Discovery of cofactor-specific, bactericidalMycobacterium tuberculosis InhA inhibitors usingDNA-encoded library technologyHolly H. Souttera,1, Paolo Centrellab, Matthew A. Clarkc, John W. Cuozzoa, Christoph E. Dumelind, Marie-Aude Guiee,Sevan Habeshianb, Anthony D. Keefef, Kaitlyn M. Kennedyb, Eric A. Sigele, Dawn M. Troastg, Ying Zhangb,Andrew D. Fergusonh, Gareth Daviesi, Eleanor R. Steadi, Jason Breedj, Prashanti Madhavapeddik, and Jon A. Readj,1

aTherapeutic Discovery Sciences, X-Chem Pharmaceuticals, Waltham, MA 02453; bDiscovery Chemistry, X-Chem Pharmaceuticals, Waltham, MA 02453;cX-Chem Pharmaceuticals, Waltham, MA 02453; dNovartis Institutes for Biomedical Research, Novartis Pharma AG, 4056 Basel, Switzerland; eScientificComputing, X-Chem Pharmaceuticals, Waltham, MA 02453; fLead Discovery, X-Chem Pharmaceuticals, Waltham, MA 02453; gMorphic Therapeutic,Waltham, MA 02451; hDiscovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Waltham, MA 02451; iDiscoverySciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Macclesfield SK10 4TG, United Kingdom; jDiscovery Sciences, InnovativeMedicines and Early Development Biotech Unit, AstraZeneca, Cambridge CB4 0WG, United Kingdom; and kAstraZeneca India Private Ltd., Hebbal,Bangalore 560 024, India

Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved October 12, 2016 (received for review July 6, 2016)

Millions of individuals are infected with and die from tuberculosis(TB) each year, and multidrug-resistant (MDR) strains of TB areincreasingly prevalent. As such, there is an urgent need to identifynovel drugs to treat TB infections. Current frontline therapiesinclude the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To in-hibit InhA, isoniazid must be activated by the catalase-peroxidaseKatG. Isoniazid resistance is linked primarily to mutations in thekatG gene. Discovery of InhA inhibitors that do not require KatGactivation is crucial to combat MDR TB. Multiple discovery effortshave been made against InhA in recent years. Until recently, de-spite achieving high potency against the enzyme, these effortshave been thwarted by lack of cellular activity. We describe herethe use of DNA-encoded X-Chem (DEX) screening, combined withselection of appropriate physical properties, to identify multipleclasses of InhA inhibitors with cell-based activity. The utilization ofDEX screening allowed the interrogation of very large compoundlibraries (1011 unique small molecules) against multiple forms ofthe InhA enzyme in amultiplexed format. Comparison of the enrichedlibrary members across various screening conditions allowed theidentification of cofactor-specific inhibitors of InhA that do not re-quire activation by KatG, many of which had bactericidal activityin cell-based assays.

Mycobacterium tuberculosis | DNA-encoded X-Chem technology |DNA-encoded libraries | multidrug resistance | InhA

Tuberculosis (TB) infects millions of people per year and con-tributes to the deaths of over 1.5 million annually. It is the

second leading cause of death from infectious disease worldwide. In2012, 8.6 million people fell ill with TB, and 1.3 million died fromTB. More than 95% of TB deaths occur in developing countries,and it is among the top three causes of death for women aged 15–44y. TB is a leading killer of people living with HIV, causing onequarter of all deaths in this population. The causative agent of TB,Mycobacterium tuberculosis (Mtb), has been increasingly observedto possess resistance to the frontline therapies rifampicin, and iso-niazid commonly used to treat TB. For this reason, new therapeuticmodalities to fight Mtb infection are desperately needed.The enoyl-acyl-carrier protein (ACP) reductase, InhA, thought

to be the primary target of the anti-Mtb drug isoniazid, catalyzesthe NADH-dependent reduction of the 2-trans double bond of thelipid-modified ACP via an enoyl intermediate forming part ofthe fatty acid biosynthetic pathway essential for the formation of theouter membrane of Mtb (1, 2). Isoniazid is used as part of a com-bination therapy for the treatment of Mtb but is a prodrug thatrequires activation by KatG. Upon activation by KatG, isoniazidforms a covalent adduct with the cofactor NADH (Fig. 1). The

isoniazid–NADH adduct acts an inhibitor of InhA by competingwith NADH (Table 1) (3, 4). Many multidrug-resistant (MDR) TBstrains exhibit resistance to isoniazid associated with mutations in atleast five genes linked to isoniazid prodrug conversion, and themajority of those mutations are linked to defects in the katG geneand its upstream promoter (5–7). Direct inhibitors of InhA wouldprovide TB drugs for the isoniazid-resistance strains without cross-resistance to isoniazid; however, until recently, discovery of InhAinhibitors with cellular activity has been challenging. The lack ofbioactive compounds with cellular activity has thwarted efforts todevelop InhA lead compounds with appropriate in vivo properties.The successful use of DNA-encoded library technologies to

discover novel chemical matter against a variety of target classeshas been reported, including the discovery of novel inhibitors of

Significance

The increasing prevalence of multidrug-resistant strains of tuber-culosis has created an urgent need for novel therapies to treattuberculosis infections. Herewe have demonstrated the successfulutilization of the DNA-encoded X-Chem technology for the dis-covery inhibitors ofMycobacterium tuberculosis enoyl–acyl-carrierprotein (ACP) reductase, InhA, a validated target for the treatmentof tuberculosis. The identified inhibitors are cofactor specific andhave activity in multiple cellular assays. Crystal structures of rep-resentative compounds from five chemical series revealed that thecompounds bind adjacent to the NADH cofactor and adopt avariety of conformations, including two previously unreportedbinding modes. The compounds identified may serve as usefulleads in the development of new antibacterial drugs with efficacyagainst multidrug-resistant tuberculosis.

Author contributions: H.H.S., P.C., M.A.C., J.W.C., C.E.D., A.D.K., K.M.K., E.A.S., D.M.T., Y.Z., A.D.F.,G.D., E.R.S., J.B., P.M., and J.A.R. designed research; H.H.S., P.C., C.E.D., S.H., K.M.K., D.M.T.,G.D., E.R.S., J.B., P.M., and J.A.R. performed research; P.C., M.-A.G., S.H., and E.A.S. con-tributed new reagents/analytic tools; H.H.S., C.E.D., and D.M.T. analyzed data; and H.H.S.,M.A.C., J.W.C., A.D.K., Y.Z., and J.A.R. wrote the paper.

