High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis...

Sharma, Sudipto Saha and Ramandeep SinghShubhra Mandal, Arpit Gupta, Deepak Garima Arora, Prabhakar Tiwari, Rahul Phosphatase

PhosphoserineMycobacterium tuberculosisMolecule Inhibitors Specific for High Throughput Screen Identifies SmallEnzymology:

doi: 10.1074/jbc.M114.597682 originally published online July 18, 20142014, 289:25149-25165.J. Biol. Chem.

10.1074/jbc.M114.597682Access the most updated version of this article at doi:

.JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/289/36/25149.full.html#ref-list-1

This article cites 57 references, 24 of which can be accessed free at

at Cam

bridge University L

ibrary on Novem

ber 18, 2014http://w

ww

.jbc.org/D

ownloaded from

at C

ambridge U

niversity Library on N

ovember 18, 2014

http://ww

w.jbc.org/

Dow

nloaded from

http://affinity.jbc.org/

http://enzyme.jbc.org

http://metabolism.jbc.org

http://www.jbc.org/lookup/doi/10.1074/jbc.M114.597682

http://affinity.jbc.org

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;289/36/25149&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/36/25149

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=289/36/25149&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/36/25149

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/289/36/25149.full.html#ref-list-1

http://www.jbc.org/

http://www.jbc.org/

High Throughput Screen Identifies Small Molecule InhibitorsSpecific for Mycobacterium tuberculosis PhosphoserinePhosphatase*

Received for publication, July 17, 2014 Published, JBC Papers in Press, July 18, 2014, DOI 10.1074/jbc.M114.597682

Garima Arora‡§, Prabhakar Tiwari‡, Rahul Shubhra Mandal¶, Arpit Gupta�, Deepak Sharma�, Sudipto Saha**,and Ramandeep Singh‡1

From the ‡Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Gurgaon122016, Haryana, India, the §Symbiosis School of Biomedical Sciences, Symbiosis International University, Lavale, Maharashtra412115, India, the ¶Biomedical Informatics Center, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal700010, India, the �CSIR-Institute of Microbial Technology, Chandigarh, 160036, India, and the **Bioinformatics Centre, BoseInstitute, Kolkata, West Bengal 700054, India

Background: Phosphoserine phosphatase (PSP) is an essential enzyme involved in L-serine biosynthesis.Results: High throughput screen was performed to identify specific PSP inhibitors with activity against intracellular bacteria.Conclusion: Validation of PSP as a drug target would lead to identification of scaffolds with a novel mechanism.Significance: This is the first report demonstrating selective inhibition of M. tuberculosis PSP enzyme.

The emergence of drug-resistant strains of Mycobacteriumtuberculosis makes identification and validation of newer drugtargets a global priority. Phosphoserine phosphatase (PSP), akey essential metabolic enzyme involved in conversion ofO-phospho-L-serine to L-serine, was characterized in this study.The M. tuberculosis genome harbors all enzymes involved inL-serine biosynthesis including two PSP homologs: Rv0505c(SerB1) and Rv3042c (SerB2). In the present study, we have bio-chemically characterized SerB2 enzyme and developed mala-chite green-based high throughput assay system to identifySerB2 inhibitors. We have identified 10 compounds that werestructurally different from known PSP inhibitors, and few ofthese scaffolds were highly specific in their ability to inhibitSerB2 enzyme, were noncytotoxic against mammalian celllines, and inhibited M. tuberculosis growth in vitro. Surfaceplasmon resonance experiments demonstrated the relativebinding for these inhibitors. The two best hits identified inour screen, clorobiocin and rosaniline, were bactericidal inactivity and killed intracellular bacteria in a dose-dependentmanner. We have also identified amino acid residues criticalfor these SerB2-small molecule interactions. This is the firststudy where we validate that M. tuberculosis SerB2 is a drug-gable and suitable target to pursue for further high through-put assay system screening.

Tuberculosis (TB)2 caused by Mycobacterium tuberculosis isan enormous health burden in developing countries. TheWorld Health Organization currently estimates that �1.8 bil-lion people are latently infected with M. tuberculosis, there are9 million new incident rates and �2 million annual deaths aris-ing from TB infections (1). The current DOTS (directlyobserved treatment short course) regimen involves drugs thatinhibit either DNA replication or transcription or cell wall bio-synthesis but is very complex and lengthy. Numerous factorssuch as poor patient compliance or treatment failure have led toemergence of various drug-resistant (multidrug-resistant,extensively drug-resistant, and totally drug-resistant) TBstrains (2). The major challenge in the field of TB drug devel-opment is to identify drug targets that are vulnerable in vitroand to identify scaffolds (i) with a novel mechanism of action,(ii) that have the potential to shorten chemotherapy, (iii) thattarget drug-resistant and latent bacteria, and (iv) that arecompatible with current TB and anti-retroviral therapy (3).In the past decade, substantial progress has been made indevelopment of genetic tools to identify and biochemicallycharacterize metabolic pathways that are essential forM. tuberculosis growth in vitro. These pathways are poten-tial drug targets to identify scaffolds with a novel mechanismof action (4 –7).

L-Serine biosynthetic pathway represents one such mechanism,and enzymes involved in L-serine biosynthesis have been predictedto be essential for M. tuberculosis growth in vitro (4–7). In bacte-ria, there are two distinct pathways involved in L-serine biosynthe-sis (8, 9). The first pathway involves serine hydroxy methyl trans-ferase that catalyzes simultaneous reversible conversion of glycineand 5,10-methylenetetrahydrofolate to serine and 5,6,7,8-tetrahy-

* This work was supported by intramural funding to Translational Health Sci-ence and Technology Institute from the Department of Biotechnology(DBT) of the Government of India, Department of Biotechnology Ramalin-gaswamy fellowships BT/HRD/35/02/18/2009 (to R. S.) and BT/RLF/Re-en-try/11/2011 (to S. S.), DBT Research Fellowship BT/HRD/35/02/18/2009 (toG. A.), Indian Council of Medical Research (ICMR) fellowship (IRIS ID 2013:19440 to G. A.), DBT Research Fellowship BT/PR15074/GBD/27/297/2011(to P. T.), and ICMR Grant IRIS ID: 2013–1551G (to R. S. M.).

1 To whom correspondence should be addressed: Vaccine and InfectiousDisease Research Centre, Translational Health Science and TechnologyInstitute, 496 UdyogVihar, Phase III, Gurgaon 122016, Haryana, India.Tel.: 91-124-2876501; Fax: 91-124-2876502; E-mail: [email protected].

2 The abbreviations used are: TB, tuberculosis; PSP, phosphoserine phospha-tase; HTS, high throughput screening; PSAT, phosphoserine aminotrans-ferase; HAD, haloacid dehalogenase; HPSP, human PSP; IPTG, isopropyl�-D-galactopyranoside; SPR, surface plasma resonance; RU, resonanceunits; RPMI, Roswell Park Memorial Institute; MBP, maltose-binding pro-tein; r.m.s.d., root mean square deviation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 36, pp. 25149 –25165, September 5, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

SEPTEMBER 5, 2014 • VOLUME 289 • NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 25149

at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

drofolate, respectively (10). In an alternative pathway, 3-phospho-glycerate dehydrogenase (PGDH) oxidizes 3-phosphoglycerate to3-phosphohydroxy pyruvate in a NAD�/NADH-dependent man-ner. Phosphoserine aminotransferase (PSAT), a PLP (pyridoxal-5�-phosphate)-dependent enzyme converts 3-phosphohydroxypyruvate to O-phospho-L-serine that is subsequently dephos-phorylated by phosphoserine phosphatase (PSP) into L-serine (11).Both 3-phosphoglycerate dehydrogenase and PSAT homologs inM. tuberculosis have been extensively biochemically character-ized, and their crystal structures have also been determined (12–14). In a recent study, it has been shown that intracellular cyclicAMP regulates the levels of PSAT enzyme, and extracellular addi-tion of L-serine restores the growth defect of M. tuberculosis crpmutant in vitro (15).

PSP enzymes belong to the haloacid dehalogenase (HAD)superfamily of enzymes that are known to regulate diverse cel-lular functions such as membrane transport, metabolism, signaltransduction, and nucleic acid repair (16). The HAD family ofenzymes are characterized by the presence of three specificmotifs: motif I, DXDX(T/V); motif II, (S/T)XX; and motif III,KX18 –30(G/S)(D/S)XXX(D/N) (17, 18). These enzymes have anabsolute requirement for divalent metal ion, and the conservedAsp residue in motif I forms a phosphoenzyme intermediatewith the substrate via nucleophilic attack (19, 20). HAD familyof enzymes possess the Rossmannoid �/� fold and an inserted“cap” that regulates substrate access to its active site (21–23).PSP enzymes have been extensively biochemically character-ized in various microorganisms and have been demonstrated tofacilitate entry of Porphyromonas gingivalis into host cells bymodulating host cytoskeletal architecture, innate immuneresponses, and dephosphorylating colicin and NF-�� (24 –26).

Despite the importance of PSP enzymes in L-serine biosyn-thesis, biochemical characterization of mycobacterial PSPhomologs has not been reported so far. In the present study,we have biochemically characterized SerB2 enzyme in vitroand developed a high throughput screening (HTS) assay sys-

tem to identify novel SerB2 specific inhibitors. These iden-tified new scaffolds that were (i) structurally different fromknown PSP inhibitors, (ii) selective in their ability to inhibitSerB2 enzyme in comparison with human PSP (HPSP)enzyme, and (iii) inhibited M. tuberculosis growth in vitro ina dose-dependent manner.

EXPERIMENTAL PROCEDURES

Chemicals, Strains, and Growth Conditions—Most of thechemicals used in the present study unless mentioned werepurchased from Sigma-Aldrich. Various strains and plasmidsused in the study are shown in Table 1. Escherichia coli strainsXL-1 Blue and BL-21 (�DE3, plysS) were used for cloning andexpression studies, respectively. M. tuberculosis H37Rv andMycobacterium bovis BCG strains were used for growth inhibi-tion and macrophage infection studies. Various E. coli andmycobacterial strains were cultured in LB and Middlebrookmedium, respectively, as per manufacturer’s standard proto-cols. The antibiotics were used in the following concentrations:ampicillin (50 �g/ml), kanamycin (25 �g/ml), tetracycline (10�g/ml), and chloramphenicol (34 �g/ml).

