C to 90 ◦C in 0.5 ◦C increments. Thefluorescence of the sample was measured at each temperaturestep, with excitation/emission wavelengths of 490/575 nm. Theseexperiments were performed using the iCycler iQ Real-Time PCRmachine (Bio-Rad Laboratories).

Abbreviations: HTH, helix–turn–helix; MIC, minimum inhibitory concentration; REMA, resazurin reduction microplate assay; RU, resonance unit; SPR,surface plasmon resonance.

1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).

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388 S. Surade and others

SPR assay

The SPR assay was carried out on a BIAcore T100 machine,designed to measure the interaction of EthR with ethA promoterDNA (106 bp) immobilized, via a biotin–streptavidin linkage, onto a CM5 Sensor Chip (BIAcore). DNA from pUC19 (113 bp)was used as the control against non-specific binding. TheseDNA fragments were produced as described previously [14,15].Streptavidin was attached to the surface of the CM5 chipaccording to the instructions provided with the amine couplingkit (BIAcore). Biotinylated control and promoter DNA fragmentswere then flowed over different channels of the chip to achievestable fixation levels of 170 and 174 RUs (resonance units) forpromoter and control DNA respectively.

For screening, EthR/fragment solution [2 μM EthR and theindicated concentration of fragment made up in running buffer(2 mM MgCl2, 10 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 200 mMNaCl and 2 % DMSO)] was flowed over the chip at 20 μl/minfor 120 s. The dissociation time was 150 s. To determine bindinglevels, the response of the control channel at steady-state wassubtracted from that of the experiment channel. The chip was re-generated between samples by passing 20 μl/min 0.03% SDS inrunning buffer for 60 s over binding channels.

For IC50 calculations, the response of EthR binding to theimmobilized DNA was measured at various concentrations ofcompounds. The resulting RUs were used to fit the data in XLfitsoftware (ID Business Solutions) and concentrations necessaryto inhibit 50% of maximal interaction were calculated. Arepresentative example of data fitting is shown for fragmenthit 1 (Supplementary Figure S4 at http://www.biochemj.org/bj/458/bj4580387add.htm).

EthR crystallization and crystal structure solution

Crystallization of EthR was carried out by the hanging-drop vapour-diffusion method using conditions based on thosedescribed previously [7]. The best crystals were obtained bymixing 2 μl of protein solution [>20 mg/ml EthR, 500 mM NaCl,20 mM Tris/HCl (pH 8.0) and 10% (v/v) glycerol] with 4 μl ofreservoir [1.8–2.2 M ammonium sulfate, 100 mM Mes-Na (pH 6–7), 5–10% (v/v) glycerol and 7–10% 1,4-dioxane] at 16 ◦C.

Fragments (100 mM in DMSO) were mixed with motherliquor [1.8 M ammonium sulfate, 100 mM Mes-Na (pH 6.75)and 12.5% (v/v) glycerol] at 1–10 mM concentrations to makecrystal-soaking solutions. The crystals were then washed in orderto remove the dioxane from the binding site by placing them inmother liquor devoid of dioxane for a few hours. The washedEthR crystals were then placed in fragment-containing solutionsfor soaking for between 1 and 16 h.

Crystals were cryoprotected by passing them briefly throughmother liquor supplemented with 20% (v/v) ethylene glycol,before freezing in liquid nitrogen. Data collection was carriedout at the European Synchrotron Radiation Facility (Grenoble,France), Diamond Light Source (Harwell, U.K.), Swiss LightSource (Villigen, Switzerland) and at the in-house source (×8Proteum, Bruker AXS). Diffraction data were analysed usingprograms within the CCP4 suite [16] run with its graphical userinterface [17]. Diffraction images were indexed and integratedusing Mosflm [18] and then scaled with Scala [19]. Molecularreplacement was carried out using Phaser [20] with PDB structure1T56 [7] as the initial model and structure refinement was carriedout using Refmac5 [21] for maximum-likelihood restrainedrefinement. Model fitting, and water and ligand fitting into thedifference map were done manually with Coot [22]. Ligandtopology and parameter files were generated either by the Dundee

PRODRG2 server [23] or with libcheck [24]. Final Figures wereprepared using PyMOL (http://www.pymol.org).

Determination of the ethionamide MIC boosting effect

Determination of ethionamide MIC boosting was performed usingthe REMA (resazurin reduction microplate assay) as describedpreviously [26]. Serial dilutions (2-fold) of ethionamide wereprepared in 96-well plates, alone or in combination with the EthRinhibitors at a fixed concentration of 1 μM. Frozen aliquots ofM. tuberculosis H37Rv in mid-exponential cultures were thawedand diluted to a D600 of 0.0025 in liquid 7H9 medium (Difco) toobtain a total volume of 100 μl. Plates were incubated for 6 daysat 37 ◦C before the addition of resazurin (0.025 %, 10 μl). Afterovernight incubation, fluorescence of the resazurin metaboliteresorufin was determined (excitation at 560 nm and emission at590 nm, measured using a Tecan infinite M200 microplate reader).The MIC was defined when the level of fluorescence is equivalentto the highest concentration of ethionamide where all cell growthis inhibited.

RESULTS AND DISCUSSION

A thermal-shift screen [13] was carried out using a libraryof 1250 fragment molecules. Any fragment displaying thermalstabilization (�Tm) of more than 1 ◦C was classified as a hit. Intotal, 86 fragment hits were identified giving a hit rate of 7%.Of these hits, 22 raised the melting temperature by more than3 ◦C, and five increased the melting temperature by more than5 ◦C. Temperature shifts of approximately 1–2 ◦C are routinelyobserved for fragment-sized ligands. In the case of EthR, thethermal shifts are quite pronounced with a significant number ofhits obtained displaying thermal shifts of more than 2 ◦C. Theselarge thermal shifts probably have resulted from the nature of theligand-binding site, which is enclosed by the protein and flankedby a number of hydrophobic residues and some hydrogen-bondinginteractions.

All 86 hits from the thermal-shift screening were then testedusing a functional assay measuring their ability to inhibit theDNA–EthR interaction, using the SPR technique as describedpreviously [14]. In addition to the 86 hits, 45 fragments thatshowed no increase in melting temperature were included in theSPR assay as negative controls. In order to rank the compounds,a single concentration screen was carried out using fragmentsat 500 μM. The resultant relative decrease in the SPR RUvalue was then used to calculate the percentage inhibition. Anycompound inhibiting the EthR–DNA interaction by more than10% was classified as a validated hit. Of the 86 fragment hitsfrom the thermal-shift assay, 45 compounds showed inhibitionof more than 10%, giving a final hit rate of 3.6 %. Out of45 negative controls from the thermal-shift assay, all exceptone exhibited inhibition of less than 10% in the SPR assay,validating the use of thermal shift as a primary screening technique(see the Supplementary Online Data at http://www.biochemj.org/bj/458/bj4580387add.htm). Hits with novel chemical scaffoldswere then further characterized by performing dose–responsetitrations in the SPR assay and the resultant percentage inhibitionswere used to calculate the IC50 values.