Conflict of interest statement: H.H.S., P.C., M.A.C., J.W.C., M.-A.G., S.H., A.D.K., K.M.K.,E.A.S., and Y.Z. are employees of X-Chem Pharmaceuticals. DNA-encoded X-Chem tech-nology (DEX) is a proprietary drug discovery platform discovered and developed by em-ployees of X-Chem. A.D.F., G.D., E.R.S., J.B., P.M., and J.A.R. are employees of AstraZeneca.

This article is a PNAS Direct Submission.

Data deposition: Crystallography, atomic coordinates, and structure factors reported inthis paper have been deposited in the Protein Data Bank database (ID codes 5G0S, 5G0T,5G0U, 5G0V, and 5G0W).1To whom correspondence may be addressed. Email: hsoutter@x-chemrx.com or Jon.Read@astrazeneca.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1610978113/-/DCSupplemental.

E7880–E7889 | PNAS | Published online November 18, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1610978113

Dow

nloa

ded

by g

uest

on

July

11,

202

0

InhA (8–14). DNA-encoded library technologies have providedthe ability to interrogate large compound libraries (109–1011

unique small molecules) against a protein target in solution veryrapidly and requires only microgram quantities of protein. Targetscan be interrogated under multiple experimental conditions in par-allel. These conditions could include different target concentrations,target bound to different cofactors/inhibitors, and off-targets,among others. Multiple conditions are included with the goal ofidentifying small molecules with desired mechanisms of action.Recently, we reported on inhibitors with activity in cellularassays that bind tightly to the InhA:NADH complex (Fig. 1 andTable 1) (15). Here we describe the use of the DNA-encodedX-Chem technology (DEX) for the discovery of both NADH-and NAD+-specific InhA inhibitors with activity in multiplecellular assays.

ResultsInhA Selections. A pool of 11 DNA-encoded libraries comprisingmore than 66 billion on-DNA compounds was used for selectionsagainst various forms of InhA including apo InhA, InhA:NAD+,and InhA:NADH. The chemical libraries were constructed usingstandard split-and-pool methodology with concomitant DNAencoding for synthetic steps (8, 16–18). InhA containing anN-terminal 6xHis tag was mixed with the library pool in thepresence or absence of NADH or NAD+. Complexes of the targetand bound small molecules then were captured on His-select nickelaffinity resin via the protein affinity tag. The complexes then werewashed to remove nonbinders, and the bound molecules were

eluted by heat denaturation of the protein. This selection processwas repeated using the eluted library members and fresh protein torefine the population of small molecule binders further. The finaleluted fraction then was amplified and sequenced to determine theidentities of the small molecule binders.Eight individual selection conditions were run in parallel to

identify library compounds with the desired mechanisms of ac-tion, in this case, binding selectively to the apo, NADH, or NAD+

complexes of InhA. The isoniazid-resistant mutant of InhA, S94A,was also included to aid in the identification of small molecules withactivity against isoniazid-resistant TB. The selection conditions wereas follows: no protein; 10 μM apo InhA; or 10 μM InhA plussaturating amounts of NADH, NAD+, or NADH in combinationwith a known NADH-dependent, tight binding InhA inhibitor(IC50 = 0.4 μM). A second tight-binding inhibitor with a knownbinding mode (IC50 = 0.8 μM) was conjugated to DNA and in-cluded in the library pool at a low concentration. Inclusion of theon-DNA positive control allowed validation of the selection con-ditions by assessment of enrichment by sequencing.Analysis of the selection output resulted in four general pro-

files of enriched library members: (i) enriched only in the pres-ence of apo InhA, (ii) enriched only in the presence of the InhA:NAD+ complex, (iii) enriched only in the presence of the InhA:NADH complex but not in the presence of the included tight-binding small molecule, and (iv) enriched in the presence ofInhA:NAD+ and InhA:NADH but not in the presence of the in-cluded tight-binding small molecule. The on-DNA control com-pound was significantly enriched in the selections against InhA:NADH, including the S94A mutant of InhA, but was not enrichedin the selections containing apo protein or InhA:NAD+. In theselection containing the WT InhA:NADH complex, the on-DNAcontrol compound was enriched by a factor of 1,100-fold over twocycles of selection when normalized to a corresponding unfunc-tionalized oligonucleotide. As expected, the on-DNA controlcompound was competed away by the NADH-dependent smallmolecule which was included in selections. The majority (70%) ofthe enriched families (groups of structurally related compoundswith the same profile) and, correspondingly, off-DNA compoundswe chose to synthesize, fell into the third profile. In each of theprofile classes, the majority of the families showed equivalent en-richment against both the WT and S94A mutant of InhA. Com-pounds that showed a significant reduction in enrichment againstthe S94A mutant relative to the WT protein were not synthesized.Representative compounds from highly enriched families wereresynthesized without the DNA tag and tested for their activity inin vitro and cell-based assays. Approximately 50 representativesmall molecules across each of the four profiles were chosen for

Fig. 1. InhA inhibitors showing cellular activity in Mtb previously describedin the literature. (1) Isoniazid adduct (23). (2) PT70 (24, 25). (3) Pyridomycin(26). (4) Methyl thiazole (15). (5) Pyrazole ELT hit (13). (6) Pyridine dione (27).

Table 1. Biochemical and cellular activity of InhA inhibitors described in the literature (Fig. 1) that showcellular activity in Mycobacterium tuberculosis

Compound Reported affinity, nMCellular activity: MIC50,

μM (H37Rv) cLogP LLE Predominant InhA binding

1 0.79* 0.158 −0.7† 9.8 Apo2 0.02‡ 10.000 7.0 3.7 NAD+

3 6309.57§ 0.398 3.1 2.1 Apo4 3.16§ 0.199 2.3 6.2 NADH5 3.98¶ 0.501# 1.4 7.0 ND6 630.96¶ 0.079 5.3 0.9 NADHk

cLogP, the calculated logarithms of water-octanol partition coefficients; LLE, log10 of reported affinity − cLogP; ND, not determined.*Ki measurement.†cLogP calculated for prodrug isoniazid rather than active drug INH-NAD adduct.‡K1 measurement.§Kd measurement.¶IC50.#MIC90.kA close analog, NITD-529, showed binding only to NADH form of InhA.