Multiple Sequence Alignment and Phylogenetic Analysis of PSPHomologs—Protein sequences of PSP homologs from variousorganisms were retrieved from the National Center for Biotech-nology Information protein database. Multiple sequence align-ment analysis was performed using the Clustal Omega (version1.2.0) alignment tool and edited using GeneDoc (27). The evo-lutionary history was inferred using the neighbor joiningmethod (28, 29).

Construction and Validation of SerB1 and SerB2 HomologyModels—The best templates for homology modeling of SerB1and SerB2 proteins were identified using Position-Specific Iter-ative Basic Local Alignment Search Tool (PSI-BLAST) analysisin the Protein Data Bank. The homology models for SerB1 andSerB2 were built using Discovery Studios 2.5 (Accelrys). Thebuilt models were further refined with repetitive loop modeling

TABLE 1List of bacterial strains and plasmids used in the present study

Description Reference

StrainsH37Rv Virulent strain of M. tuberculosis ATCC 27294M. bovis BCG Danish Vaccine strain against tuberculosis A kind gift from Prof. Anil K. Tyagi

PlasmidspET28b T7 based expression system used to generate NH2-terminus His6-tagged proteins NovagenpET-28b-serB1 pET28b carrying serB1 This workpET-28b-serB2 pET28b carrying serB2 This workpMAL-c2x E. coli tac based expression system used to generate NH2-terminal MBP-tagged proteins New England BiolabspMAL-c2x-serB1 pMAL-c2X carrying serB1 This workpMAL-c2x-serB2 pMAL-c2X carrying serB2 This workpMAL-c2x-D341G serB2 pMAL-c2X carrying D341G serB2 This workpMAL-c2x-D185G serB2 pMAL-c2X carrying D185G serB2 This workpET28b-S273A serB2 pET28b carrying S273A serB2 This workpET28b-S188A serB2 pET28b carrying S188A serB2 This workpET28b-V186Q serB2 pET28b carrying V186Q serB2 This workpMAL-c2x-K318E serB2 pMAL-c2X carrying K318E serB2 This workpMAL-c2x-K361A serB2 pMAL-c2x carrying K361A serB2 This workpMAL-c2x-R365A serB2 pMAL-c2x carrying R365A serB2 This workpMAL-c2x-E197A serB2 pMAL-c2x carrying E197A serB2 This workpMAL-c2x-D187A serB2 pMAL-c2x carrying D187A serB2 This workpMAL-c2x-E214A serB2 pMAL-c2x carrying E214A serB2 This workpMAL-c2x-V191A-serB2 pMAL-c2x-carrying V191A serB2 This workpGEX-4T1 E. coli tac based expression system used to generate GST fusion proteins GE HealthcarepGEX-4T1-serB1 pGEX-4T1 carrying serB1 This workpET-DUET-pstPcat pETDuet1 vector carrying His6-tagged PstP cytosolic domain (PstPcat) A kind gift from Dr. Y. Singh

High Throughput Screen Identifies PSP Specific Inhibitor

25150 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 36 • SEPTEMBER 5, 2014

at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

and energy minimization studies. The refined homology mod-els were finally validated with PROCHECK, Verify_3D, andErrat programs (30 –32).

Expression and Purification of PSP Proteins—For expres-sion studies, both serB1 and serB2 were PCR-amplified andcloned into either pET28b or pMALc2x or pGEX4T-1. Var-ious active site point mutants of SerB2 enzyme were gener-ated by two-step PCR using gene specific primers having thedesired mutations. E. coli BL-21 (�DE3, plysS) transformedwith either wild type or mutant constructs were grown inLB medium at 37 °C. Protein expression was induced atA600 nm � �0.8 with the addition of isopropyl �-D-galacto-pyranoside (IPTG) at a final concentration of 1 mM. Thecultures were grown overnight at 18 °C, pelleted by centrif-ugation, and frozen at �20 °C for later use. Purification ofHis6-tagged and MBP (maltose-binding protein)-taggedproteins was performed by affinity chromatography usingnickel-nitrilotriacetic acid or amylose resin, respectively, asper the manufacturer’s recommendations. Purified proteinswere visualized on 10% SDS-PAGE by Coomassie BrilliantBlue staining. The purified fractions were dialyzed, concen-trated using Amicon Ultra-15 centrifugal units (Millipore),and stored as aliquots in enzyme storage buffer (50 mM Tris,pH 7.4, 100 mM NaCl, and 10% glycerol). Protein concentra-tion was estimated using Coomassie Plus protein assay rea-gent (Thermo Fisher).

Steady State Kinetics of SerB2 Enzyme—To determine kineticparameters for SerB2 enzyme, assays were performed in 25 �lof reaction volume (100 mM Tris, pH 7.4, 5 mM MgCl2, 5 mM

D��) using varying concentration of O-phospho-L-serine (20 –320 �M) and 1 �M His6-SerB2 enzyme at 37 °C for 10 min. Theformation of inorganic phosphate (Pi) in enzyme reaction wasmonitored by measuring absorbance at 630 nm using Quan-tichrome phosphate assay kit (Bioassay Systems) as per man-ufacturer’s recommendations. The initial velocities inenzyme reactions (rate of Pi release in �M/min) were plottedagainst concentration of O-phospho-L-serine using nonlin-ear regression to the Michaelis-Menten equation usingPrism 5 software (GraphPad Software, Inc., version 5.01).The apparent Km, Vmax, and kcat/Km for SerB2 enzyme wasdetermined from the plotted area. The substrate specificityfor SerB2 enzyme was determined by performing assays inthe presence of varying concentration of either O-phospho-L-serine or O-phospho-L-threonine. To determine optimumconditions for SerB2 activity, assays were performed usingbuffers in the pH range 6.0 –9.0 in the presence of variouscations and nonionic detergent (0.01% Triton X-100) using100 �M O-phospho-L-serine. For identification of catalyti-cally important residues, enzyme assays were performedusing 100 �M of O-phospho-L-serine in the presence ofeither 1 �M wild type or mutant SerB2 protein.

Far Ultraviolet Circular Dichroism Studies—CD spectra ofvarious proteins were recorded on a JASCO-J-815 spectropola-rimeter using a 1-mm-path length cuvette with a scan rate of 10nm/min and averaged over five scans. The raw CD data wereconverted into molar ellipticity (M) as follows,

M � 100*obs�/�d*C (Eq. 1)

where obs is the observed ellipticity (in degrees), d is the pathlength (in centimeters), and C is the protein concentration(molar). The M of MBP was calculated and subtracted fromthe M of MBP-SerB2 fusion protein to obtain molar ellipticityof free SerB2. M was converted to mean residue ellipticity(MRE) as follows,

MRE � M/n � 1� (Eq. 2)

where n is the total number of amino acids in the protein.High Throughput Screen to Identify PSP Inhibitors—Inor-

ganic phosphate release was adapted for a high throughputscreen to identify novel PSP inhibitors. This end point assay wasperformed in a final volume of 50 �l and validated using varyingconcentrations (10 �M to 100 mM) of known PSP inhibitorssuch as DL-AP3 (DL-2-amino-3-phosphonopropionic acid) andsodium orthovandate in the presence of 1 �M His6-SerB2 (33,34). The contents of small molecule library of the National Can-cer Institute Developmental Therapeutic Program comprising2300 structurally diverse compounds were transferred to96-well plate (Nunc) at a final concentration of 100 �M in assaybuffer (containing 1 �M His6-SerB2) for preliminary screening.All reaction plates included proper controls such as buffer only,no substrate, and DL-AP3 control. The enzyme-scaffold mix wasincubated at room temperature for 10 min, and the reactionwas initiated by the addition of 200 �M O-phospho-L-serine.After incubation for a further 10 min, the enzyme activity wasmeasured as described above, and the percentage of inhibitionwas calculated. Half-maximal inhibitory (IC50) concentrationdetermination experiment was performed in the presence ofincreasing concentration of compounds in triplicates. IC50 val-ues were determined by nonlinear regression analysis usingPrism 5 software.

Determination of Specificity of Primary Hits—The hits iden-tified in our HTS were also evaluated for their ability to inhibitmycobacterial Ser/Thr phosphatase (PstP, Rv0018c), alkalinephosphatase (Bangalore Genei, Bangalore, India) and HPSPenzyme (Calbiochem) in vitro. PstP and alkaline phosphataseinhibition studies were performed in the presence of 100 �M ofphoshopeptide (K-R-pT-I-R-R; Millipore) and 200 �M p-nitro-phenyl phosphate, respectively, as per standard protocols.HPSP inhibition studies were performed in conditions similarto those standardized for SerB2 protein.