Table 1 shows four examples of molecules that are capable ofbinding to EthR and inhibit the EthR–DNA interaction, threeof which, 2, 3 and 4, have a similar scaffold. These fourfragment hits were selected for further characterization using X-ray crystallography by soaking into preformed crystals of EthR,as described in the Experimental section of the Supplementary


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Fragment-based screening and design of allosteric EthR inhibitors 389

Table 1 Fragment hits from the screening campaign

�T m, relative change in melting temperature of protein in complex with the hit. �T m wasdetermined using the thermal-shift screen. The percentage inhibition was determined using anSPR assay [14].

Fragment hit Structure �T m (◦C)Percentageinhibition (%)

1 5.5 78

2 1.75 36

3 1.5 50

4 4.5 82

Online Data. Binding of a fragment was judged by thepresence of difference electron density (Fo − Fc) correspondingto the fragment molecules in the hexadecyl octanoate-binding

site. All four fragments showed excellent electron densityallowing unambiguous determination of the binding mode of thefragment molecules. The detailed crystallographic informationand refinement statistics are presented in Supplementary TableS2 at http://www.biochemj.org/bj/458/bj4580387add.htm.

As shown in Figure 1, the EthR ligand-binding site isan elongated hydrophobic tunnel flanked by a number ofhydrophobic residues (Phe110, Phe114 and Phe184; Trp103, Trp138,Trp145 and Trp207; Leu87 and Leu183; Ile107; Met142) with only threepolar, but uncharged, amino acids (Asn176, Asn179 and Thr149). Twodioxane molecules, an important component of the crystallizationconditions, occupy the allosteric site. The binding channel can beroughly divided into three parts, a larger central tunnel, and tworelatively small cavities that are termed here ‘cavity 1’, ‘cavity 2’and ‘cavity 3’ respectively.

All four fragments described in the present study were ableto bind Asn179 in the central binding pocket of EthR. The sameregion of the binding pocket, cavity 1, has been targeted by aprevious study [15], and has been shown to exhibit conformationalplasticity [27]. Fragment hit 1 was observed to bind in cavity 2of the channel, in addition to cavity 1, with associated side-chainmovements. Fragments 3 and 4 were also found to bind the centralcavity 1 twice in the same crystal structure (see SupplementaryOnline Data), whereas only one molecule of fragment 2 was foundbound in cavity 1

Fragments 2, 3 and 4, all containing a sulfonyl group, displayedslightly different activities. Analyses of crystal structures of thesefragments bound to EthR indicated clear hydrogen bonding andhydrophobic interactions. As shown in Figure 2, key interactingpartners for hydrogen bonding in this series of compoundswere Asn176 and Asn179, whereas Phe110 exhibited π-stackinginteractions with the aromatic component. Fragment 2 formed

Figure 1 The EthR physiological dimer structure shown in complex with two dioxane molecules, PDB code 1T56 (the dioxane is shown in stick representation)

The region corresponding to the binding channel is encircled by a magenta line. The amino acids forming the central binding channel are shown in stick representation and coloured using the CPKscheme.




Figure 2 Interaction of sulfonyl-containing fragments with EthR

(A) Fragment hit 2 forms hydrogen bonds (shown as yellow broken lines, all shorter than 3.5 A) with Asn176 and Asn179, whereas (B) fragment hit 3 only forms a hydrogen bond with Asn179, but itsp-chloro-phenyl ring extends into the hydrophobic pocket. (C) Fragment hit 4 forms hydrogen bonds with Asn176 and Asn179, as well as interactions of the p-chloro-phenyl ring in the hydrophobicchannel. (D) A second molecule of fragment 4 forms additional hydrogen bonds with Tyr148, Leu90 and a water molecule. This additional interaction may account for its higher affinity.

hydrogen bonds with Asn176 and Asn179, and fragment 3 formed aπ-stacking interaction with Phe110. Among these three, fragment 4formed the most interactions. It interacted with Asn176 and Asn179

through hydrogen bonding, as well as π -stacking with Phe110.The p-chloro substituents in fragments 3 and 4 were positionedin a hydrophobic environment. Interestingly the thioamide offragments 2 and 4 formed an additional hydrogen bond with thebackbone of Asn176. A second molecule of fragment 4 in the samecrystal structure formed additional hydrogen bonds with Tyr148,Leu90 and a water molecule. Comparing the modes of interactionof these three fragments and their ability to inhibit the DNA–protein interaction, it appears that the thioamide group and thep-chloro-phenyl substituent both make important contributions.

The crystal structure of fragment 1 in complex with EthRreveals an interesting mode of interaction and novel insights into

the conformational plasticity of EthR. Fragment 1 occupies thehydrophobic channel at two positions, even though the preformedcentral binding region of EthR, cavity 1, is large enough toaccommodate only a single molecule of fragment 1. The secondmolecule is able to bind to the previously unexploited cavity 2by bringing about changes in the conformation of side chainsof Phe184 and Gln125. A significant movement of the Phe184 sidechain allows access to the smaller second cavity (Figure 3B). Thisinduction of a conformational change was necessary to bind thetwo molecules of fragment 1 that together are able to spanthe entire binding pocket of EthR.

Interestingly the two molecules of fragment 1 adopt slightlydifferent conformations in the same crystal structure. Fragment1 binds in both cavity 1 and cavity 2, close to the DNA-bindingHTH (helix–turn–helix) motif. It has an IC50 of 280 μM and a



Figure 3 Conformation change upon fragment binding and retention of the initial conformation and interactions during fragment linking

(A) Phe184, encircled in magenta, separates the central cavity 1 of the binding channel from cavity 2 in the dioxane-bound EthR structure (PDB code 1T56). (B) The Phe184 side-chain movementand key hydrogen bonds between fragment hit 1 and Asn179 and a water molecule. Upon binding to fragment hit 1 the Phe184 side chain moves away, adopting a different conformation (encircled inmagenta), allowing the second molecule of fragment 1 to bind to cavity 2, thus creating a larger continuous cavity, displayed in blue. This movement is also accompanied by movement of the Gln125

side chain. This significant change in Phe184 conformation, the binding mode and key interactions are maintained by compound 5 (C) and disulfide-linked compound 9 (D). All protein residues aredisplayed as lines, ligands are in stick representation and the EthR ligand-binding channel surface is shown in blue. All atoms follow the CPK colouring scheme. Hydrogen bonds are represented byyellow broken lines.

unique binding mode. In addition, the high sp3 content and numberof rotatable bonds distinguishes it from planar aromatic startingpoints of many fragment-based campaigns.