Soutter et al. PNAS | Published online November 18, 2016 | E7881

APP

LIED

BIOLO

GICAL

SCIENCE

SPN

ASPL

US

Dow

nloa

ded

by g

uest

on

July

11,

202

0

off-DNA synthesis based on potential mechanism of action, en-richment level, and their physicochemical properties.

Assay Results. Off-DNA compounds were tested in in vitro assayswhich monitored the WT InhA-dependent conversion of NADHto NAD+ by reading the fluorescence at 420 nm. Two forms ofthe assay were used, one in which the protein was incubated inthe presence of NADH (NADH-dependent) and another inwhich the protein was incubated with a mixture of NADH andNAD+ in which NAD+ was in excess (NAD+-dependent). Of theroughly 50 compounds synthesized, 25 had detectable activitywith four having an IC50 in the 10- to 30-μM range, seven havingan IC50 in the 1- to 10-μM range, and 14 having an IC50 less than1 μM in one of the two assays. These compounds representedfamilies observed in multiple DNA-encoded libraries.Compounds with IC50 values less than 20 μM in either of the in

vitro assays were sent for testing in the TB panel provided by In-fectious Disease Research Institute (IDRI), which is part of theMicrobiology and Infectious Disease Resources supported by theNational Institute of Allergy and Infectious Disease (NIAID).This panel of assays includes determinations of minimum inhibitoryconcentration (MIC); IC50; the concentration that results in 90%inhibition of growth (IC90) against Mtb H37Rv under both aerobicand anaerobic conditions; Mtb H37Rv minimum bacterial con-centration (MBC); IC50 and IC90 in an intracellular activity assaymeasuring the ability of compounds to kill Mtb-infected humancells; and MIC against a set of five drug-resistant strains of Mtb.Multiple chemical series were identified with an MIC less than50 μM in the aerobic Mtb H37Rv assay. Although multiple repre-sentatives of the NADH- and NAD+-dependent profiles had cel-lular activity, no compound that was uniquely enriched against apoInhA and was active in one of the in vitro assays showed activity inthe MIC panel.Compounds with an MIC value less than 100 μM in the aerobic

Mtb H37Rv assay were submitted for affinity determination insurface plasmon resonance (SPR) experiments under three con-ditions: binding against apo InhA, binding to InhA in the presenceof NAD+, and binding to InhA in the presence of NADH. No-tably, the NADH and NAD+ cofactor-dependent profiles ob-served in selection corresponded to the behavior observed in invitro and biophysical assays. Compounds that were enriched onlyin the presence of NAD+ were significantly more potent in theNAD+-dependent assay (Table 2). Likewise, compounds with apreference for NADH- or NAD+-bound forms of InhA in selec-tion showed increased affinities for their respective complexes inSPR experiments (Table 2). Structure–activity relationships andcrystal structures of the most potent of the series with cell-basedactivity are described subsequently.

Series 1–3. Series 1–3 were identified from a capped dipeptidelibrary containing 225 million on-DNA compounds. These seriesare defined by a conserved cyclohexyl core, a variety of benzyl orheterocyclic substituents at the position proximal to the methylamide, which serves as the chemical handle for DNA attachment,and most often a benzyl triazole or methylbenzothiophene at theposition distal to the methyl amide (Table 3). Crystal structures ofthe two parent compounds, compound 1a (IC50 = 0.065 μM) andcompound 2a (IC50 = 0.057 μM), in complex with InhA:NADHwere obtained (Fig. 2). These structures revealed that the amidelinking the substituent at the hydrophobic pocket, known as “siteII,” to the cyclohexyl core formed the warhead of the moleculebinding into the catalytic site, site I, adjacent to the nicotinamidering of NADH. In the substrate-bound state, site II is occupied bythe lipophilic chain of ACP. The protein active site adopts a Tyr158-in conformation in which the carboxylic oxygen of the warheadforms a pair of bridging hydrogen bonds between the cofactor andside-chain oxygen atom of Tyr158. The cyclohexyl core formspacking interactions with the main-chain carbon atoms near Phe97(site III). In both cocrystal structures, the methyl amide and theproximal benzyl substituents were disordered, and the solvent wasexposed. Those substituents were modeled as occupying site II. Theactive site loop of InhA was also disordered in these structures.Multiple analogs were synthesized in an attempt to improve cell-based activity. The parent compounds along with their analogs wereevaluated in the NADH-dependent in vitro assay, and compoundswith an IC50 <1 μM were submitted for testing in the TB panel(Table 3). Compound 1a had an MIC of 13 μM under aerobicconditions and an IC50 of 4 μM in the intracellular activity assay.The intracellular activity did not appear to be caused by nonspecificcytotoxicity, as demonstrated by an MIC >50 μM in the cytotoxicityassay (Table S1). Compound 1c, a closely related analog of com-pound 3, demonstrated cellular activity comparable to that observedfor the parent compound, and both compounds had MIC’s in the12- to 31-μM range against a panel of isoniazid and rifampicin re-sistant strains (Table S2).

Series 4 and 5. Series 4 and 5 were identified from a pyridine corelibrary with two points of diversity containing 1 million on-DNAcompounds. The off-DNA parent compound, compound 4a,contains fluorophenoxybenzyl and piperidinyl pyridine substitu-ents. The crystal structure of InhA:NADH in complex withcompound 4a (IC50= 0.310 μM;MIC= 25 μM)was obtained (Fig. 3)and revealed that the piperidinyl pyridine substituent formed thewarhead of the molecule binding into site I and that the hydro-phobic pocket at site II was occupied by the fluorophenoxybenzylsubstituent, which forms hydrophobic packing interactions withresidues in the active site loop including Ile202. The pyridinecore formed the linker portion of the molecule, packing against the

Table 2. Comparison of selection profiles, in vitro assay potencies, and SPR-binding constants for InhA inhibitors

Compound Series Profile classInhA NAD+

assay IC50, μMInhA NADH

assay IC50, μM InhA NAD+ SPR Kd, μM InhA NADH SPR Kd, μM InhA Apo SPR Kd, μM

11 11 1 0.389 ± 0.007 0.168 (n = 1) NT NT NT10a 10 2 0.038 ± 0.006 0.198 ± 0.009 0.26 ± 0.12 5 ± 1.20 12.4 ± 1.4112 12 2 0.682 ± 0.207 6.838 ± 0.175 NT NT NT2a* 1–3 3 0.060 ± 0.004 0.057 ± 0.006 46.7 ± 11.6 0.094 ± 0.06 >10013 13 3 0.791 ± 0.008 0.609 ± 0.114 NT NT NT8a* 8 and 9 3 NT 0.130 ± 0.006 49 ± 2.90 0.055 ± 0.03 >1006a* 6 and 7 3 NT 5.917 ± 1.22 >100 36.8 ± 3.25 >1004a* 4 and 5 3 NT 0.297 ± 0.053 >100 0.25 ± 0.11 >1001a* 1–3 3 NT 0.065 ± 0.008 13.4 ± 4.30 0.34 ± 0.22 >10014 14 3 NT 5.568 ± 0.777 >100 6.3 >100

Profile classes: 1, enriched only in the presence of apo InhA; 2, enriched only in the presence of the InhA:NAD+ complex; 3, enriched only in the presence ofthe InhA:NADH complex but not in presence of small molecule. NT, not tested. Chemical structures for compounds 11–14 are given in Fig. S1.*WT InhA cocrystal structures reported herein.