Surface Plasmon Resonance Studies—Surface plasmon reso-nance (SPR) experiments were performed at 25 °C usingBIACORE T200 (GE Healthcare). SerB2 was diluted to a con-centration of 400 �g/ml in 10 mM sodium acetate buffer, pH 4.0.The protein was immobilized on CM5 sensor chip by the use ofamine coupling chemistry as per standard protocols. The freesurface was blocked with 1 M ethanolamine-HCl (pH 8.5) andwashed with 50 mM NaOH to remove free SerB2. The immobi-lization levels ranged from 8,000 to 10,000 resonance units(RU). For binding studies drugs at concentration of 1 mM inrunning buffer (20 mM Tris, pH 7.4, 200 mM NaCl, 0.005%Tween 20, 2% DMSO) were injected at a flow rate of 30 �l/minfor 2 min over the immobilized protein or a reference surfacewithout protein. The surfaces were than washed with the run-ning buffer and regenerated twice using 10 mM glycine, pH 2.5.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Cytotoxicity of Primary Hits—THP-1 (human acute mono-cytic leukemia) or Vero (green monkey kidney epithelial cellline) were cultured in Roswell Park Memorial Institute (RPMI)medium containing 10% FBS. These cell lines were procuredfrom the National Cell Repository of the National Centre forCell Science (Pune, India). THP-1 monocytes were differenti-ated into macrophages by the addition of 30 nM phorbol 12-my-ristate 13-acetate. For cytotoxicity assays, cells were diluted to adensity of 5 � 105/ml in fresh medium, and 100 �l was ali-quoted into 96-well flat bottom plates (Nunc). After 24 h ofincubation at 37 °C, cells were overlaid with RPMI mediumcontaining drugs (0.625–50 �M) along with DMSO control intriplicate wells. After 96 h postincubation, cellular viability wasassayed by measuring lactate dehydrogenase activity in culturesupernatants using Cytotox96 nonradioactive cytotoxicity kitas per manufacturer’s recommendations (Promega Corpora-tion, Madison, WI). These assays were performed three inde-pendent times, and concentration causing 50% cytotoxicity(TC50) was calculated.

Mycobacterial Growth Inhibition Assays—Minimum inhibi-tory concentration (MIC99) against M. tuberculosis H37Rv andM. bovis BCG for these primary hits was determined usingstandard procedures. Bacterial cells were incubated at 37 °C inthe presence of varying concentration of drugs for 14 days.MIC99 was determined as the lowest concentration of druginhibiting visible growth. To determine whether the observedgrowth inhibition by SerB2 specific inhibitors was bactericidalor bacteriostatic, mycobacterial cultures were grown till anA600 nm of 0.1 and subsequently exposed to drugs at 10� MIC99concentration. For bacterial enumeration, samples were with-drawn at designated time points, and 100 �l of 10-fold serialdilutions were plated on MB-7H11 plates and incubated at37 °C for 3 weeks. For intracellular killing experiments, THP-1cells were seeded at a density of 5 � 105/well in a 24-well plate(Nunc) in RPMI medium supplemented with 10% FBS. Macro-phages were infected with bacteria at a multiplicity of infectionof 1:10 in triplicate wells for 4 h, washed with antibiotic freeRPMI medium, and overlaid with RPMI medium containing200 �g/ml amikacin for 2 h. Subsequently, macrophages werewashed twice with antibiotic free RPMI medium and overlaidwith RPMI medium supplemented with 10% FBS. After 24 hpostinfection, cells were overlaid with RPMI medium contain-ing drugs at either 4� or 16� MIC99 concentration for 4 days.For bacterial enumeration, macrophages were lysed by theaddition of 1� PBS, 0.1% Triton X-100, and the number ofintracellular bacteria was determined by plating 100 �l of10-fold serial dilutions on MB-7H11 plates.

Molecular Docking Studies—All programs used for molecu-lar docking studies were from Suite 2012 from Schrodinger.SerB1 and SerB2 models and HPSP crystal structure (ProteinData Bank code 1L8L) were optimized for docking studies withprotein preparation wizard. These structures were finally min-imized by converging it to a root mean square deviation(r.m.s.d.) tolerance of 0.3 Å using the OPLS 2005 force field.Three-dimensional ligand structures were generated using Lig-Prep module. Ligands were fitted to SerB1 and SerB2 modelsand HPSP crystal structure using the GLIDE extra precision

method (35). Images were obtained and visualized through theMaestro interface.

RESULTS

Bioinformatic and Homology Modeling Studies—The enzymesinvolved in L-serine biosynthesis are highly conserved in vari-ous mycobacterial species (Fig. 1A and Table 2). M. tuberculosisPGDH and PSAT enzymes catalyzing the first two steps ofL-serine biosynthesis have been biochemically well character-ized (13–15). Phylogenetic analysis among PSP proteins fromvarious organisms revealed that PSP proteins from actinobac-ter and helicobacter species were more similar to each other incomparison with enterobacterial and human PSP enzymes (Fig.1B). As shown in Fig. 1B, both SerB1 and SerB2 proteins weredistantly related to each other and HPSP enzyme, with whichthey shared an overall sequence similarity of 18 and 27%,respectively. Multiple sequence alignment among PSP enzymesfrom various microorganisms revealed that these proteinsshare an overall homology of 15% among themselves, and bothSerB1 and SerB2 possessed HAD specific motifs (motifs I–III;Fig. 2A). In these enzymes, motif I DXDX(T/V) is responsiblefor phosphoprotein intermediate formation with the substratealong with Mg2� ion binding, whereas motif (II) (S/T)XX andmotif III KX18 –30(G/S)(D/S)XXX(D/N) are involved in coordi-nation of the Mg2� ion and phosphoprotein intermediate(17–20).

The best templates for homology modeling for SerB1 andSerB2 proteins were identified by PSI-BLAST analysis usingProtein Data Bank. The closest homolog for SerB1 protein wasPSP enzyme from Bordetella pertussis (Protein Data Bank code3FVV) with 34% sequence identity, 41% query coverage (con-sisting only the HAD superfamily domain region), and anE-value of 7 e�16. We were unable to build full-length structureof SerB1 because homologous template with high sequencecoverage was not available. The closest homolog for SerB2 pro-tein was SerB protein from Mycobacterium avium (ProteinData Bank code 3P96) with 84% sequence identity, 99% querycoverage, and an E value of 0.0. The superimpositions of SerB1-and SerB2-modeled proteins over 3FVV and 3P96, respectively,resulted in backbone r.m.s.d. of 0.449 and 0.451 Å, respectively.The superimpositions of SerB1 and SerB2 models over HPSPcrystal structure resulted in r.m.s.d. of 6.216 and 2.565 Å,respectively (Fig. 2B). These computational studies predicted11 �-helices and 7 �-sheets for SerB1 domain region and 16�-helices and 15 �-sheets for SerB2 protein (Fig. 2B). The Ram-achandran plot, a measure of the stereochemical parameters ofmodeled structures, revealed that 87 and 92% of the amino acidresidues were in the favored region for SerB1 and SerB2 builtmodels, respectively (data not shown). In addition, Verify_3Dand Errat scores for SerB1 model were 81.02 and 85.85%,respectively, whereas these values were 96.21 and 94.06%,respectively, for SerB2 model. Overall, these results suggestedthat both models were of acceptable quality and suitable formolecular docking studies. Molecular docking of O-phospho-L-serine using Discovery Studios 2.5 revealed that amino acidresidues Asp-127 (motif I), Thr-234 (motif II), Lys-279, andAsp-302 (motif III) in the case of SerB1 protein; residues Asp-185 (motif I), Ser-273 (motif II), Lys-318, and Asp-341 (motif



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

III) in the case of SerB2 protein; and residues Asp-20 (motif I),Ser-109 (motif II), Lys-158, and Asp-179 (motif III) in the caseof HPSP are part of their respective substrate binding pockets(Fig. 2C). In addition to these above mentioned critical inter-acting residues, docking studies also revealed that Val-186and Ser-188 of SerB2 enzyme might also interact with O-phospho-L-serine.

Expression and Purification of M. tuberculosis PhosphoserinePhosphatases—To biochemically characterize PSP enzymes,pET28b-serB1 and pET28b-serB2 were transformed into BL-21(�DE3, plysS), and expression of recombinant protein in trans-formants was induced by addition of 1 mM IPTG. As shown inFig. 3A, His6-SerB2 was expressed at high levels in soluble frac-tion and purified at levels �95% using nickel-nitrilotriaceticacid chromatography with a total yield of 8 mg/liter. However,we observed that expression of His6-SerB1 in E. coli transfor-mants was very minimal and only detectable by immunoblotanalysis using �-His6 antibody. As expected, both His6-SerB1and His6-SerB2 migrated at their predicted molecular masses of42.0 and 45.0 kDa, respectively, on 10% SDS-PAGE (data notshown). To achieve better expression, serB1 was also cloned in

other prokaryotic expression vectors such as pMALc2x orpGEX4T-1. However, as observed in the case of His6-SerB1,expression of both MBP-SerB1 and GST-SerB1 in IPTG-in-duced transformants was very minimal and nondetectable inCoomassie Brilliant Blue-stained 10% SDS-PAGE (data notshown).

Biochemical Characterization of SerB2 Enzyme—To deter-mine kinetic parameters for SerB2 enzyme, steady state kineticswas performed by varying O-phospho-L-serine concentrationin the presence of 1 �M His6-SerB2 enzyme. Conversion ofO-phospho-L-serine to L-serine by SerB2 followed Michaelis-Menten kinetics with a Km of 92.68 �M and kcat of 8.83 min�1

(Fig. 3B). The catalytic efficient constant (kcat/Km) for SerB2enzyme was 0.0952 min�1 �M�1. These kinetic constants wereobserved to be lower in comparison with those obtained for PSPenzymes characterized from either Hydrogenobacter thermo-philes (Km of 1.6 mM), P. gingivalis (Km of 2.0 mM and 2.6 mM forphosphoserine peptides), or Pseudomonas aeruginosa (Km of207 �M) (26, 36, 37). We also observed that maximal SerB2activity was observed in the initial 5 min and that inclusion of0.01% Triton X-100 enhanced SerB2 activity by 15–20% (Fig.

FIGURE 1. A, schematic representation of L-serine biosynthetic pathway in M. tuberculosis. The gene identifier and genetic essentiality of enzymes involved inL-serine biosynthesis in M. tuberculosis have been mentioned. PGM, phosphoglyceratemutase; PGDH, phosphoglycerate dehydrogenase. B, phylogeneticanalysis of PSP proteins. The evolutionary history was inferred using the neighbor joining method using MEGA5 software, and distance was computed usingthe POISSON correction method and is shown as the units of number of amino acid substitutions per site. The branches are labeled with the protein accessionnumber along with organism name. The bootstrap consensus tree inferred from 100 replicates is taken to represent the evolutionary history of the taxa.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3C). As expected, SerB2 displayed substrate preference for O-phospho-L-serine over O-phospho-L-threonine (Fig. 3D). Wedid not observe any Pi release even at 80 �M of O-phospho-L-threonine (Fig. 3D). The amino acid residues of SerB2 enzymepredicted to interact with O-phospho-L-serine were mutated,cloned into either pET28b or pMALc2x, and purified as His6-tagged or MBP-tagged proteins. We observed that mutation ofaspartic acid at position 185 and lysine at position 318 com-pletely abolished SerB2 activity (Fig. 3E). As shown in Fig. 3E,mutation of aspartic acid at position 341, serine at position 273,and valine at position 186 abolished SerB2 activity by 80, 60,and 50%, respectively, as compared with wild type proteinactivity.