For chemical elaboration of fragment hit 1, fragment linking aswell as fragment growing were considered. As the two moleculesof fragment 1 together could span the entire binding channel,the fragment-linking approach was the more attractive option.However, before fragment linking, steps were taken to modify

fragment 1 with the aim of improving the binding affinity andmaking the subsequent chemical synthesis more straightforward.The compounds made and their respective activities are reportedin Table 2. Analogues 5 and 6 of fragment 1 were synthesizedby varying ring A and ring B, keeping intact the importantcarbonyl oxygen that forms key hydrogen bonds. Although theyshowed a slight reduction in activity, they offered better prospectsfor subsequent chemical elaboration. The crystal structure of



Table 2 IC50 of structural analogues of 1, and corresponding linked compounds

The IC50 was determined using an SPR assay [14].

Compound Structure IC50 (μM)

1 280

5 350

6 310

7 1.10

8 25

9 0.97

10 1200

11 150

compounds 5 (Figure 3C) and 6 in complex with EthR showedthat they maintained the binding mode of the initial fragmenthit 1, including occupying two sites in the binding channeland maintaining the hydrogen-bonding pattern, i.e. ability tointeract with Asn179 and to form a hydrogen bond with a watermolecule (Supplementary Figure S3 at http://www.biochemj.org/bj/458/bj4580387add.htm).

In order to link the two modified compounds 5 and 6, severaldifferent linkers were explored in silico. The designed moleculeswere docked into the fragment hit 1-bound crystal structure ofEthR using GOLD [28,29]. The two separate linkers, a disulfidefor compound 5 and amide for compound 6, were found to offer asuitable geometry allowing individual ligands to be able to adoptthe starting fragment conformation. The linked compounds weresynthesized (see Supplementary Online Data) and tested using theSPR functional assay. The structure and activities of these linkedcompounds are also reported in Table 2.

As can be seen from Table 2, the compounds with the disulfidelinker showed significantly higher activity than the amide-linked

compounds. This could be due to the structural rigidity of theamide bond preventing individual arms of the molecule fromadopting a conformation that is able to exploit the hydrophobicand hydrogen-bond interactions. In addition, an energy cost inburying the polar amide hydrogen atoms in the binding channelmay have contributed to its reduced activity.

Crystal structures of the disulfide-linked compounds 7, 8 and9 bound to EthR were obtained by soaking the compounds intoEthR crystals. Compounds 7, 8 and 9, the most potent ligands,maintain the same ligand-binding mode as that of the startingfragment and are able to form a key hydrogen bond with Asn179 aswell as with a water molecule. The retention of binding mode andinteractions of compounds 1, 5 and 9 during the fragment linkingis shown in Figure 3.

EthR ligands, post-binding, are thought to bring about achange in the EthR conformation, notably leading to changesin the distance between the HTH motifs of the EthR dimer[15]. The changes in the cell dimensions and the separation ofthe HTH domain of EthR are suggestive of the mechanism





Figure 4 Evaluation of the ethionamide boosting effect by REMA [26]

EthR inhibitors were tested in combination with ethionamide (ETH) at 1 μM.

of EthR inhibition. Ligands from the present study were ableto induce changes in conformation of proteins as measured by thedistance between HTH motifs of the two protomers withinthe crystallographic dimers. The distance ranged between 49.5 Å(1 Å = 0.1 nm) for the apo-form crystal structure to 52.3 Å forthe compound 9-bound EthR structure (Supplementary Figure S5at http://www.biochemj.org/bj/458/bj4580387add.htm). Crystalstreated similarly with soaking and cryo-solutions that are devoidof any ligands did not reveal any changes in cell dimensions. Thisindicates that the changes in cell dimensions were due to ligandinteraction and not because of backwashing of crystallizationreagent, dioxane or other experimental conditions. The analysisof all of the X-ray structures with or without ligands did not revealany noticeable changes, other than the side-chain conformationsas described above.

The appreciable activity of the final linked compound 9 inthe in vitro SPR assay was encouraging, prompting evaluationof its effectiveness in combination with ethionamide in live M.tuberculosis cultures. In addition, the starting fragment hit 1 wasalso selected to check whether it had any EthR boosting activity.These compounds were tested using the REMA (Figure 4) [26] incombination with ethionamide at a fixed concentration of 1 μMsince these compounds were found to display some bactericidalactivity at 10 μM but none at 1 μM. The MIC for ethionamide onits own was found to be 15 μM.

Compound 9 reduced the MIC of ethionamide from 15 μMto 1.9 μM at 1 μM concentration, an effective 8-fold reduction(Figure 4). It is worth noting that the IC50 of compound 9is approximately 280-fold that of fragment 1, whereas at a1 μM concentration the ethionamide MIC boosting activity ofcompound 9 is similar. The large discrepancy between the IC50

and the boosting activity of fragment 1 and compound 9 could beattributed to the relative permeability of individual compounds. Itis also possible that compound 9 serves as a prodrug and is reducedin M. tuberculosis to the thiol, which is the active species. Thispossibility will be explored in future studies by testing the reducedcompound in the SPR assay as well as by REMA.

Transcription factors have previously been considered to beundruggable [30]. In the present study we have targeted EthR asan example of a challenging drug target, and several observationsconcerning the fragment-based approach are likely to be ofwider application. Using the thermal-shift assay as a screeningtechnique allowed rapid screening of a library of compounds. Thesubsequent SPR assay was used as a functional assay to assessthe ability of compounds to inhibit the EthR–DNA interaction. Byusing a cascade of two complementary techniques a final set of

fragments was obtained, which functionally inhibited the EthR–DNA interaction by specifically interacting with EthR.

The crystal structures of EthR in complex with fragmenthits 2, 3 and 4 demonstrated that the fragments wereable to form key hydrogen bonds, even though the highlylipophilic hexadecyl octanoate, a potential natural regulatorof EthR, interacts with EthR primarily through hydrophobicinteractions (Supplementary Figure S1 at http://www.biochemj.org/bj/458/bj4580387add.htm). As shown here, even in a veryhydrophobic environment fragment-based methods are able toidentify hydrogen-bonding opportunities that are not exploitedin Nature. The observed conformations of the two molecules offragment 1 (which has a number of rotatable bonds) in the thesame crystal structure differ from each other (rmsd of 1.1 Å for a14 atom fragment). The same observation was made for fragment4, showing that conformational flexibility allows the fragmentto achieve subtly different binding interactions/orientations,emphasizing the need to have the right balance betweenflat aromatic molecules and molecules with increased three-dimensionality and rotatable bonds in screening collections. Thecrystal structures of EthR bound to fragments demonstrated thatthe fragments were able to induce conformational changes in theprotein, allowing them to occupy different pockets of the protein.In this structure-guided work it is shown that these fragment hitscould be readily linked, resulting in higher activity molecules,while retaining the initial conformational changes and bindingmode and preserving key interactions.