E7882 | www.pnas.org/cgi/doi/10.1073/pnas.1610978113 Soutter et al.

Dow

nloa

ded

by g

uest

on

July

11,

202

0

side chain of Phe97 in site III. The active site loop is ordered, andTyr158 adopts the “out” conformation, rotated away from thenicotinamide ring of NADH and toward the side chains of Ile215and Leu218. Most members of the series have bicyclic or biarylsubstituents at both positions. Although most modifications of theparent compound led to a loss in potency, the replacement of theether linkage of the fluorophenoxybenzyl group with an amine incompound 4b did lead to a 10-fold improvement in potency in thein vitro assay (IC50 = 0.026 μM), possibly because of the formationof an additional hydrogen-bonding interaction with the main-chain carbonyl oxygen atom of Ala198, and a threefold improve-ment in bactericidal activity (MIC = 7.9 μM) (Table 4).

Series 6 and 7. Series 6 and 7 were identified from a two-cyclelibrary containing 3.8 million on-DNA compounds. The off-DNA compounds that comprise these series contain a tertiaryamine core decorated with three substituents, typically theN-methylacetamide and two bicyclic groups. The parent com-pound of series 6 and 7, compound 6a (IC50 = 6 μM), has apiperidinyl pyridine moiety that, in the cocrystal structure withInhA:NADH, forms the warhead of the molecule binding adja-cent to the nicotinamide ring of NADH (Fig. 4) in a fashionsimilar to that of the piperidinyl pyridine of compound 4a fromseries 4 and 5. The phenyl pyrazole of compound 6a appears tobypass the typical stacking interaction with Phe97 and instead

reaches into a previously unobserved pocket defined by the sidechains of Phe41 and Arg43. The active site loop is disordered,and the nearby hydrophobic pocket is unoccupied. In this crystalstructure Tyr158 adopts the out conformation. Compound 6a hasan MIC of 50 μM under aerobic conditions. Multiple analogswere synthesized in an attempt to improve cell-based activity;however, the majority of the changes made to the molecule re-duced or eliminated activity. One analog, compound 7, had im-proved activity in the in vitro assay (IC50 = 0.5 μM) but was lessactive in the cell-based assay (MIC = 57 μM), potentially becauseof an increased cLogP (the calculated logarithms of water-octanolpartition coefficients) (Table 5).

Series 8 and 9. Series 8 and 9 were identified from the samelibrary as series 1–3 described previously. These series are exem-plified by compound 8a (IC50 = 0.130 μM), which has a conservedpyrrolidine core and three substituents: benzoyl and 1-t-Butoxyethyl groups that are directly connected to the pyrrolidine coreand a pyrazole benzaldehyde connected via the t-Butoxy ethyl.The compound 8a cocrystal structure showed the pyrazole groupforming the warhead by binding into site I adjacent to the nic-otinamide ring of NADH (Fig. 5). The t-Butoxy ethyl groupforms packing interactions with the active site loop, which isordered. Tyr158 adopts the out conformation. The benzoyl formsa previously unobserved π–π stacking interaction with Phe97.

Table 3. Series 1–3 structure–activity relationships

NT, not tested.

Soutter et al. PNAS | Published online November 18, 2016 | E7883

APP

LIED

BIOLO

GICAL

SCIENCE

SPN

ASPL

US

Dow

nloa

ded

by g

uest

on

July

11,

202

0

The methyl amide forms a pair of hydrogen bonds with the side chainof Arg43, also a previously unobserved interaction. When tested inthe TB panel, compound 8a had an MIC of 19 μM (Table 6).

Series 10. Series 10 was identified from the same library as series1–3 described previously. This series of compounds was based oncompound 10a, which was identified from a family of on-DNAcompounds uniquely enriched in the presence of NAD+. Com-pound 10a had an IC50 of 0.198 μM in the NADH-dependentassay, but the potency improved fivefold to 0.038 μM whentested in the NAD+-dependent assay, confirming the preferenceof the compound for the InhA:NAD+ complex (Table 2). SPRexperiments further confirmed this preference. The Kd of com-pound 10a for the InhA:NADH complex is 5 μM, whereas the Kd

for the InhA:NAD+ complex is 260 nM. The series 10 com-

pounds contain a 4-phenylpiperidine-4-carboxylate core. Com-pound 10a has a pyrazole connected to the nitrogen atom of thepiperidine ring and a trifluorobenzamide at the 4 position (Table7). Repeated attempts to cocrystallize InhA:NADH and InhA:NAD+ in complex with compound 10a were unsuccessful. TheNAD+-dependent compound 10a had an MIC of 12 μM underaerobic conditions.

DiscussionA pool of 11 DNA-encoded libraries comprising more than 66billion unique small molecules was interrogated against InhA inthe apo-, NAD+-, NADH-, and NADH-inhibitor–bound forms.Comparison of the profiles of enriched library members acrossselections allowed the discovery of small molecules that in-hibited the enzyme in a cofactor-specific manner as demon-strated in the in vitro and biophysical assays. Fourteen of thenearly 50 library members synthesized off-DNA had IC50 valuesof less than 1 μM in the in vitro assays (Table 2). Seven of the off-DNA compounds initially evaluated had MIC values less than50 μM in the aerobic Mtb assay. Of those seven, three com-pounds—compound 1a from series 1–3, compound 8a from se-ries 8 and 9, and compound 10a from series 10—had MICsof 12–13 μM.The crystal structures obtained revealed a diversity of binding

modes in which site I, adjacent to the nicotinamide ring ofNADH, could be occupied by a variety of substituents: a triazole(series 1–3), a piperidinyl pyridine (series 4, 5, and 10), or apyrazole (series 8 and 9). Compounds from series 1–3 and fromseries 4 and 5 formed the previously observed packing interac-tions with main chain atoms near Phe97. In three of the fourcrystal structures, Tyr158 adopted the out conformation, rotatedaway from the nicotinamide ring of NADH and toward the sidechains of Ile215 and Leu218, under the active site loop. Theparent compound of series 8 and 9 forms a unique π–π stackinginteraction with the side chain of Phe97, whereas the series 6 and7 compound bypassed Phe97 altogether, adopting an unusualbinding mode in which the phenyl pyrazole binds into a pre-viously unobserved pocket defined by the side chains of Phe41and Arg43.