To examine the changes in secondary structural content(such as �-helix, � sheet, or random coil) of SerB2 upon aminoacid changes, both wild type and mutant proteins were analyzedusing far-UV CD spectroscopy between wavelength range of195 and 250 nm. As shown in Fig. 3F, SerB2 showed character-istic spectra of a protein consisting of a mixture of secondarystructure elements such as � helix and � sheet. We observedthat mutant proteins S273A and D341G showed similar spectraas that of the wild type protein. However, D185G and K318Eproteins showed decreased and increased secondary structuralcontent, respectively, in comparison with that of the wild typeprotein (Fig. 3F). These data suggest that reduced enzymaticactivity of S273A and D341G proteins was not due to changes intheir secondary structures but could be due to their alteredinteraction with O-phospho-L-serine as predicted by moleculardocking. However, a decrease in activity of both D185G andK318E proteins could be due to the combined effect of bothaltered structure and interaction with O-phospho-L-serine.

Optimization of Assay Conditions for HTS—To optimize theassay conditions for HTS to identify novel SerB2 inhibitors, theinfluence of various parameters such as cations and buffer pHon its activity was evaluated in saturating O-phospho-L-serineconcentration. We observed that inclusion of divalent ions suchas Mg2� and Mn2� significantly enhanced SerB2 activity incomparison with inclusion of Ca2�, Zn2�, and Fe3� in the assaybuffer. The optimum SerB2 activity was observed upon inclu-sion of 5 mM Mg2� or Mn2� ions in the assay buffer (Fig. 4A). Inour assay conditions, increasing Fe3� ion concentration in thebuffer did not affect SerB2 activity, whereas significant reduc-tion in activity was observed upon inclusion of Zn2� ion inassay buffer in a dose-dependent manner (Fig. 4A). We alsoobserved a slight decrease in SerB2 dephosphorylating activityupon increasing concentration of Ca2� ion in the assay buffer(Fig. 4A). To determine pH optima for SerB2 enzyme, assayswere performed in buffers of pH ranging from 6.0 –9.0 and opti-mum activity was achieved at pH 7.5 (Fig. 4B). Therefore, theoptimum conditions for HTS assays were 100 mM Tris, pH 7.5,5 mM MgCl2, 5 mM DTT, and 0.01% Triton X-100. For furtheroptimization and validation of HTS assays, SerB2 activity wasdetermined in the presence of increasing concentration of knownPSP inhibitors such as DL-AP3 and sodium orthovandate (26, 33,34). We observed that both DL-AP3 and sodium orthovandateinhibited SerB2 activity by 50% at the concentration of 458 and 670�M, respectively (data not shown). The IC50 values obtained forT

AB

LE2

The

locu

so

fgen

esfo

ren

zym

esin

volv

edin

L-se

rin

eb

iosy

nth

esis

inva

rio

us

myc

ob

acte

rial

gen

om

es.

Myc

obac

teri

umsp

p.PG

MPG

DH

PSA

TPS

PG

pm1/

Gpm

AG

pm2

SerA

/Ser

A1

SerA

2Se

rCSe

rB1

SerB

2

M.t

uber

culo

sisRv

0489

Rv32

14Rv

2996

cRv

0728

cRv

0884

cRv

0505

cRv

3042

cM

.bov

isst

rain

BCG

BCG

_053

0BC

G_3

241,

BCG

_333

4BC

G_3

017c

BCG

_077

8cBC

G_0

936c

BCG

_054

8cBC

G_3

066c

M.m

arin

umM

MA

R_08

14,M

MA

R_37

47M

MA

R_13

43M

MA

R_17

15M

MA

R_10

65M

MA

R_46

48M

MA

R_08

33M

MA

R_16

52M

.lep

rae

ML2

441c

ML1

692

ML1

837

(Pse

udog

ene)

ML2

136

ML2

424

ML1

727c

M.u

lcer

ans

MU

L_36

91M

UL_

2536

MU

L_19

52M

UL_

0823

MU

L_02

66M

UL_

4577

MU

L_18

88M

.afr

ican

umM

AF_

0493

0M

AF_

3223

0M

AF_

3001

0M

AF_

0738

0M

AF_

0893

0M

AF_

0512

0M

AF_

3049

0M

.sm

egm

atis

MSM

EG_0

935,

MSM

EI_0

912

MSM

EG_1

926,

MSM

EI_1

885

MSM

EG_2

378,

MSM

EI_2

318

MSM

EG_5

684,

MSM

EI_5

534

MSM

EG_0

949

MSM

EG_2

321,

MSM

EG_1

041,

MSM

EI_1

012,

MSM

EI_2

263

M.x

enop

iM

XEN

_048

33,M

XEN

_111

51,

MX

EN_0

5750

MX

EN_1

3801

MX

EN_0

3119

,M

XEN

_089

29M

XEN

_189

04M

XEN

_143

11M

XEN

_165

62

M.c

anet

tiiM

CA

N_0

4891

MC

AN

_323

11M

CA

N_3

0181

MC

AN

_073

31M

CA

N_0

8851

MC

AN

_306

71M

.par

atub

ercu

losis

MA

P_39

81M

AP_

3033

cM

AP_

0823

MA

P_39

97c

MA

P_30

90c

M.c

olom

bien

seM

CO

L_V

2009

05,M

CO

L_V

2040

95,

MC

OL_

V21

1600

MC

OL_

V22

1796

MC

OL_

V21

1770

,M

CO

L_V

2166

14M

CO

L_V

2255

17M

CO

L_V

2221

16

M.i

ntra

cellu

lare

OC

U_4

5260

,OC

U_2

2160

�Ser

A4

OC

U_0

3090

OC

U_0

8880

OC

U_3

7410

M.a

bsce

ssus

MA

B_16

23,M

AB_

1905

,M

AB_

0133

,MA

B_39

36c,

MA

B_21

09,M

AB_

1419

,M

AB_

0254

c

MA

B_33

04c

MA

B_09

28c

MA

B_33

88c



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

these two inhibitors for SerB2 enzyme were comparable withthose obtained for other PSP enzymes (26, 33, 34).

HTS Assays and Identification of Inhibitors for SerB2Enzyme—In our preliminary screening, we determined the per-centages of inhibition for each compound (belonging to eitherNCI diversity set or mechanistic set or natural product set) at100 �M concentration, and the compounds that inhibited SerB2activity by at least 50% were considered to be primary hits. Inour initial assays, the majority of the compounds were inactive,whereas 21 compounds inhibited SerB2 activity by �50% invitro (Fig. 4, C and D). We observed that SerB2 enzymatic activ-ity was inhibited by �80, 60 – 80, and 40 – 60% in the presenceof 5 compounds, 4 compounds, and 12 compounds, respec-tively (Fig. 4, C and D). However, in our repeat in vitro assays,only 10 of these 21 compounds inhibited SerB2 enzyme by atleast 50% at 100 �M concentration. These 10 active scaffoldswere structurally different from known PSP inhibitors (Fig. 5).

As expected, these primary hits inhibited SerB2 activity in adose-dependent manner with at least 50% inhibition observedat the highest concentration. As shown in Table 3, CID 4 andCID 8 were most potent in our in vitro SerB2 inhibition assayswith an IC50 value of 4.12 � 1.2 and 3.89 � 1.2 �M, respectively.The IC50 values for remaining primary hits varied between 10and 40 �M with CID 2 showing least inhibitory activity in our invitro enzymatic assays (Table 3). To determine specificity ofthese primary hits, we next evaluated their ability to inhibiteither M. tuberculosis PstP (Rv 0018c) or alkaline phosphataseenzyme. Both PstP and alkaline phosphatase were suitable con-trol for these assays because they belong to different phospha-tase families. Alkaline phosphatase is a hydrolase responsiblefor removing phosphate group from various molecules includ-ing nucleotides, proteins, and alkaloids. PstP belongs to PP2Cphosphatase family that strictly requires Mn2� ion for binding.We observed that CID 1, CID 5, CID 6, CID 7, CID 8, CID 9, and

FIGURE 2. A, multiple sequence alignment among PSP proteins. Multiple sequence alignment among PSP proteins from various microorganisms was per-formed using Clustal Omega software. Highly conserved residues among PSP enzymes from various bacterial species are shaded in black, whereas light grayshading denotes a level of conservation among these proteins. The accession numbers for these proteins are EFC04663, Staphylococcus aureus; YP_177732,M. tuberculosis SerB1; EDN61796, Saccharomyces cerevisiae; AAA97284, E. coli; ZP_18026828, Vibrio cholerae; YP_005780390, Helicobacter pylori; NP_217558,M. tuberculosis SerB2; YP_002342576, Neisseria meningitidis; YP_004510496, P. gingivalis; and NP_253647, P. aeruginosa. B and C, superimposition of modeledstructures of SerB1, SerB2, and HPSP (Protein Data Bank code 1L8L). B, the built SerB1 model (green), SerB2 model (pink), and HPSP (yellow) were superimposedand visualized using PyMOL. C, the binding site for O-phospho-L-serine in superimposed models is magnified. The critical amino acid residues of SerB1 (green),SerB2 (pink), and HPSP (yellow) predicted to interact with O-phospho-L-serine have been highlighted.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

CID 10 failed to significantly inhibit PstP enzyme even at 100�M concentration, whereas CID 2, CID 3, and CID 4 displayedequal inhibitory activity (�80 –90%) for both SerB2 and PstPenzymes, in vitro (Fig. 6A). We observed that CID 1, CID 5, CID6, CID 7, CID 8, CID 9, and CID 10 inhibited enzymatic activityof SerB2 and PstP enzymes by �70 and 0 –20%, respectively.We also observed that none of these inhibitors were able toinhibit alkaline phosphatase activity even at 100 �M concentra-tion (Fig. 6A). We next performed SPR experiments to confirmbinding of CID 1, CID 7, CID 8, CID 9, and CID 10 with SerB2enzyme. The protein immobilization over the flow cell variedbetween 8,000 and 10,000 RU, and binding responses wererecorded at 1 mM drug concentration. At this tested concentra-tion, all the compounds showed increase in RU indicative ofinteraction with SerB2; however, the increase in RU varied fordifferent compounds (Fig. 6B). We observed that CID 1, CID 7,and CID 8 displayed higher binding to SerB2 enzyme in com-parison with CID 9 and CID 10 (Fig. 6B).