In summary, the crystal structures of EthR complexes show thatfragments are able to exploit key hydrogen bonds in a hydrophobicenvironment that the natural ligand does not exploit. The newlydiscovered hydrogen-bonding interactions are maintained ininhibitors designed from linking the fragments and give rise tohigher affinity and presumably more selective leads for drugdiscovery. Also, the observations in the present study argue for theuse of flexible lipophilic fragments with hydrogen-bond donorsor acceptors for targeting lipophilic sites. Such fragments may bevaluable in probing difficult targets.

AUTHOR CONTRIBUTION

Sachin Surade and Tom Blundell envisaged the project; Sachin Surade established theearly protocols in protein purification, crystallization, thermal-shift screening and the SPRassay, and designed and supervised the fragment screening; Narin Hengrung performedthe fragment screening and determined the crystal structure of fragment hits; NancyTy and Chris Abell designed compounds 5–11; Nancy Ty carried out in silicoprioritization and performed synthesis of compounds 5–11. Sachin Surade determinedthe crystal structures of EthR in complex with ligands. Benoit Lechartier perfomed theREMA under the supervision of Stewart Cole. Tom Blundell and Chris Abell supervisedthe operation of all of the project. All authors contributed to the development of the paper.

ACKNOWLEDGEMENTS

We are grateful to our many colleagues in the Department of Biochemistry, Universityof Cambridge including Dr Marko Hyvonen, Dr Marcio Dias, Dr Leo Silvestre, DrMichal Blaszczyk and Dr Vitor Mendes for stimulating discussions. We would alsolike to acknowledge Dr Dima Chirgadze and Dr Katherine Stott for maintaining the X-rayCrystallographic and Biophysics Facility in the department.

FUNDING

This work was supported by the Bill and Melinda Gates Foundation, the EuropeanCommunity’s Seventh Framework Programme [grant number 260872] and the Universityof Cambridge. B.L. is a recipient of a grant from Fondation Jacqueline Beytout.






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19 Evans, P. R. (2011) An introduction to data reduction: space-group determination, scalingand intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292

20 McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. andRead, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674

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Resazurin microtiter assay plate: simple and inexpensive method for detection of drugresistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 46,2720–2722

27 Willand, N., Desroses, M., Toto, P., Dirie, B., Lens, Z., Villeret, V., Rucktooa, P., Locht, C.,Baulard, A. and Deprez, B. (2010) Exploring drug target flexibility using in situ clickchemistry: application to a mycobacterial transcriptional regulator. ACS Chem. Biol. 5,1007–1013

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Received 22 August 2013/2 December 2013; accepted 6 December 2013Published as BJ Immediate Publication 6 December 2013, doi:10.1042/BJ20131127


Biochem. J. (2014) 458, 387–394 (Printed in Great Britain) doi:10.1042/BJ20131127

SUPPLEMENTARY ONLINE DATAA structure-guided fragment-based approach for the discovery of allostericinhibitors targeting the lipophilic binding site of transcription factor EthRSachin SURADE*1, Nancy TY†, Narin HENGRUNG*, Benoit LECHARTIER‡, Stewart T. COLE‡, Chris ABELL† andTom L. BLUNDELL*1

*Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.‡Global Health Institute, Ecole Polytechnique Federale de Lausanne-EPFL, Lausanne, Switzerland

EXPERIMENTAL

General procedure 1 for the coupling between arylalkylcarboxylicacids and secondary amines

To a solution of acid (0.67 mmol) in anhydrous dichloromethane(5 ml) under nitrogen was added anhydrous dimethylformamide(two drops) and oxalyl chloride (85 μl, 1.00 mmol) at roomtemperature (25 ◦C). The reaction mixture was stirred for 1 h andconcentrated in vacuo. To the residue dissolved in anhydrousdichloromethane (2 ml) was added, under nitrogen, triethylamine(120 μl, 0.87 mmol) and a solution of the secondary amine(0.87 mmol) in dichloromethane (3 ml) at room temperature.After stirring for 1 h, the reaction mixture was diluted withdichloromethane (15 ml), then washed successively with water(2×10 ml) and brine (10 ml). The organic layer was dried overmagnesium sulfate, filtered and concentrated in vacuo. The crudeproduct was purified by flash chromatography.

3-Phenyl-1-(pyrrolidin-1-yl)propan-1-one (5)

General procedure 1 was performed with 3-phenylpropanoic acidand pyrrolidine. After purification of the crude product by flashchromatography using a linear gradient (n-hexane/ethyl acetate,65:35–50:50) as eluent, compound 5 was obtained as a colourlessoil (102 mg, 75%). Rf (n-hexane/ethyl acetate, 60:40): 0.16.1H-NMR (400 MHz, [2H]chloroform): δ 7.30–7.19 (m, 5 H, Ar.H), 3.46 (m, 2 H, CH2N), 3.29 (m, 2 H, CH2N), 2.99 (m, 2 H,CH2Ph), 2.57 (m, 2 H, CH2CO), 1.85 p.p.m. (m, 4 H, CH2CH2);13C-NMR (100 MHz, [2H]chloroform): δ 170.9 (CO), 141.6(C), 128.5, 126.2 (aromatic CH×5), 46.7 (CH2N), 45.9 (CH2N),36.9 (CH2CO), 31.4 (CH2Ph), 26.2, 24.6 (CH2CH2); MS: m/z204.2 [M + H]+ , 226.3 [M + Na]+ ; HRMS (high-resolution MS)(ESI + ): calculated for C13H18NO [M + H]+ 204.1383, found204.1377.

3-Phenyl-1-(piperidin-1-yl)propan-1-one (6)

General procedure 1 was performed with 3-phenylpropanoic acidand piperidine. After a purification of the crude product by flashchromatography using a linear gradient (n-hexane/ethyl acetate,

75:25–55:45) as eluent, compound 6 was obtained as a colourlessoil (116 mg, 80%). Rf (n-hexane/ethyl acetate, 75:25): 0.22. 1H-NMR (400 MHz, [2H]chloroform): δ 7.21–7.08 (m, 5 H, Ar.H), 3.47 (t, J 5.5 Hz, 2 H, CH2N), 3.24 (t, J 5.5 Hz, 2 H,CH2N), 2.88 (m, 2 H, CH2Ph), 2.53 (m, 2 H, CH2CO), 1.51(m, 2 H, CH2), 1.45–1.33 p.p.m. (m, 4 H, CH2×2); 13C-NMR(100 MHz, [2H]chloroform): δ 171.0 (CO), 141.9 (C), 128.8,128.7, 126.4 (aromatic CH×5), 46.4 (CH2N), 42.5 (CH2N), 34.9(CH2CO), 31.3 (CH2Ph), 26.1, 25.2, 24.2 (CH2×3); MS: m/z218.5 [M + H]+ , 240.5 [M + Na]+ ; HRMS (ESI + ): calculatedfor C14H20NO [M + H]+ 218.1545, found 218.1534.