A

B

Fig. 2. (A) Crystal structure of compound 1a (green carbon atoms) bound toInhA (yellow carbon atoms) (PDB ID code 5G0S). The inhibitor occupies threedistinct subsites, which are depicted by colored ovals; red oval: catalytic site(site I); yellow oval: hydrophobic site (site II); green oval: hydrophilic site (siteIII). Selected residues are displayed as sticks. NADH is displayed with pinkcarbon atoms. The active site loop of InhA is displayed in orange. (B) Overlayof all five series [compounds 1a (green carbon atoms) (PDB ID code 5G0S); 2a(yellow) (PDB ID code 5G0T); 4a (orange) (PDB ID code 5G0U); 6a (cyan) (PDBID code 5G0V); and 8a (red) (PDB ID code 5G0W)]. The proteins from thecomplexes were superimposed, and only the bound compounds and NADHfrom the complex of InhA with compound 1a are shown for clarity. Thecompounds occupy the three sites marked in A, and compounds 6a and 8aoccupy an additional subsite above the adenine of NADH marked in cyan. Aand B are shown in same orientation.

Fig. 3. X-ray crystal structure of InhA:NADH in complex with compound 4a(PDB ID code 5G0U) from series 4. Carbon atoms for compounds are shownin green. The protein backbone cartoon is represented in yellow. Selectedatoms for the InhA side chains including F97, M98, and Ile202 in the activesite loop are shown as sticks. NADH is shown as sticks with magenta carbonatoms. Refined (2fo-fc) electron density contoured at 1.0 σ is represented as awire mesh. Some atoms of the active-site covering loop represented in or-ange have been removed for clarity.

E7884 | www.pnas.org/cgi/doi/10.1073/pnas.1610978113 Soutter et al.

Dow

nloa

ded

by g

uest

on

July

11,

202

0

InhA was discovered to be the molecular target of isoniazid in1996 (19). Since then ongoing efforts to develop novel InhAinhibitors as drugs for the treatment of TB have met with nosuccess. Here, we have further demonstrated the successful ap-plication of DEX technology in identifying drug leads against adifficult protein target. Using the DEX technology, we haveidentified five unique, cofactor-specific chemical series withdemonstrated activity in cell-based assays. Structure-guideddrug design was combined with structure–affinity relationshipsrevealed from selection in the design and synthesis of analogsfrom multiple chemical series that showed cell-based activity.The most broadly explored series were series 4 and 5 with 32analogs made. These compounds showed a range of activities inthe TB panel as described in Table 4. Analogs from series 4 and5 made in an attempt to lower the molecular weight of themolecule (removal of methyl amide, cyclization, and replace-ment of phenyl rings with smaller isosteres) did not improveactivity. Replacement of the ether linkage of the fluorophenox-ybenzyl group with an amine did lead to a 10-fold improvementin potency in the in vitro assay and to a threefold improvement inthe MIC. Although 12 analogs were made in series 6 and 7, onlyone resulted in improvement in the IC50, and that compound wasless potent in the TB panel. The parent compound of the series8 and 9 had an MIC of 12 μM. Five analogs in this series weresynthesized, all of which were less potent than the parent in thein vitro assay, and none was evaluated in the TB panel. Series 10was the only NAD+-dependent series explored. In contrast toprevious reports of cell-based activity for NADH-dependentcompounds (13), the parent compound of the series 10, com-pound 10a, had an MIC of 12 μM, equivalent to the two mostpotent compounds from NADH-dependent series reportedherein, demonstrating that bactericidal compounds can bind toeither reduced or oxidized cofactor-bound forms of InhA. Thisresult suggests that further efforts to identify InhA inhibitors thatbind specifically to the NAD+-bound form of the enzyme couldyield bactericidal compounds.Utilization of the DEX technology allowed the identifica-

tion of multiple classes of Mtb InhA inhibitors, many of which

had cell-based activity, directly from the primary affinity-basedscreen. Compounds were identified as cofactor-specific bind-ers of Mtb InhA with direct target engagement further dem-onstrated by both in vitro activity assays and SPR experiments.Of the 50 initial compounds synthesized, 25 inhibited in vitroenzyme activity. Seven compounds with in vitro activity in-hibited Mtb bacterial growth under aerobic conditions. Series1–3 both inhibited bacterial growth in Mtb MIC assays andkilled Mtb-infected THP-1 cells. Further efforts are needed to

Table 4. Series 4 and 5 structure–activity relationships

NA, not applicable; NT, not tested.

Fig. 4. X-ray crystal structure of InhA:NADH in complex with compound 6a(PDB ID code 5G0V) from series 6. Carbon atoms for compounds are shown ingreen. The protein backbone cartoon is represented in yellow. Selectedatoms for the InhA side chains including the catalytic residue Y158, F97, andM98 are shown as sticks. NADH is shown as sticks with magenta carbonatoms. Refined (2fo-fc) electron density contoured at 1.0 σ is represented as awire mesh. Some atoms of the active-site covering loop represented in or-ange have been removed for clarity.

Soutter et al. PNAS | Published online November 18, 2016 | E7885

APP

LIED

BIOLO

GICAL

SCIENCE

SPN

ASPL

US

Dow

nloa

ded

by g

uest

on

July

11,

202

0

understand how these compounds enter bacteria and whetherthey are subject to efflux. This understanding would allow thedevelopment of a next generation of lead compounds thatare effective therapeutics for the treatment of MDR Mtbinfections.