Cell Cytotoxicity, Antimycobacterial Activity, and HPSP Inhi-bition Studies—In our lactate dehydrogenase-based cell viabil-ity assays, we observed that primary hits CID 1, CID 5, CID 6,CID 7, CID 8, and CID 10 that specifically inhibited SerB2enzyme in vitro were noncytotoxic to THP-1 macrophageseven at 50 �M concentration (Table 3). Scaffolds such as CID 2,CID 3, and CID 4 that inhibited both PstP and SerB2 activity by

�90% were highly cytotoxic to THP-1 macrophages with TC50values of 5, 5, and 2.5 �M, respectively (Table 3). A similar pat-tern of cell cytotoxicity for these scaffolds was also observed inVero cell lines (Table 3). Next, these inhibitors were also eval-uated for their ability to inhibit mycobacterial growth in vitro.In our MIC99 determination assays, most of these compoundspossessed modest activity (ranging from 2 to 25 �M) againstboth M. tuberculosis H37Rv and M. bovis BCG Danish strains(Table 3). As shown in Table 3, CID 5, CID 6, CID 8, and CID 10were less active against mycobacteria in vitro in our whole cell-based assays, which might be attributed to lower intracellularconcentration of drugs because of (i) their poor penetration, (ii)their effluxing out by various pumps, or (iii) their modificationby intracellular enzymes. The most potent inhibitors in ourwhole cell-based assays were CID 1 and CID 7, both of whichdisplayed MIC99 values of 2 �M against both M. tuberculosisand M. bovis BCG in vitro.

Next we performed kill kinetics assays in vitro by exposingactively growing mycobacteria to 10� MIC99 concentration ofeither CID 1 or CID 7 or isoniazid. As shown in Fig. 6C, bothCID 1 and CID 7 were bactericidal in their mode of killing with�10-fold killing observed after exposure of mycobacteria for 3days. In our time kill experiments, 7 days of exposure to CID 1and CID 7 led to approximately �80- and 20-fold killing,respectively (Fig. 6C). As expected, no significant growth inhi-

FIGURE 3. Protein expression, purification, and biochemical characterization. A, the expression of recombinant proteins was analyzed in IPTG-inducedBL-21 (�DE3, plysS) cells transformed with either pET28b (lane 1), pET28b-serB1 (lane 2), or pET28b-serB2 (lane 3). Purified fractions, purified elution fractions ofHis6-SerB2 using nickel-nitrilotriacetic acid chromatography. B, Michaelis-Menten plot for SerB2 enzyme activity. The conversion of O-phospho-L-serine toL-serine was monitored at 630 nm using the Quantichrome phosphate assay kit. The values represent the means � S.E. of initial velocities of �M Pi release/minobtained from three independent experiments. C, time course analysis of SerB2 enzymatic activity in the presence and absence of 0.01% Triton X-100. Thevalues depicted are means � S.E. of �M Pi release obtained from three independent experiments. D, substrate specificity of SerB2 enzyme. To determinesubstrate specificity, enzyme assays were performed using varying concentration of either O-phospho-L-serine or O-phospho-L-threonine or O-phospho-DL-serine. Pi release was measured in enzyme reactions, and data depicted are the means � S.E. obtained from three independent experiments. E, phosphoserinephosphatase activity of wild type and mutant SerB2 proteins. Pi release assays were performed using either 1 �M of wild type or mutant SerB2 enzyme in thepresence of 100 �M of O-phospho-L-serine. Pi release was measured in enzyme assays, and the data are depicted as the means � S.E. obtained from threeindependent experiments. F, secondary structural analysis of wild type and mutant SerB2 proteins using far-UV CD spectroscopy. The spectra was recordedusing 10 �M protein in the wavelength range between 195 and 250 nm. Both wild type and mutant proteins show CD spectra characteristic of a mixture of�-helix and �-sheet containing protein. M.R.E., mean residue ellipticity.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

bition was observed in the presence of DMSO during the courseof experiment (Fig. 6C). Because M. tuberculosis is an intracel-lular pathogen, we next evaluated the ability of both CID 1 andCID 7 to kill bacteria in THP-1 macrophages at either 4� or16� MIC99 concentration. In our macrophage experiments,both CID 1 and CID 7 were able to arrest M. bovis BCG repli-cation in a dose-dependent manner. As shown in Fig. 6D, at 4days after drug exposure, �100- and 50-fold intracellular kill-

ing was observed in the presence of 16� MIC99 concentrationof CID 7 and CID 1, respectively.

Cross-reactivity of these inhibitors with the human homo-log would be a major concern in further validation of SerB2as a drug target. Therefore, we compared the ability of theseactive and noncytotoxic primary hits to inhibit HPSPenzyme in vitro at 100 �M concentration. As shown in Fig.7A, both CID 7 and CID 10 were nonselective PSP inhibitors,

FIGURE 4. A, effect of ions on SerB2 enzymatic activity. SerB2 enzymatic assays were performed in the presence of varying concentration of either CaCl2, MgCl2,ZnCl2, FeCl3, MnCl2 using 100 �M of O-phospho-L-serine. The data depicted in each bar are the means � S.E. values obtained from two independent experi-ments. B, effect of buffer pH on SerB2 enzyme activity. SerB2 activity assays were performed in buffers of pH ranging from 6.0 to 9.0 in the presence of 100 �M

of O-phospho-L-serine. Pi release in enzymatic reactions was determined, and the data depicted are the means � S.E. values obtained from three independentexperiments. C and D, preliminary inhibition studies of SerB2 activity using the National Cancer Institute Developmental Therapeutic Program library. Theentire compounds in the National Cancer Institute Developmental Therapeutic Program library belonging to either NCI diversity set (C) or mechanistic andnatural product set (D) were evaluated for their ability to inhibit SerB2 enzyme at 100 �M concentration. The data depicted are averages of percentages ofinhibition obtained from two independent reactions.

FIGURE 5. Chemical structures of novel phosphoserine phosphatase inhibitors and novobiocin used in the study.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

inhibiting both HPSP and SerB2 enzymes to a similar extentof 70% at 100 �M concentration. However, the rest of thecompounds CID 1, CID 5, CID 8, and CID 9 were highlyspecific in their ability to inhibit SerB2 enzyme in compari-

son with HPSP enzyme even at 100 �M concentration (Fig.7A). These results suggest that despite the presence of ahuman homolog, subtle differences exist between secondarystructures of these two enzymes, which could be explored

TABLE 3Chemical properties and activities of SerB2 inhibitors identified in the study

CID No.NSC

numberMolecular

weight IC50 MIC99 TC50 (THP-1/Vero)T-i

valuesGLIDE

binding energy

�M �M kcal/mol1 NSC227186 697 16.84 � 1.6 2.34 � 0.4 �50 �21 �6.02 NSC104129 455 37.57 � 1.8 9.37 � 1.8 5 0.53 �4.93 NSC672904 354 11.68 � 1.4 25 5 0.2 �4.54 NSC693172 414 4.12 � 1.2 12.5 2.5 0.2 NDa

5 NSC71948 1171 25.73 � 1.4 50 � 14.3 �50 �1 �5.46 NSC204665 376 25.37 � 2.4 �200 �50 ND ND7 NSC93739 338 31.49 � 1.4 2.34 � 0.4 �50 �21 �5.148 NSC76027 1058 3.89 � 1.2 150 � 28.8 �50 �0.3 �4.449 NSC305798 404.3 33.0 � 1.2 12.5 3.12 0.25 �5.010 NSC165701 295.4 29.7 � 2.8 200 �50 �0.4 �4.0

a ND, not determined.

FIGURE 6. A, SerB2, PstP, and alkaline phosphatase inhibition by primary hits. SerB2, PstP, and alkaline phosphatase activity assays in the presence of primaryhits were performed as described under “Experimental Procedures.” The values depicted in this panel are the means � S.E. from three independent experi-ments. B, SPR experiments to confirm binding of inhibitors to SerB2 enzyme. The binding of CID 1, CID 7, CID 8, CID 9, and CID 10 was evaluated by SPR. Theexperiment was done in duplicate, and the data shown are representative of two separate experiments. The inset shows the sensorgram obtained uponpassing various inhibitors over the SerB2 immobilized surface. C, time kill curves of CID 1, CID 7, and INH against mycobacteria in liquid cultures. Earlylogarithmic cultures of M. bovis BCG were exposed to either CID 1, CID 7, or isoniazid at 10� MIC99 concentrations, and bacterial enumeration was performedby plating 100 �l of 10-fold serial dilutions on MB-7H11 plates at days 3 and 7 post-exposure. D, intracellular activity of CID 1 and CID 7 against bacteria ininfected macrophages. Intracellular bacterial numbers in THP-1 macrophages after 4 days post-exposure to either CID 1 or CID 7 at 4� or 16� MIC99concentration were determined by lysing macrophages in 1� PBS, 0.1% Triton X-100 and plating 100 �l of 10-fold serial dilutions on MB-7H11 plates.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

further for identification of inhibitors with more potencyand specificity toward SerB2 enzyme.