(S)-N-tert-butyloxycarbonyl-3-pyrrolidinol (14) [1]

To a solution of (S)-3-pyrrolidinol (523 mg, 6.00 mmol) inanhydrous dichloromethane (4 ml) under nitrogen at 0 ◦C wassuccessively added triethylamine (1 ml, 7.2 mmol) then a solutionof di-tert-butyl dicarbonate (1.57 g, 7.2 mmol) in anhydrousdichloromethane (4 ml). The reaction mixture was stirred for1 h, then at room temperature for an additional 30 min. Thendichloromethane (50 ml) was added, followed by water (25 ml).The aqueous layer was thereafter washed with dichloromethane(10 ml). The combined organic layers were washed with water(30 ml) and brine (30 ml), dried over magnesium sulfate, filteredand concentrated under reduced pressure. The crude productwas purified by flash chromatography [first elution with n-hexane/ethyl acetate, 65:35 during three CV (column volumes),then a linear gradient of ethyl acetate from 35 to 80% over 4CV and 2 CV at 80%] to give compound 14 as a colourlessoil (1.05 g, 94%). Rf (n-hexane/ethyl acetate, 50:50): 0.31. 1H-and 13C-NMR; + 22.2 (c 0.49, chloroform) [ + 13.06 (c 0.49,chloroform)].

(R)-N-tert-butyloxycarbonyl-3-acetylthiopyrrolidine (15)

To a solution of triphenylphosphine (2.42 g, 9.22 mmol) inanhydrous tetrahydrofuran (30 ml) under nitrogen at 0 ◦C was

1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).


S. Surade and others

added diethylazodicarboxylate (1.45 ml, 9.22 mmol) and theresulting reaction mixture was stirred for 30 min at the sametemperature. Then a solution of protected 3-pyrrolidinol 14(864 mg, 4.61 mmol) in anhydrous tetrahydrofuran (5 ml) wasadded dropwise, followed by thioacetic acid (660 μl, 9.22 mmol).The reaction mixture was stirred for an additional 1 h at 0 ◦Cthen allowed to warm at room temperature and stirred overnight.Concentration of the reaction mixture followed by crystallizationupon addition of diethyl ether and removal of triphenylphosphineoxide gave a crude product. This latter was thereafter subjected topurification by flash chromatography (elution with n-hexane/ethylacetate, 95:5 3 CV, then a linear gradient of ethyl acetate from 5 to80% over 16 CV and 2 CV at 80%), affording a mixture of thedesired thioester 15 and triphenylphosphine oxide (1.42 g) thatwas directly used for the next step without further purification.Analyses of thioester 15 were performed on a pure batch ofproduct recovered after the synthesis of disulfide 17. Rf (n-hexane/ethyl acetate, 80:20): 0.32. 1H- and 13C-NMR; MS: m/z268.4 [M + Na]+ ; + 22.1 (c 1.0, chloroform).

(R)-N-tert-butyloxycarbonyl-3-mercaptopyrrolidine (16)

To a solution of the mixture of thioester 15 and triphenylphosphineoxide in dimethylformamide (40 ml) was successively addedhydrazine hydrate (405 μl, 8.16 mmol) and, 5 min later, aceticacid (470 μl, 8.16 mmol) at room temperature. The reactionmixture was stirred for 10 min. The reaction mixture wasquenched with water (40 ml) and extracted with ethyl acetate(2×40 ml). The combined organic layers were successivelywashed with water (50 ml) and brine (50 ml), dried overmagnesium sulfate, filtered and concentrated under reducedpressure. The resulting crude product was purified by flashchromatography (elution with n-hexane/ethyl acetate, 95:5 3 CV,a linear gradient of ethyl acetate from 5 to 20% over 7 CV toremove triphenylphosphine oxide, then a linear gradient of ethylacetate from 20 to 50% over 7 CV and 3 CV at 50%) to givea mixture of the desired thiol 16 and thioester 15 (728 mg) thatwas directly used for next step. Rf (n-hexane/ethyl acetate, 80:20):0.32.

1,2-di[(R)-N-tert-butyloxycarbonylpyrrolidin-3-yl]disulfane (17)

To a solution of the mixture of thiol 16 and thioester 15 (728 mg)in ethyl acetate (10 ml) was successively added sodium iodide(5.4 mg, 0.036 mmol) and a 30% solution of hydrogen peroxide(430 μl, 3.58 mmol). The reaction mixture was stirred at roomtemperature for 1 h then quenched with a saturated aqueoussolution of sodium thiosulfate (40 ml) and extracted with ethylacetate (3×30 ml). The combined organic phases were dried overmagnesium sulfate, filtered and concentrated in vacuo, affordinga crude product which was purified by flash chromatography

(elution with n-hexane/ethyl acetate, 80:20 2 CV, a linear gradientof ethyl acetate from 20 to 40% over 9 CV, and then alinear gradient of ethyl acetate from 40 to 100% over 5 CV).The desired disulfide compound 17 was hence obtained as acolourless oil that crystallized rapidly into a white amorphoussolid (223 mg, 24% yield over three steps). The thioester 15was recovered as a colourless oil and submitted again to adeprotection–oxidation sequence as described previously. Thisallowed the collection of an additional 290 mg of the disulfide(0.72 mmol), the overall yield finally reaching 55%. Rf (n-hexane/ethyl acetate 70:30): 0.29. 1H- and 13C-NMR; HRMS(ESI + ) for C18H33N2O4S2 [M + H]+ : calculated 405.1882, found405.1875; for C18H32N2O4S2Na [M + Na]+ : calculated 427.1701,found 427.1704; − 65.0 (c 1.0, chloroform).

Synthesis of disulfide 17 from a pure batch of thioester 15:deprotection–oxidation sequence

To a solution of the recovered thioester 15 (523 mg, 2.13 mmol)in dimethylformamide (20 ml) was successively added hydrazinehydrate (190 μl, 3.77 mmol) and, 5 min later, acetic acid (215 μl,3.77 mmol) at room temperature. The reaction mixture was stirredfor 22 h at room temperature. The reaction mixture was quenchedwith water (30 ml) and extracted with ethyl acetate (2×30 ml).The combined organic layers were successively washed withwater (40 ml) and brine (40 ml), dried over magnesium sulfate,filtered and concentrated under reduced pressure. The resultingcrude product was purified by flash chromatography (same elutionconditions as described previously) to give the desired thiol 16with traces of thioester 15 (399 mg). The mixture was submittedto the next step without further purifications.