Experimental MethodsProtein Expression/Purification.Mtb InhAWTwas cloned in the pET15b vectorand expressed in the Escherichia coli BL21 Star (DE3) strain. The cell pelletwas suspended in 50 mL lysis buffer [50 mM Tris·HCl (pH 8.0), 150 mM NaCl,10 mM imidazole, 1 mM PMSF, 50 μg/mL lysozyme, and one tablet of EDTA-free protease inhibitor mixture], incubated on ice for 30 min, and sonicated.The cell lysate was centrifuged at 100,000 × g at 4 °C for 1 h, and the su-pernatant was collected. Qiagen Ni-NTA resin was equilibrated with fivecolumn volumes of lysis buffer, and the clarified lysate was passed throughthe column. The column was washed with five column volumes of washbuffer [50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 50 mM imidazole] to 12.5%(vol/vol) of buffer B [50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 400 mM im-idazole]. Ni-NTA–bound proteins were eluted using buffer B [12.5 to 100%(vol/vol) B] over seven column volumes. Peak fractions were pooled anddialyzed into 30 mM Pipes (pH 6.8), 150 mM NaCl, 1 mM EDTA, and 10%(vol/vol) glycerol. Precipitation was seen in both pools after dialysis. Sam-ples were centrifuged to remove precipitated protein, and supernatant wasloaded onto the gel-filtration column. Peak fractions were pooled, con-centrated, and evaluated for activity.

Expression and purification of Mtb inhA (S94A) were identical to that ofthe WT InhA with the exception of the lysis buffer, which was comprised of50 mM Tris HCl (pH 8.0), 10 mM imidazole, 1 mM PMSF, 50 μg/mL lysozyme,and one tablet of EDTA-free protease inhibitor mixture.

Affinity-Mediated Selection of DNA-Encoded Libraries for InhA-Binding SmallMolecules. All reagents were acquired from Sigma-Aldrich unless otherwisenoted. Eleven different DNA-encoded chemical libraries comprising in total∼100 billion different encoded building-block combinations were combinedin solution, and affinity-mediated selection for InhA binders was initiated bymultiple incubations in 60 μL of a model cytosol incubation buffer con-taining Hepes (20 mM), potassium acetate (134 mM), sodium acetate (8 mM),sodium chloride (4 mM), magnesium acetate (0.8 mM), sheared salmonspermDNA (1mg/mL; Invitrogen), imidazole (5mM), and Tween 20 [0.02% (vol/vol)]at pH 7.2. Different incubation samples contained different combinations ofInhA (10 μM), NAD+ (5 mM), NADH (500 μM), and a known NADH-dependent

InhA inhibitor (50 μM). Individual libraries were included in the library mixture ata concentration of 50 nM. The on-DNA positive control compound was includedin each 60-μL selection at a final concentration of 1.67 pM. An unsubstitutedheadpiece was included as a negative control (subsequently ligated) at a con-centration of 167 nM (10 pmol). After 1 h of incubation the mixture was flowedover a 5-μL bed of nickel affinity matrix (His-Select High-Flow Nickel Affinity Gel;Phynexus) with 20 passages followed by eight washes with 200-μL incubationbuffer aliquots. Retained library members were eluted by incubation with 60 μLof incubation buffer at 85 °C for 5 min followed by a further incubation with asecond 5-μL resin bed of His-Select High-Flow Nickel Affinity Gel to remove any

Table 5. Series 6 and 7 structure–activity relationships

NA, not applicable; NT, not tested.

Fig. 5. X-ray crystal structure of InhA:NADH in complex with compound 8a(PDB ID code 5G0W) from series 8. Carbon atoms for compounds are shownin green. The protein backbone cartoon is represented in yellow. Selectedatoms for the InhA side chains including Phe41, Arg43, F97, and M98 areshown as sticks. NADH is shown as sticks with magenta carbon atoms. Re-fined (2fo-fc) electron density contoured at 1.0 σ is represented as a wiremesh. Some atoms of the active-site covering loop represented in orangehave been removed for clarity.

E7886 | www.pnas.org/cgi/doi/10.1073/pnas.1610978113 Soutter et al.

Dow

nloa

ded

by g

uest

on

July

11,

202

0

eluted protein. This entire selection protocol was repeated with a fresh additionof InhA and cofactor where appropriate to half of the round-one eluate to re-generate a 10-μM InhA concentration. Encoding oligonucleotides present in theoutput of the second selection round were amplified using Platinum PCRSupermix (Invitrogen) with denaturation at 94 °C, annealing at 55 °C, and ex-tension at 72 °C for 24 cycles using 5′- and 3′-oligonucleotides (each at 0.5 μM)that each incorporate sequences complementary to the tailpiece or headpiecealong with Illumina READ1 or READ2 sequences required to support clusteringand subsequent single-read 100-bp sequencing on an Illumina HiSeq 2500

system. Sequencing also was performed for PCR-amplified samples of thenaive (unselected) library and the output of a no-target selection performed inthe absence of InhA. From the combined samples, 398 million sequence readswere determined. Sequence data were converted back into encoded chemicalinformation computationally, and demographic and statistical informationwas calculated for individual building-block combinations.

In Vitro Assays. In the NADH-dependent assay, the 2-trans-dodecenoyl-CoA(DDCoA) substrate and NADH cofactor were prepared in the assay buffer [30 mM

Table 6. Series 8 and 9 structure–activity relationships

Me, methyl; NA, not applicable; NT, not tested.

Table 7. Series 10 structure–activity relationships

CF3, trifluoromethyl; Br, bromine; nPr, n-propyl; NT, not tested.

Soutter et al. PNAS | Published online November 18, 2016 | E7887

APP

LIED

BIOLO

GICAL

SCIENCE

SPN

ASPL

US

Dow

nloa

ded

by g

uest

on

July

11,

202

0

Pipes (pH 6.8), 50 mM NaCl, 0.005% (vol/vol) Brij detergent, 2 mM DTT, 0.1 mMEDTA]. InhA was diluted in a buffer comprised of 30 mM Pipes (pH 6.8), 150 mMNaCl, and 1 mM EDTA. Compounds were prepared at 90× the final concentrationin 100% (vol/vol) DMSO and then were diluted to 3× in assay buffer. A 3× solutionof InhA:NADH was prepared by further dilution of the enzyme in 3× NADH.Positive control wells contained DMSO in lieu of the compound. Negative controlwells did not contain enzyme. The InhA:NADH was preincubated with the com-pound for 1 h at room temperature followed by the addition of 3× DDCoA. Thefinal concentrations of the components were 10 nM InhA, 50 μM NADH, and150 μMDDCoA. Upon the addition of DDCoA, the reaction was run for 30 min at30 °C. End-point fluorescence (F) was read at excitation λ = 340 nM and emissionλ = 420 nM. Compounds were tested at a top concentration of 30 μM seriallytitrating down threefold for a total of 11 doses. The percent inhibition of enzymewas calculated as 100*(1−(Fcompound well − Fneg control)/(Fpos control − Fneg control))