Molecular Docking Studies of Scaffolds on M. tuberculosisSerB2 Model Protein and HPSP Enzyme—Molecular dockingstudies were performed for CID 1 (clorobiocin), novobiocin,CID 5, CID 7 (rosaniline), CID 8, CID 9, and CID 10 using SerB2built model and HPSP protein as described under “Experimen-tal Procedures.” The binding free energies for interaction ofthese inhibitors with SerB2 protein ranged from �4.44 to�5.14 kcal/mol, which is comparable with the binding energyfor interaction of O-phospho-L-serine with SerB2 (�6.89 kcal/mol; Table 3). Because amino coumarins are clinically utilizedantibiotics with tolerable toxicity, we were particularlyintrigued by the ability of clorobiocin to inhibit SerB2 enzymein vitro. This class of compounds that includes novobiocin, clo-robiocin, and coumermycin is known to inhibit DNA topo-isomerase and heat shock proteins by binding to their nucleo-tide binding pockets (38, 39). Interestingly, novobiocin, acompound structurally similar to clorobiocin, did not inhibit

SerB2 activity even at 10-fold higher concentration in our invitro assays (Fig. 7B). Molecular docking of a SerB2 built modelwith clorobiocin and novobiocin predicted that the differencein ability of these structural analogs to inhibit SerB2 enzymecould be attributed to (i) interaction of the Asp-341 residue ofSerB2 enzyme with clorobiocin via hydrogen bond formationand (ii) better fit of the pyrrole ring of clorobiocin in compari-son with the substituted amino group of novobiocin in the sub-strate binding pocket. In addition, Lys-361 and Arg-365 resi-dues of SerB2 enzyme might interact with clorobiocin viahydrogen bond formation and Asp-187 and Glu-197 residueswere observed to be closely associated (2.2 and 2.8 Å, respec-tively) with clorobiocin (Fig. 7, C and D). In concordance withour in vitro activity results, molecular docking of clorobiocin inHPSP revealed that Asp-179 (identical to Asp-341 in SerB2) isinteracting with O-phospho-L-serine but not with clorobiocin(Figs. 2C and 7E). In addition, further analysis revealed that theconserved FDVDST motif forms a different secondary struc-ture (right-handed helix in case of HPSP) as compared with

FIGURE 7. A, HPSP and SerB2 inhibition studies in the presence of CID 1, CID 5, CID 7, CID 8, CID 9, and CID 10. HPSP and SerB2 inhibition assays were performedas described under “Experimental Procedures.” The data depicted in this panel are mean � S.E. for percentages of inhibition obtained from three independentexperiments. B, inhibition studies of SerB2 enzyme in the presence of novobiocin and clorobiocin. SerB2 enzymatic assays were performed in the absence andpresence of either novobiocin or clorobiocin. The data depicted in each bar represent the means � S.E. for percentages of inhibition obtained from threeindependent experiments. C and D, molecular docking of clorobiocin and novobiocin in the SerB2 modeled structure. Docking of clorobiocin (C) and novo-biocin (D) in the modeled structure of SerB2 protein was performed as described under “Experimental Procedures.” The H-bond interactions between aminoacid residues of SerB2 enzyme and aminocoumarins have been highlighted with a yellow dotted line. The pyrrole ring of clorobiocin has been highlighted bya red circle. Identical residues in both panels have been highlighted. E, molecular docking of HPSP with clorobiocin. Molecular docking of HPSP with clorobiocinwas performed as described under “Experimental Procedures.” The conserved motif FDVDST in HPSP blocking access of clorobiocin to substrate bindingpocket is highlighted in black.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

SerB2 protein, which might block the accessibility of clorobio-cin to the substrate binding pocket of HPSP (Fig. 7E).

Molecular docking studies revealed that CID 5, CID 7, CID 8,CID 9, and CID 10 also interact with critical Asp-341 and Asp-187 residues of SerB2 protein (Fig. 8, A–E). In addition to theseinteractions, CID 5 is also interacting with Val-186 and Glu-197via hydrogen bond and salt bridge interactions. Moleculardocking studies revealed that CID 7 might also interact withGlu-197 and Val-186 residues of SerB2 protein, and there mightbe some electrostatic interactions with Glu-214, because itresides in close proximity of 3.8 Å (Fig. 8B). As shown in Fig. 8C,we observed that CID 8 might also interact with the Lys-361residue of SerB2 enzyme through -cation interaction, whereasCID 9 might interact with Glu-194, Ser-188, and Val-186 resi-dues of SerB2 protein through hydrogen bond formation (Fig.8D). As shown in Fig. 8E, CID 10 might also be possibly inter-acting with Glu-197 and Glu-214 residues through electrostaticinteraction because these two residues seem to be in close con-tact (2.23 and 1.99 Å, respectively). Despite the lack of struc-tural similarities among these primary hits, these scaffolds pos-sess a substructure that fits well in the SerB2 modeled protein.Molecular docking of CID 7 with HPSP crystal structurerevealed that Asp-20, Asp-22, Asp-179, and Ala-71 might inter-act with rosaniline via hydrogen bond formation, and binding

free energy for this interaction was �5.7 kcal/mol (Fig. 9B). Inconcordance with our in vitro activity assays, we observed thatCID 5, CID 8, and CID 9 are not interacting with known HPSPcritical residues, thereby explaining their inability to inhibitHPSP enzyme (Fig. 9, A, C, and D). Molecular docking of CID 10with HPSP revealed that it might interact with Val-56 and Thr-182 residues via hydrogen bond formation and with Lys-158through cation- interaction (Fig. 9E). Even though Val-56 andThr-182 of HPSP are not conserved with SerB2 enzyme, Lys-158 is a conserved active site residue between SerB2 and HPSP(as per pair wise sequence alignment studies).

Validation of SerB2 Small Molecule Interactions Predicted byMolecular Docking Studies—To validate SerB2-small moleculeinteractions predicted by molecular docking, Lys-361, Arg-365,Glu-214, and Asp-187 were mutated to alanine residue asdescribed under “Experimental Procedures.” As shown in Fig.10A, mutation of Asp-187 and Glu-214 reduced SerB2 activityby 50% as compared with wild type protein, whereas the enzy-matic activity of K361A-SerB2, R365A-SerB2, and E197A-SerB2 were almost similar to that of wild type protein. Far-UVCD studies revealed that mutation of these amino acids did notsignificantly alter the folded state of these mutant proteinsexcept for E197A-SerB2 where we observed increase in second-ary structure content (Fig. 10B). These results suggest that

FIGURE 8. Molecular docking of primary hits CID 5, CID 7, CID 8, CID 9, and CID 10 in SerB2 model. Molecular docking of SerB2 built model with CID 5 (A),CID 7 (B), CID 8 (C), CID 9 (D), and CID 10 (E) was performed as described under “Experimental Procedures.” The H-bond interactions between amino acidresidues of SerB2 enzyme and various scaffolds have been highlighted with yellow dotted lines.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

reduction in activity of D187A-SerB2 and E214A-SerB2 couldbe due to their altered interaction with O-phospho-L-serine(Fig. 10, A and B). In our enzymatic assays, we observed thatmutation of Asp-187 and Asp-341 prevented efficient bindingof CID 1, CID 5, CID 7, CID 8, CID 9, and CID 10 to the bindingpocket of these mutant proteins (Fig. 10, C–H). We alsoobserved that mutation of Glu-197 to alanine residue reducedthe ability of CID 1, CID 5, and CID 7 to inhibit dephosphoryl-ation activity of SerB2 enzyme. As shown in Fig. 10 (E and F),Val-191 and Lys-361 were important for interaction of SerB2with CID 7 and CID 8, respectively. In concordance with ourdocking studies, we report that Val-186 is important for inter-action of SerB2 with CID 9, whereas Glu-214 is critical for inter-action of CID 7 and CID 10 with SerB2 enzyme (Fig. 10, E, G,and H).

DISCUSSION

The development of novel inhibitors for essential and con-served M. tuberculosis pathways is one plausible solution toshorten duration of TB chemotherapy and eradicate drug-re-sistant TB. The advent of new computational methods, combi-natorial synthetic chemistry approach and whole cell- and tar-get-based HTS assays, have led to identification of severalantitubercular scaffolds that are currently being evaluated indifferent stages of clinical trial. Numerous studies have shownthat M. tuberculosis strains deficient in enzymes involved invarious amino acid biosynthetic pathways are compromised intheir ability to infect mice in comparison with the ability of

parental strain (40 – 42). In addition, several of these enzymesinvolved in amino acid biosynthesis are being currentlyexplored for development of more potent antitubercular scaf-folds (43– 48). L-Serine biosynthesis is an attractive and unex-plored anti-microbial drug target because L-serine is not onlyinvolved in protein synthesis but also acts as precursor for var-ious cellular metabolites (15, 49 –54). The enzymes involved inL-serine biosynthetic pathway are widely conserved across var-ious mycobacterial species including Mycobacterium leprae, anorganism that has undergone massive gene decay, thereby sug-gesting that these enzymes are indispensable and are essentialfor survival of bacteria in the host.