To a solution of the mixture of thiol 16 and thioester 15 (399 mg)in ethyl acetate (5 ml) was successively added sodium iodide(3 mg, 0.02 mmol) and a 30 % solution of hydrogen peroxide(235 μl, 1.96 mmol). The reaction mixture was stirred at roomtemperature for 2 h then quenched with a saturated aqueoussolution of sodium thiosulfate (25 ml) and extracted with ethylacetate (3×15 ml). The combined organic phases were driedover magnesium sulfate, filtered and concentrated under reducedpressure. The crude product was purified by flash chromatography(same elution conditions as described previously), affording thedesired disulfide compound 17 as a white amorphous solid(290 mg, 68% yield over two steps). Nevertheless, some thioester15 was still recovered (62 mg, 0.25 mmol).

1,2-di[(R)-pyrrolidin-3-yl]disulfane hydrochloride (18)

To a solution of disulfide 17 (148 mg, 0.37 mmol) in anhydrousmethanol (3 ml) under nitrogen at 0 ◦C was added dropwiseacetylchloride (530 μl, 7.40 mmol). The reaction mixture wasstirred for 10 min at 0 ◦C, then at room temperature for 20 minbefore being concentrated under reduced pressure. The desiredhydrochloride 18 was obtained quantitatively as an ochre solidand was used for the next step without further purification. Thiscompound being highly hygroscopic, it must be stored undernitrogen. 1H- and 13C-NMR; MS: m/z 205.4 [M − 2HCl + H]+ ;HRMS (ESI + ): calculated for C8H17N2S2 [M + H]+ 205.0828,found 205.0823.


Fragment-based screening and design of allosteric EthR inhibitors

(R)-1-(tert-butyloxycarbonyl)-3-piperidinecarboxylic acid (20)

For both enantiomers, see [2], but not described. For compound20, see [3], but done in chloroform instead of methanol and at‘room temperature’.

To a suspension of (3R)-3-piperidinecarboxylic acid(1.01 g, 7.61 mmol) in a mixture of dioxane/acetonitrile/water(7.5 ml/7.5 ml/2 ml) was added 1 M aqueous sodium hydroxide(7.60 ml, 7.60 mmol) then a solution of di-tert-butyldicarbonate [dissolved in a mixture of dioxane/acetonitrile (1:1)(2.5 ml/2.5 ml)]. The reaction mixture was stirred overnight atroom temperature, then concentrated under reduced pressure and10% aqueous citric acid (50 ml) was added. The mixture wasextracted with ethyl acetate (3×60 ml) and the combined organiclayers were dried over MgSO4, filtered and concentrated underreduced pressure. The resulting crude product was recrystallizedin ethyl acetate/n-hexane to provide compound 20 as whitecrystals (1.68 g, 97%). 1H- and 13C-NMR; HRMS (ESI + ) forC11H20NO4 [M + H]+ : calculated 230.1392; found 230.1398;− 27.3 (c 1.0, methanol).

(S)-1-(tert-butyloxycarbonyl)-3-piperidinecarboxylic acid (24)

Compound 24 was obtained from (3S)-3-piperidinecarboxylicacid (1.03 g, 7.7 mmol) according to the same procedure as for 20and was recrystallized in ethyl acetate/n-hexane as white crystals(1.34 g, 75%). 1H- and 13C-NMR; HRMS (ESI + ): calculated forC11H20NO4 [M + H]+ 230.1392; found 230.1386; + 30.9 (c 1.0,methanol).

(3R)-N-[(3S)-1-tert-butyloxycarbonylpiperidin-3-yl]-1-tert-butyloxycarbonyl-piperidine-3-carboxamide (21)

To a solution of Boc acid 20 (917 mg, 4 mmol) in anhydrousdichloromethane (15 ml) under nitrogen was added triethylamine(620 μl, 4.4 mmol) then PyBOP (2.29 g, 4.4 mmol) at roomtemperature. After 10 min, a solution of the commercial (S)-( + )-3-amino-1-Boc-piperidine (908 mg, 4.4 mmol) in anhydrousdichloromethane (10 ml). The reaction mixture was stirredovernight at room temperature then quenched with an aqueoussaturated solution of sodium bicarbonate (35 ml). The mixturewas extracted with dichloromethane (3×25 ml) and the combinedorganic layers were successively washed with water (50 ml)and brine (50 ml), dried over magnesium sulfate, filtered andconcentrated under reduced pressure. Purification of the crudeproduct by flash chromatography (n-hexane/ethyl acetate, 50:50)gave the desired amide 21 as a white foam (1.06 g, 64%). Rf

(n-hexane/ethyl acetate, 50:50): 0.30. 1H- and 13C-NMR; HRMS(ESI + ) for C21H38N3O5 [M + H]+ : calculated 412.2811, found412.2805; for C21H37N3O5Na [M + Na]+ : calculated 434.2631,found 434.2634; − 12.1 (c 1.0, chloroform).

(3S)-N-[(3R)-1-tert-butyloxycarbonylpiperidin-3-yl]-1-tert-butyloxycarbonyl-piperidine-3-carboxamide (25)

Compound 25 was obtained from the commercial (R)-( − )-3-amino-1-Boc-piperidine (908 mg, 4.4 mmol) according to thesame operating procedure as 21 as a white foam (1.05 g, 64%). Rf

(n-hexane/ethyl acetate, 50:50): 0.30. 1H- and 13C-NMR; HRMS(ESI + ) for C21H38N3O5 [M + H]+ : calculated 412.2811, found412.2800; for C21H37N3O5Na [M + Na]+ : calculated 434.2631,found 434.2619; + 17.8 (c 1.0, chloroform).

(3R)-N-[(3S)-piperidin-3-yl]piperidine-3-carboxamidehydrochloride (22)

To a solution of amide 21 (412 mg, 1 mmol) in anhydrousmethanol (6 ml) under nitrogen at 0 ◦C was added dropwiseacetylchloride (1.42 ml, 20 mmol). The reaction mixture wasstirred for 10 min at 0 ◦C, then at room temperature for 20 minbefore being concentrated under reduced pressure, affording thedesired compound 22 as a white foam in quantitative yield thatwas used for the next step without further purification. Thiscompound being highly hygroscopic, it must be stored undernitrogen. 1H- and 13C-NMR; MS: m/z 212.5 [M + H]+ ; HRMS(ESI + ): calculated for C11H22N3O [M + H]+ 212.1757, found212.1752.

(3S)-N-[(3R)-piperidin-3-yl]piperidine-3-carboxamidehydrochloride (26)

Compound 26 was obtained from 25 according to the sameoperating procedure as 22 as a white foam in quantitativeyield. Compound 26 was used for the next step without furtherpurification. This compound being highly hygroscopic, it mustbe stored under nitrogen. 1H- and 13C-NMR; MS: m/z 212.5[M + H]+ ; HRMS (ESI + ): calculated for C11H22N3O [M + H]+

212.1757, found 212.1752.

General procedure 2 of peptidic coupling between linkers andpropanoic acid derivatives

To a solution of carboxylic acid (0.65 mmol) in anhydrousdichloromethane (4 ml) was added successively at room temper-ature dimethylformamide (two drops) and oxalyl chloride (83 μl,0.98 mmol). After stirring for 1 h, the reaction mixture was con-centrated under reduced pressure to provide crude acid chloride.