In the NAD+-dependent assay, the DDCoA substrate and stock solution ofInhA were prepared as described for the NADH-dependent assay. A mixture ofNADH and NAD+ cofactors were prepared in the assay buffer [30 mM Pipes (pH6.8), 50 mM NaCl, 0.005% Brij, 2 mM DTT, and 0.1 mM EDTA]. Compounds wereprepared as described for the NADH-dependent assay. A 3× solution of InhA wasprepared by further dilution of the enzyme in 3× NADH:NAD+. Positive controlwells contained DMSO in lieu of the compound. Negative control wells did notcontain enzyme. The InhA:NADH:NAD+ mixture was preincubated with thecompound for 1 h at room temperature followed by the addition of 3× DDCoA.Upon the addition of DDCoA, the reaction was run for 30 min at 30 °C. The finalconcentrations of the components were 10 nM InhA, 50 μMNADH, 2 mMNAD+,and 150 μMDDCoA. End-point fluorescence (F) was read at excitation λ = 340 nMand emission λ = 420 nM. The percent inhibition of enzyme was calculated asdescribed for the NADH-dependent assay.

SPR Assay. A BIAcore 4000 instrument (GE Healthcare) was used to monitorbinding interactions using a direct binding assay format. Before activation, theresearch-grade CM5 chip surface was preconditioned using two 50-μL injectionseach of 10 mM HCl, 50 mM NaOH, 0.1% SDS, and 0.085% H3PO4, at a flow rateof 100 μL/min. Full-length InhA was immobilized on the sensor surface usingstandard amine coupling. Amine coupling was achieved by activating the sensorsurface using 7-min injections of a mixture of 11.5 mg/mL N-hydroxysuccinimidewith 75 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.Protein immobilization was accomplished using a 10-min injection of full-lengthInhA (100 μg/mL) in 100 mM sodium acetate (pH 5.5) buffer. Remaining reactiveesters were blocked using a 7-min injection of 100 mM Tris·HCl (pH 8.5) at a flowrate of 10 μL/min. Immobilization levels typically were around 5,000 resonanceunits. Reference flow cells were prepared without the protein. All bindingmeasurements were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.005%(vol/vol) T20, and 1% DMSO at a flow rate of 50 μL/min. Compound concen-trations were injected over the active protein and reference surface with at least90-s association and 600-s dissociation times. Solvent calibration and double-referencing subtractions were made to eliminate changes in the refractive indexand injection noise using Biacore Evaluation software (GE Healthcare). Surfaceregeneration was achieved using dissociation for a time period, allowing theresponse to return to baseline. Control injections of a fixed, saturating com-pound concentration of 100 nM and buffer were interspersed with injections ofcompound to allow monitoring of the functionality of the immobilized proteinsurface. Compounds were tested at a top concentration of 100-μM threefolddilution series at nine concentrations. Compounds were injected either alone,in the presence of 2 mM NAD+, or in the presence of 100 μM NADH + 1 mMTris(2-carboxyethyl)phosphine (TCEP). Excess cofactor was included in the com-pound injections to ensure that the protein remained saturated with cofactor.NADH injections (50 μM) were interspersed with compound injections to en-sure that the chip surface had not degraded. SPR equilibrium binding data,consisting of Req values from eight-point concentration series, were analyzed

by fitting a simple 1:1 binding model to yield Rmax and Kd values using Grafit(Erithacus software).

Crystallography. Crystals of InhA with compounds were grown using thehanging-drop method at 293 K. The reservoir solution contained 12% (wt/vol)PEG 4000, 0.1 M N-(2-acetamido)iminodiacetic acid (ADA) (pH 6.8), 6 mM DMSO,0.1 M ammonium acetate, 1% glycerol, and 4.5 mM NAD+. Drops were set upwith 1.5 μL protein and 1 μL reservoir. Trays were incubated at 20 °C, and crystalsappeared after 3–6 d. Crystals were transferred to a solution containing 5 mMcompound and 12% (wt/vol) PEG 4000, 0.1 M ADA (pH 7.2), 6 mM DMSO, 0.1 Mammonium acetate, 1% glycerol, and 4.5 mM NADH and were incubated for24 h. Crystals then were cryo-protected after increasing the concentrations ofPEG 4000 to 15% (wt/vol) and glycerol to 20% (vol/vol). Crystals were frozendirectly into a cryo-stream at 100 K.

Diffraction datawere collected at Diamond beam-line I04-1 equippedwitha Dectris Pilatus 6M X-ray detector, using a Si111 monochromatic wavelengthof 0.92 Å. Data were processed using MOSFLM and AIMLESS and were re-duced using CCP4 software (20). The structures were solved by molecularreplacement using coordinates Mtb InhA in complex [Protein Data Bank(PDB) ID code 4D0R] as a trial model using CCP4 software. Protein and in-hibitor were modeled into the electron density using COOT (21). The modelwas refined using BUSTER (22). Crystallographic statistics are reported inTable S3.

IDRI TB Panel.MIC under aerobic conditions. The MICs of compounds were determined bymeasuring bacterial growth after 5 d in the presence of test compounds.Compounds were prepared as 10-point twofold serial dilutions in DMSO andwere diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSOconcentration of 2% (vol/vol). The highest concentration of compound was200 μM where compounds were soluble in DMSO at 10 mM. For potentcompounds, assays were repeated at lower starting concentrations. Eachplate included assay controls for background (medium/DMSO only, no bac-terial cells), zero growth (100 μM rifampicin), and maximum growth (DMSOonly) as well as a rifampicin dose–response curve. Plates were inoculatedwith Mtb and incubated for 5 d; growth was measured by OD590 and fluo-rescence (excitation 560/emission 590) using a BioTek Synergy 4 plate reader.Growth was calculated separately for OD590 and relative fluorescence units(RFU). To calculate the MIC, the 10-point dose–response curve was plotted aspercent of growth and was fitted to the Gompertz model using GraphPadPrism 5. The MIC was defined as the minimum concentration at whichgrowth was completely inhibited and was calculated from the inflectionpoint of the fitted curve to the lower asymptote (zero growth). In additiondose–response curves were generated using the Levenberg–Marquardtalgorithm, and the IC50 and IC90 were determined.MBC. Mtb was grown aerobically to logarithmic phase and inoculated intoliquid medium containing four different compound concentrations with afinal maximum concentration of 2% (vol/vol) DMSO. For compounds with anMIC <20 μM (from task group 1 assay), the concentrations selected were 10×MIC, 5× MIC, 1× MIC, and 0.25× MIC. For compounds with MIC >20 μM, thehighest concentration possible was tested (200, 100, 20, and 5 μM). Cultureswere exposed to compounds for 21 d, and cell viability was measured byenumerating colony-forming units on agar plates on days 0, 7, 14, and 21.The MBC was defined as the minimum concentration required to achieve a2-log kill in 21 d. For compounds with >1-log kill, an assessment of time- and/orconcentration-dependence was determined from the kill kinetics. DMSO wasused as a positive control for growth.