This is the first study where a detailed biochemical charac-terization of PSP homolog from M. tuberculosis has been per-formed. Phylogenetic and sequence alignment analysis revealedthat both SerB1 and SerB2 proteins are distantly related to eachother and share an identity of 18 and 27%, respectively, withHPSP enzyme. Multiple sequence alignment analysis revealedthat HAD specific motifs responsible for Mg2� ion binding,phosphoprotein formation, and stabilization are present inboth SerB1 and SerB2. Despite several attempts, we wereunable to express SerB1 in detectable amounts as either His6-tagged, GST-tagged, or MBP-tagged proteins. We observedthat SerB2 had a substrate preference for O-phospho-L-serineover O-phospho-L-threonine and displayed lower kinetic con-stants in comparison with PSP enzymes previously character-ized from H. thermophiles, P. gingivalis, and P. aeruginosa (26,

FIGURE 9. Molecular docking of primary hits CID 5, CID 7, CID 8, CID 9, and CID 10 in HPSP protein. Molecular docking of HPSP with CID 5 (A), CID 7 (B), CID8 (C), CID 9 (D), and CID 10 (E) was performed as described under “Experimental Procedures.” We did not observe any significant interactions between CID 5, CID8, or CID 9 and HPSP enzyme. The H-bond interactions between CID 7, CID 10, and amino acid residues of HPSP enzyme have been highlighted with yellowdotted lines.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

36, 37). The optimum SerB2 activity was observed in the pHrange of 7.0 – 8.5 and in the presence of 5 mM Mg2� or Mn2�

ions. The activity of SerB2 enzyme was unaltered by the inclu-sion of Fe3� ion in the assay conditions. However, inclusion ofZn2� ion in the assay buffer abolished SerB2 enzymatic activityin a dose-dependent manner that might be attributed to disrup-tion of the SerB2 secondary structure, thereby preventing bind-ing of O-phospho-L-serine to its active site. We also observedreduction in SerB2 activity upon inclusion of Ca2� ion in theassay buffer, which might be attributed to disruption of nucleo-philic attack by the conserved Asp residue (motif I) on the phos-phate group of O-phospho-L-serine as reported earlier in thecase of HPSP (22). The activity of SerB2 enzyme was enhancedby 15–20% by inclusion of nonionic detergent in assay condi-tions that might be attributed to stabilization of SerB2 second-ary structure. Molecular docking and in vitro enzymatic assaysusing purified mutant proteins revealed that amino acid resi-

dues Asp-185, Asp-187, Val-186, Ser-273, Lys-318, Glu-214,and Asp-341 are critical for SerB2 dephosphorylation activity.

Despite the importance for PSP enzymes in biosynthesis ofL-serine, these enzymes have not been extensively studied as anantimicrobial drug target except for a report where dihydro-quinolinone derivatives were shown to inhibit SerB enzymesfrom P. gingivalis (55). In the present study, HTS was per-formed using 2300 compounds belonging to a library receivedfrom National Cancer Institute Developmental TherapeuticProgram, and we identified 10 scaffolds that inhibited dephos-phorylation activity of SerB2 enzyme. The interaction of someof these chemical entities with SerB2 protein was further con-firmed using SPR studies. A subset of these primary hits inhib-ited M. tuberculosis growth in vitro and displayed low cytotox-icity toward both THP-1 and Vero cell lines. The best twochemical entities in our primary hits, clorobiocin and rosanilinewith a therapeutic index (Ti, ratio of TC50/MIC99 values) of

FIGURE 10. A, phosphoserine phosphatase activity of wild type and mutant SerB2 proteins. Pi release assays were performed using either 1 �M of wild type ormutant SerB2 proteins in the presence of 100 �M of O-phospho-L-serine. Pi release was measured in enzymatic reactions, and the data are depicted as themeans � S.E. obtained from three independent experiments. B, secondary structural analysis of wild type and mutant SerB2 protein using far-UV CD spectros-copy. The CD studies were performed as described above in the legend for Fig. 3F. We observed that except E197A, other mutant proteins did not show muchsignificant alterations in their secondary structure as compared with the wild type protein. C–H, inhibition assays in the presence of SerB2 specific inhibitors.Activity assays for wild type and mutant proteins were performed in the absence and presence of CID 1 (C), CID 5 (D), CID 7 (E), CID 8 (F), CID 9 (G), or CID 10 (H)at 200 �M concentration. The data presented in each panel are the means � S.E. of percentages of inhibition obtained from three independent experiments.



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

�21, were evaluated for their ability to kill mycobacteria inliquid cultures and in infected macrophages. Both clorobiocinand rosaniline were bactericidal in their mode of killing andinhibited bacterial growth in infected macrophages in a dose-dependent manner. NSC 76027, the most potent compound inour in vitro enzymatic assays with an IC50 value of 3.8 �M, washighly specific for SerB2 enzyme but displayed MIC99 value of150 �M against both M. tuberculosis H37Rv and M. bovis BCG,which might be attributed to its poor penetration or intracellu-lar stability. NSC 693172 displayed an IC50 value of 4 �M in ourenzymatic assays but was highly cytotoxic in THP-1 cells, whichmight be due to its ability to inhibit other phosphatases in anonspecific manner. Therefore, future experiments wouldinvolve designing of their structural analogs with more potentin vitro SerB2 specific and intracellular activity.

To the best of our knowledge, this is the first study where weshow that clorobiocin inhibits SerB2 enzyme of M. tuberculosis,a non-ATP binding protein in addition to its other known bac-terial targets such as DNA gyrase, heat shock protein, and UDP-galactose-4�-epimerase (38, 39, 56). Interestingly, novobiocin, aclose structural analog of clorobiocin failed to inhibit SerB2enzymatic activity even at 10-fold higher concentration, whichmight be attributed to hydrogen bond formation between Asp-341 residue of SerB2 protein with clorobiocin and a better fit ofits pyrrole ring in the SerB2 substrate binding pocket. Similarsubtle differences were also observed in inhibition of UDP-ga-lactose 4�-epimerase by clorobiocin and novobiocin, respec-tively (56). Further experiments to solve crystal and co-crystalstructure of SerB2 enzyme either alone or with clorobiocinwould be useful to understand such subtle differences in theability of these aminocoumarins to inhibit SerB2 enzyme. Wepropose that use of scaffolds like clorobiocin rather than novo-biocin might be more effective for eradication of drug-resistantTB. Molecular docking of CID 5, CID 7, CID 9, and CID 10 inthe SerB2 built model revealed that these scaffolds interact withcommon critical residues Asp-187 and Asp-341 of SerB2 pro-tein via hydrogen bond formation. Because we did not observemuch difference in the far-UV CD spectra for K361A-SerB2,E214A-SerB2, D187A-SerB2, D341G-SerB2, and wild typeprotein, we speculate that the observed reduced inhibition forthese mutant proteins could be due to loss of interaction ofthese mutant proteins with their respective scaffolds. However,the altered folded state of E197A-SerB2 could have contributedto the loss of inhibition observed in the case of CID 1, CID 5,and CID 7.

Validation of enzymes with human homologs is hampered bylack of safe and highly specific inhibitors. Interestingly exceptCID 7 and CID 10 all other scaffolds were highly specific in theirability to inhibit M. tuberculosis SerB2 enzyme. As expected, wedid not observe any significant interactions between CID 5, CID8, and CID 9 with HPSP enzyme, therefore explaining theirability to specifically inhibit SerB2 enzyme. Because RNAi-me-diated inhibition of PSAT and PSP enzymes reduces tumor for-mation in breast cancer, we propose that nonspecific inhibitors(NSC 93739 and NSC 165701) could be exploited further fortreating such disorders or infections caused by P. gingivalis(57). Based on our observations, future experiments wouldinvolve (i) screening of more libraries to identify novel PSP

inhibitors and (ii) structure-activity relationship studies involv-ing these SerB2 specific scaffolds in an attempt to design ana-logs that display enhanced potency, specificity toward SerB2enzyme, and better intracellular activity. These findings suggestthat despite being widely conserved, enzymes involved inenergy metabolism can be targeted to combat the problem ofdrug resistance in intracellular pathogens. Collectively, theseresults demonstrate feasibility of HTS to obtain novel PSPinhibitors that would be useful for development of anti-myco-bacterial agents.

Acknowledgments—We acknowledge Dr. Rohan Dhiman and Dr.Shubhra Ghosh Dastidar for critical reading of the manuscript. Dr.Rohan Dhiman and Sakshi Agarwal are acknowledged for help withmacrophage experiments. Dr. Ujjini Manjunatha is acknowledged forscientific discussions during the course of study. We acknowledgeNational Cancer Institute-Developmental Therapeutic Program forproviding small molecule libraries. We thank Saqib Kidwai for excel-lent technical assistance. Lab attendant Kumar Amarender Bharti isacknowledged for assistance. We acknowledge Prof. Anil K. Tyagi(Department of Biochemistry, University of Delhi) for access to theBSL-3 facility. We thank Dr. Sailesh Bajpai (GE Healthcare) and Dr.Sanjay Kapoor (Department of Plant Molecular Biology, University ofDelhi) for help with Biacore experiments.

REFERENCES1. Dye, C., and Williams, B. G. (2010) The population dynamics and control

of tuberculosis. Science 328, 856 – 8612. Green, K. D., and Garneau-Tsodikova, S. (2013) Resistance in tuberculo-

sis: what do we know and where can we go? Front. Microbiol. 4, 2083. Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K. (2011)

The challenge of new drug discovery for tuberculosis. Nature 469,483– 490

4. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D.,Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., 3rd, Tekaia, F., Badcock,K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Dev-lin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels,K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J.,Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton,J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S., andBarrell, B. G. (1998) Deciphering the biology of Mycobacterium tubercu-losis from the complete genome sequence. Nature 393, 537–544

5. Griffin, J. E., Pandey, A. K., Gilmore, S. A., Mizrahi, V., McKinney, J. D.,Bertozzi, C. R., and Sassetti, C. M. (2012) Cholesterol catabolism by My-cobacterium tuberculosis requires transcriptional and metabolic adapta-tions. Chem. Biol. 19, 218 –227

6. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Genes required formycobacterial growth defined by high density mutagenesis. Mol. Micro-biol. 48, 77– 84

7. Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobac-terial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100,12989 –12994

8. Helgadottir, S., Rosas-Sandoval, G., Soll, D., and Graham, D. E. (2007)Biosynthesis of phosphoserine in the Methanococcales. J. Bacteriol. 189,575–582

9. Snell, K. (1984) Enzymes of serine metabolism in normal, developing andneoplastic rat tissues. Adv. Enzyme Regul. 22, 325– 400

10. Schirch, V., Hopkins, S., Villar, E., and Angelaccio, S. (1985) Serine hy-droxymethyltransferase from Escherichia coli: purification and properties.J Bacteriol. 163, 1–7