To a suspension of the hydrochloride salt (0.3 mmol) indichloromethane (1.5 ml) was successively added triethylamine(175 μl, 1.25 mmol) and a solution of the crude acid chloride inanhydrous dichloromethane (3.5 ml). After stirring for 24 h atroom temperature, the reaction mixture was quenched with water(10 ml) and was extracted with dichloromethane (3×10 ml). Thecombined organic layers were washed with a saturated aqueoussolution of sodium bicarbonate (2×15 ml), dried over magnesium



sulfate, filtered and concentrated under reduced pressure. Apurification of the crude product by flash chromatography gavethe desired compound.

1,1′-(3R,3′R)-3,3′-disulfanediylbis(pyrrolidine-3,1-diyl)bis[3-(4-methoxyphenyl)propan-1-one] (7)

Compound 7 was obtained according to general procedure 2from 4-methoxyphenylpropionic acid (0.81 mmol) and disulfidehydrochloride 18 (0.37 mmol), after a purification by flashchromatography (elution with n-hexane/ethyl acetate, 20:80 2CV, a linear gradient of ethyl acetate from 80 to 100% over5 CV, ethyl acetate 100% 3 CV, then a linear gradient of ethylacetate/methanol from 100:0 to 80:20 over 7 CV and methanol20% 6 CV), as a colourless crystallized solid (113 mg, 58%).Compound 7 can be recrystallized in acetone/n-hexane as whitecrystals. Rf (ethyl acetate): 0.16. 1H- and 13C-NMR; MS: m/z 529.7[M + H]+ ; HRMS (ESI + ) for C28H36N2O4S2Na [M + Na]+ : cal-culated 551.2009, found 551.2008; HPLC: TR = 26.20 min (purity100%); method 5–95 over 25 min; − 21.2 (c 1.0, chloroform).

1,1′-(3R,3′R)-3,3′-disulfanediylbis(pyrrolidine-3,1-diyl)bis[3-(pyridin-3-yl)propan-1-one] (8)

Compound 8 was obtained according to general procedure2 from 3-(pyridin-3-yl)propanoic acid (0.65 mmol) anddisulfide hydrochloride 18 (0.30 mmol), after a purificationby flash chromatography (elution with ethyl acetate + 0.1%triethylamine 100% 3 CV, then a linear gradient of ethylacetate/methanol + 0.1% triethylamine from 100:0 to 80:20 over5 CV and ethyl acetate/methanol + 0.1% triethylamine 80:2030 CV), as a pale yellow lacquer (88 mg, 62%) that can berecrystallized in acetone/n-hexane as white crystals. Rf (ethylacetate/methanol, 8:2): 0.11. 1H- and 13C-NMR; MS: m/z 471.6[M + H]+ ; HRMS (ESI + ) for C24H30N4O2S2Na [M + Na]+ : cal-culated 493.1702, found 493.1697; HPLC: TR = 18.30 min (purity100%); method 5–95 over 25 min; − 15.7 (c 1.0, chloroform).

1,1′-(3R,3′R)-3,3′-disulfanediylbis(pyrrolidine-3,1-diyl)bis(3-phenylpropan-1-one) (9)

Compound 9 was obtained according to general procedure 2 from3-phenylpropanoic acid (0.65 mmol) and disulfide hydrochloride

18 (0.30 mmol), after a purification by flash chromatography(elution with n-hexane/ethyl acetate, 20:80 2 CV, a lineargradient of ethyl acetate from 80 to 100% over 5 CV,ethyl acetate 100% 3 CV, then a linear gradient of ethylacetate/methanol from 100:0 to 80:20 over 7 CV and methanol20% 6 CV), as a pale yellow lacquer (50 mg, 36%) thatcan be recrystallized in acetone/n-hexane as white crystals.Rf (ethyl acetate): 0.14. 1H- and 13C-NMR; MS: m/z 469.6[M + H]+ ; HRMS (ESI + ) for C26H32N2O2S2Na [M + Na]+ :calculated 491.1797, found 491.1792; HPLC: TR = 26.91 min(purity 100%); method 5–95 over 25 min; − 21.9 (c 1.0,chloroform).

(3R)-1-(3-phenylpropanoyl)-N-[(3S)-1-(3-phenylpropanoyl)piperidin-3-yl]piperidine-3-carboxamide (10)

Compound 10 was obtained according to general procedure 2from 3-phenylpropanoic acid and diamide hydrochloride 22.After purification of the crude product by flash chromatography(elution with n-hexane/ethyl acetate 15:85 2 CV, a linear gradientof ethyl acetate from 85 to 100% over 7 CV, ethyl acetate100% 3 CV, then a linear gradient of ethyl acetate/methanolfrom 100:0 to 80:20 over 7 CV and methanol 20 % 2 CV),compound 10 was obtained as a white amorphous solid (97 mg,68%) that can be recrystallized in acetone/n-hexane as whitecrystals. Rf (ethyl acetate): 0.19. 1H- and 13C-NMR; MS: m/z 474.9[M − H]− , 476.7 [M + H]+ ; HRMS (ESI + ) for C29H37N3O3Na[M + Na]+ : calculated 498.2733, found 498.2734; HPLC: TR =24.45 min (purity 73%); method 5–95 over 25 min; − 25.4 (c1.0, chloroform).

(3S)-1-(3-phenylpropanoyl)-N-[(3R)-1-(3-phenylpropanoyl)piperidin-3-yl]piperidine-3-carboxamide (11)

Compound 11 was obtained according to general procedure2 from phenylpropionic acid and diamide hydrochloride 26.After purification of the crude product by flash chromatography(elution with n-hexane/ethyl acetate 15:85 2 CV, a linear gradientof ethyl acetate from 85 to 100% over 7 CV, ethyl acetate100% 3 CV, then a linear gradient of ethyl acetate/methanolfrom 100:0 to 80:20 over 7 CV and methanol 20 % 2 CV),compound 11 was obtained as a white amorphous solid (98 mg,69%) that can be recrystallized in acetone/n-hexane as whitecrystals. Rf (ethyl acetate): 0.19. 1H- and 13C-NMR; MS: m/z476.7 [M + H]+ ; HRMS (ESI) for C29H37N3O3Na [M + Na]+ :calculated 498.2733, found 498.2751; HPLC: TR = 24.43 min(purity 91 %); method 5–95 over 25 min; + 22.3 (c 1.0,chloroform).



Figure S1 The interaction of hexadecyl octanoate with EthR is primarilyhydrophobic in nature

Ligand is displayed in stick representation, whereas the amino acid side chains are displayedas lines.

Figure S2 Comparison of hits with respect to their �T m values from the thermal-shift assay and the percentage inhibition from the SPR assay

All negative controls, except one, exhibited inhibition of less than 10 % in the SPR assay, validating the use of thermal shift as a primary screening technique.