ACKNOWLEDGMENTS. This work was supported by NIH National Institute ofAllergy and Infectious Diseases Contract HHSN272201100009I.

1. Banerjee A, et al. (1994) inhA, a gene encoding a target for isoniazid and ethion-amide in Mycobacterium tuberculosis. Science 263(5144):227–230.

2. Dessen A, Quémard A, Blanchard JS, Jacobs WR, Jr, Sacchettini JC (1995) Crystalstructure and function of the isoniazid target of Mycobacterium tuberculosis. Science267(5204):1638–1641.

3. Rozwarski DA, Grant GA, Barton DHR, Jacobs WR, Jr, Sacchettini JC (1998) Modifi-cation of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis.Science 279(5347):98–102.

4. Chollet A, et al. (2015) Crystal structure of the enoyl-ACP reductase of Mycobacteriumtuberculosis (InhA) in the apo-form and in complex with the active metabolite ofisoniazid pre-formed by a biomimetic approach. J Struct Biol 190(3):328–337.

5. Schroeder EK, de SouzaN, Santos DS, Blanchard JS, Basso LA (2002) Drugs that inhibitmycolicacid biosynthesis in Mycobacterium tuberculosis. Curr Pharm Biotechnol 3(3):197–225.

6. Basso LA, Santos DS (2005) Drugs that inhibit mycolic acid biosynthesis in Mycobac-terium tuberculosis an update. Med Chem Res 2:393–413.

7. Hazbón MH, et al. (2006) Population genetics study of isoniazid resistance mutationsand evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob AgentsChemother 50(8):2640–2649.

8. Clark MA, et al. (2009) Design, synthesis and selection of DNA-encoded small-moleculelibraries. Nat Chem Biol 5(9):647–654.

9. Gilmartin AG, et al. (2014) Allosteric Wip1 phosphatase inhibition through flap-subdomain interaction. Nat Chem Biol 10(3):181–187.

10. Deng H, et al. (2012) Discovery of highly potent and selective small moleculeADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded librarytechnology (ELT). J Med Chem 55(16):7061–7079.

11. Ding Y, et al. (2015) Discovery of Potent and Selective Inhibitors for ADAMTS-4through DNA-Encoded Library Technology (ELT). ACS Med Chem Lett 6(8):888–893.

12. Kollmann CS, et al. (2014) Application of encoded library technology (ELT) to a protein-protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists. Bioorg Med Chem 22(7):2353–2365.

E7888 | www.pnas.org/cgi/doi/10.1073/pnas.1610978113 Soutter et al.

Dow

nloa

ded

by g

uest

on

July

11,

202

0

13. Encinas L, et al. (2014) Encoded library technology as a source of hits for the discoveryand lead optimization of a potent and selective class of bactericidal direct inhibitorsof Mycobacterium tuberculosis InhA. J Med Chem 57(4):1276–1288.

14. Yang H, et al. (2015) Discovery of a Potent Class of PI3Kα Inhibitors with Unique BindingMode via Encoded Library Technology (ELT). ACS Med Chem Lett 6(5):531–536.

15. Shirude PS, et al. (2013) Methyl-thiazoles: A novel mode of inhibition with the po-tential to develop novel inhibitors targeting InhA in Mycobacterium tuberculosis.J Med Chem 56(21):8533–8542.

16. Litovchick A, et al. (2015) encoded library synthesis using chemical ligation and thediscovery of sEH inhibitors from a 334-million member library. Sci Rep 5:10916.

17. Keefe AD, Clark MA, Hupp CD, Litovchick A, Zhang Y (2015) Chemical ligation methodsfor the tagging of DNA-encoded chemical libraries. Curr Opin Chem Biol 26:80–88.

18. Goodnow RA, Jr (2014) A Handbook for DNA-Encoded Chemistry: Theory andApplications for Exploring Chemical Space and Drug Discovery (John Wiley & Sons,New York).

19. Wheeler PR, Anderson PM (1996) Determination of the primary target for isoniazid inmycobacterial mycolic acid biosynthesis with Mycobacterium aurum A+. Biochem J318(Pt 2):451–457.

20. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: Programs for

protein crystallography. Acta Crystallogr D Biol Crystallogr 50(Pt 5):760–763.21. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta

Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132.22. Bricogne G, et al. (2011) BUSTER version 2.11.5 (Global Phasing Ltd., Cambridge, UK).23. Rawat R, Whitty A, Tonge PJ (2003) The isoniazid-NAD adduct is a slow, tight-binding

inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: Adduct affinity

and drug resistance. Proc Natl Acad Sci USA 100(24):13881–13886.24. Luckner SR, Liu N, am Ende CW, Tonge PJ, Kisker C (2010) A slow, tight binding in-

hibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuber-

culosis. J Biol Chem 285(19):14330–14337.25. Pan P, Tonge PJ (2012) Targeting InhA, the FASII enoyl-ACP reductase: SAR studies on

novel inhibitor scaffolds. Curr Top Med Chem 12(7):672–693.26. Hartkoorn RC, et al. (2014) Pyridomycin bridges the NADH- and substrate-binding

pockets of the enoyl reductase InhA. Nat Chem Biol 10(2):96–98.27. Manjunatha UH, et al. (2015) Direct inhibitors of InhA are active against Mycobac-

terium tuberculosis. Sci Transl Med 7:269ra3.

Soutter et al. PNAS | Published online November 18, 2016 | E7889

APP

LIED

BIOLO

GICAL

SCIENCE

SPN

ASPL

US

Dow

nloa

ded

by g

uest

on

July

11,

202

0