Figure S3 Compounds 5 and 6 were able to form key interactions and maintain the binding pose of starting fragment 1

The structures of EthR in complex with three ligands are superimposed. Fragment hit 1 is coloured using the CPK scheme, compounds 5 is coloured entirely in green and compound 6 is colouredentirely in blue. Ligands are displayed in stick representation, whereas the amino acid side chains are displayed as lines. For clarity, key hydrogen-bonding interactions between the ligand, Asn179

and a water molecule are shown only for fragment hit 1.

Figure S4 Representative example of fitting and IC50 calculation forinhibition of the EthR–DNA interaction by fragment hit 1

Table S1 Summary of results from fragment screening

The total fragments screened was 1250.

(a) Thermal-shift screening (total hits 86)

�T m (◦C) Hits

1–3 643–5 17>5 5

(b) SPR functional assay (hits tested 86)

Percentage inhibition by SPR assay (%) Hits

0–10 4110–30 2630–50 11>50 8

Figure S5 Relative changes in the distance between the two HTH motifs inthe EthR dimer (generated using crystallographic symmetry)

The distances between two corresponding Cα of Thr60 residues are given for each of theccomplexes. Compound 9 is shown as sticks, whereas EthR is shown in cartoon representation.The inset shows ligands, their affinities and the distances between the two corresponding Cα

of Thr60 residues from a crystallographic dimer.


Fragment-based

screeningand

designofallostericEthR

inhibitors

Table S2 Crystallographic data and refinement statistics

Values in parentheses are for the highest-resolution shell. ESRF, European Synchrotron Radiation Facility; NA, not applicable; Nref, total number of measured reflections; SLS, Swiss Light Source.

EthR complex Backwash 1 2 3 4 5 6 7 8 9

Source ESRF Diamond ESRF ESRF Diamond SLS DiamondIn-houseProteum2 Diamond Diamond

Data collection Space group P41212 P41212 P41212 P41212 P41212 P41212 P41212 P41212 P41212 P41212

Cell parameters anddata processing

a, b, c (A) 121.47, 121.47,33.71

121.73, 121.73,33.70

120.80, 120.80,33.64

122.25, 122.25,33.70

121.06, 121.06,33.72

122.23, 122.23,33.69

122.46, 122.46,33.63

122.32, 122.32,33.73

120.71, 120.71,33.86

122.41, 122.41,33.71

a, β , γ (◦) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90Resolution 40.49–1.80

(1.90–1.80)43.04–1.75

(1.84–1.75)33.64–1.86

(1.96–1.86)43.22–1.75

(1.85–1.75)29.46–1.59

(1.63–1.59)29.65–1.57

(1.66–1.57)43.30–1.90

(2.0–1.90)33.93–2.20

(2.26–2.20)33.48–1.68

(1.78–1.68)33.95–2.0

(2.11–2.0)Total Nref 307839 (43038) 233602 (33774) 160243 (20809) 205792 (27635) 216494 (7699) 485113 (63865) 274235 (39174) 199916 231662 (28547) 138182 (19830)Unique Nref 24067 (3437) 26272 (3761) 21407 (3043) 26314 (3760) 33952 (2113) 36139 (5171) 20846 (2974) 13341 (804) 28509 (4115) 17357 (2463)Rmerge 0.079 (0.431) 0.077 (0.387) 0.094 (0.554) 0.080 (0.547) 0.050 (0.547) 0.057 (0.411) 0.109 (0.396) 0.03 (0.27) 0.088 (0.276) 0.082 (0.418)I/σ I 20.3 (5.9) 19.7 (4.8) 14.7 (3.5) 14.7 (3.3) 19.7 (2.0) 27.1 (6.7) 19.2 (8.0) 16.35 (1.8) 12.5 (4.9) 14.6 (5.5)Completeness 99.9 (100) 100.0 (100.0) 99.8 (99.6) 100.0 (100.0) 98.1 (86.0) 99.9 (100.0) 99.9 (100.0) 97.9 (91.9) 98.6 (98.9) 97 (95.9)Multiplicity 12.8 (12.5) 8.9 (9.0) 7.5 (6.8) 7.8 (7.3) 6.1 (3.6) 13.4 (12.4) 13.2 (13.2) 14.67 (4.79) 8.1 (6.9) 8.0 (8.1)

Refinement Resolution 29.46–1.80 33.78–1.63 85.42–1.86 86.44–1.75 38.28–1.57 29.65–1.57 43.30–1.70 86.49–2.20 33.48–1.68 31.41–1.68Number of reflections 22783 30596 20236 24894 33339 34281 27300 12550 27002 27188Rwork/R free 0.20/0.23 0.20/0.22 0.20/0.25 0.20/0.23 0.21/0.25 0.19/0.23 0.19/0.23 0.20/0.27 0.21/0.25 0.21/0.24Rmsd

Bond lengths (A)/bond angles (◦)

0.025/2.396 0.030/2.27 0.027/2.05 0.024/1.91 0.03/2.09 0.03/2.66 0.027/2.148 0.020/1.781 0.022/2.239 0.026/2.246

Ligand B-factor NA 20.0 and 24.9 22.14 22.65 and 45.42 18.7 and 27.01 16.19 and 23.85 20.06 and 30.29 27.19 22.72 29.76

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icalSociety


Scheme S1 Synthesis of fragment analogues 5 and 6

Scheme S2 Synthesis of the disulfide hydrochloride linker 18

Scheme S3 Synthesis of the diamide hydrochloride linkers 22 and 26



Scheme S4 Synthesis of the disulfide compounds 7, 8 and 9

Scheme S5 Synthesis of the amide compounds 10 and 11

REFERENCES

1 Bhat, K. L., Flanagan, D. M. and Joullie, M. M. (1985) Synthetic routes to chiral3-pyrrolidinols. Synth. Commun. 15, 587–598

2 Hayashi, S., Hirao, A., Imai, A., Nakamura, H., Murata, Y., Ohashi, K. and Nakata, E. (2009)Novel non-peptide nociceptin/orphanin FQ receptor agonist,1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole:design, synthesis, and structure-activity relationship of oral receptor occupancy in thebrain for orally potent antianxiety drug. J. Med. Chem. 52, 610–625

3 Gessier, F., Schaeffer, L., Kimmerlin, T., Flogel, O. and Seebach, D. (2005) Preparation ofβ2-amino acid derivatives (β2hThr, β2hTrp, β2hMet, β2hPro, β2hLys,pyrrolidine-3-carboxylic acid by using DIOZ as chiral auxiliary. Helv. Chim. Acta 88,2235–2250

Received 22 August 2013/2 December 2013; accepted 6 December 2013Published as BJ Immediate Publication 6 December 2013, doi:10.1042/BJ20131127


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