11. Ho, C. L., Noji, M., and Saito, K. (1999) Plastidic pathway of serine biosyn-thesis. Molecular cloning and expression of 3-phosphoserine phosphatasefrom Arabidopsis thaliana. J. Biol. Chem. 274, 11007–11012



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12. Coulibaly, F., Lassalle, E., Baker, H. M., and Baker, E. N. (2012) Structure ofphosphoserine aminotransferase from Mycobacterium tuberculosis. ActaCrystallogr. D Biol. Crystallogr. 68, 553–563

13. Dey, S., Grant, G. A., and Sacchettini, J. C. (2005) Crystal structure ofMycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: ex-treme asymmetry in a tetramer of identical subunits. J. Biol. Chem. 280,14892–14899

14. Dey, S., Hu, Z., Xu, X. L., Sacchettini, J. C., and Grant, G. A. (2005) D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is alink between the Escherichia coli and mammalian enzymes. J. Biol. Chem.280, 14884 –14891

15. Bai, G., Schaak, D. D., Smith, E. A., and McDonough, K. A. (2011) Dys-regulation of serine biosynthesis contributes to the growth defect of aMycobacterium tuberculosis crp mutant. Mol. Microbiol. 82, 180 –198

16. Seifried, A., Schultz, J., and Gohla, A. (2013) Human HAD phosphatases:structure, mechanism, and roles in health and disease. FEBS J. 280,549 –571

17. Koonin, E. V., and Tatusov, R. L. (1994) Computer analysis of bacterialhaloacid dehalogenases defines a large superfamily of hydrolases with di-verse specificity: application of an iterative approach to database search. J.Mol. Biol. 244, 125–132

18. Wang, W., Kim, R., Jancarik, J., Yokota, H., and Kim, S. H. (2001) Crystalstructure of phosphoserine phosphatase from Methanococcus jannaschii,a hyperthermophile, at 1.8 A resolution. Structure 9, 65–71

19. Burroughs, A. M., Allen, K. N., Dunaway-Mariano, D., and Aravind, L.(2006) Evolutionary genomics of the HAD superfamily: understanding thestructural adaptations and catalytic diversity in a superfamily of phos-phoesterases and allied enzymes. J. Mol. Biol. 361, 1003–1034

20. Cho, H., Wang, W., Kim, R., Yokota, H., Damo, S., Kim, S. H., Wemmer,D., Kustu, S., and Yan, D. (2001) BeF3

� acts as a phosphate analog inproteins phosphorylated on aspartate: structure of a BeF3

� complex withphosphoserine phosphatase. Proc. Natl. Acad. Sci. U.S.A. 98, 8525– 8530

21. Kim, H. Y., Heo, Y. S., Kim, J. H., Park, M. H., Moon, J., Kim, E., Kwon, D.,Yoon, J., Shin, D., Jeong, E. J., Park, S. Y., Lee, T. G., Jeon, Y. H., Ro, S., Cho,J. M., and Hwang, K. Y. (2002) Molecular basis for the local conforma-tional rearrangement of human phosphoserine phosphatase. J. Biol. Chem.277, 46651– 46658

22. Peeraer, Y., Rabijns, A., Verboven, C., Collet, J. F., Van Schaftingen, E., andDe Ranter, C. (2003) High-resolution structure of human phosphoserinephosphatase in open conformation. Acta Crystallogr. D. Biol. Crystallogr.59, 971–977

23. Rossmann, M. G., Moras, D., and Olsen, K. W. (1974) Chemical and bio-logical evolution of nucleotide-binding protein. Nature 250, 194 –199

24. Moffatt, C. E., Inaba, H., Hirano, T., and Lamont, R. J. (2012) Porphyromo-nas gingivalis SerB-mediated dephosphorylation of host cell cofilin mod-ulates invasion efficiency. Cell. Microbiol. 14, 577–588

25. Takeuchi, H., Hirano, T., Whitmore, S. E., Morisaki, I., Amano, A., andLamont, R. J. (2013) The serine phosphatase SerB of Porphyromonas gin-givalis suppresses IL-8 production by dephosphorylation of NF-�B RelA/p65. PLoS Pathog 9, e1003326

26. Tribble, G. D., Mao, S., James, C. E., and Lamont, R. J. (2006) A Porphy-romonas gingivalis haloacid dehalogenase family phosphatase interactswith human phosphoproteins and is important for invasion. Proc. Natl.Acad. Sci. U.S.A. 103, 11027–11032

27. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez,R., McWilliam, H., Remmert, M., Soding, J., Thompson, J. D., and Higgins,D. G. (2011) Fast, scalable generation of high-quality protein multiplesequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539

28. Saitou, N., and Nei, M. (1987) The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406 – 425

29. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S.(2011) MEGA5: molecular evolutionary genetics analysis using maximumlikelihood, evolutionary distance, and maximum parsimony methods.Mol. Biol. Evol. 28, 2731–2739

30. Colovos, C., and Yeates, T. O. (1993) Verification of protein structures:patterns of nonbonded atomic interactions. Protein Sci. 2, 1511–1519

31. Laskowski, R. A. (2001) PDBsum: summaries and analyses of PDB struc-tures. Nucleic Acids Res. 29, 221–222

32. Luthy, R., Bowie, J. U., and Eisenberg, D. (1992) Assessment of proteinmodels with three-dimensional profiles. Nature 356, 83– 85

33. Hawkinson, J. E., Acosta-Burruel, M., Ta, N. D., and Wood, P. L. (1997)Novel phosphoserine phosphatase inhibitors. Eur. J. Pharmacol. 337,315–324

34. Hawkinson, J. E., Acosta-Burruel, M., and Wood, P. L. (1996) The metabo-tropic glutamate receptor antagonist L-2-amino-3-phosphonopropionicacid inhibits phosphoserine phosphatase. Eur. J. Pharmacol. 307,219 –225

35. Friesner, R. A., Murphy, R. B., Repasky, M. P., Frye, L. L., Greenwood, J. R.,Halgren, T. A., Sanschagrin, P. C., and Mainz, D. T. (2006) Extra precisionglide: docking and scoring incorporating a model of hydrophobic enclo-sure for protein-ligand complexes. J. Med. Chem. 49, 6177– 6196

36. Chiba, Y., Oshima, K., Arai, H., Ishii, M., and Igarashi, Y. (2012) Discoveryand analysis of cofactor-dependent phosphoglycerate mutase homologsas novel phosphoserine phosphatases in Hydrogenobacter thermophilus.J. Biol. Chem. 287, 11934 –11941

37. Singh, S. K., Yang, K., Karthikeyan, S., Huynh, T., Zhang, X., Phillips, M. A.,and Zhang, H. (2004) The thrH gene product of Pseudomonas aeruginosais a dual activity enzyme with a novel phosphoserine:homoserine phos-photransferase activity. J. Biol. Chem. 279, 13166 –13173

38. Anderle, C., Stieger, M., Burrell, M., Reinelt, S., Maxwell, A., Page, M., andHeide, L. (2008) Biological activities of novel gyrase inhibitors of the ami-nocoumarin class. Antimicrob. Agents Chemother. 52, 1982–1990

39. Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M., and Neckers, L. M.(2000) The heat shock protein 90 antagonist novobiocin interacts with apreviously unrecognized ATP-binding domain in the carboxyl terminusof the chaperone. J. Biol. Chem. 275, 37181–37186

40. Awasthy, D., Bharath, S., Subbulakshmi, V., and Sharma, U. (2012) Ala-nine racemase mutants of Mycobacterium tuberculosis require D-alaninefor growth and are defective for survival in macrophages and mice. Micro-biology 158, 319 –327

41. Hondalus, M. K., Bardarov, S., Russell, R., Chan, J., Jacobs, W. R., Jr., andBloom, B. R. (2000) Attenuation of and protection induced by a leucineauxotroph of Mycobacterium tuberculosis. Infect Immun. 68, 2888 –2898

42. Smith, D. A., Parish, T., Stoker, N. G., and Bancroft, G. J. (2001) Charac-terization of auxotrophic mutants of Mycobacterium tuberculosis andtheir potential as vaccine candidates. Infect. Immun. 69, 1142–1150

43. Anthony, K. G., Strych, U., Yeung, K. R., Shoen, C. S., Perez, O., Krause,K. L., Cynamon, M. H., Aristoff, P. A., and Koski, R. A. (2011) New classesof alanine racemase inhibitors identified by high-throughput screeningshow antimicrobial activity against Mycobacterium tuberculosis. PLoSOne 6, e20374

44. Kishor, C., Arya, T., Reddi, R., Chen, X., Saddanapu, V., Marapaka, A. K.,Gumpena, R., Ma, D., Liu, J. O., and Addlagatta, A. (2013) Identification,biochemical and structural evaluation of species-specific inhibitorsagainst type I methionine aminopeptidases. J. Med. Chem. 56, 5295–5305

45. Lee, Y., Mootien, S., Shoen, C., Destefano, M., Cirillo, P., Asojo, O. A.,Yeung, K. R., Ledizet, M., Cynamon, M. H., Aristoff, P. A., Koski, R. A.,Kaplan, P. A., and Anthony, K. G. (2013) Inhibition of mycobacterial ala-nine racemase activity and growth by thiadiazolidinones. Biochem. Phar-macol. 86, 222–230

46. Poyraz, O., Jeankumar, V. U., Saxena, S., Schnell, R., Haraldsson, M., Yo-geeswari, P., Sriram, D., and Schneider, G. (2013) Structure-guided designof novel thiazolidine inhibitors of O-acetyl serine sulfhydrylase from My-cobacterium tuberculosis. J. Med. Chem. 56, 6457– 6466

47. Shen, H., Wang, F., Zhang, Y., Huang, Q., Xu, S., Hu, H., Yue, J., and Wang,H. (2009) A novel inhibitor of indole-3-glycerol phosphate synthase withactivity against multidrug-resistant Mycobacterium tuberculosis. FEBS J.276, 144 –154

48. Wang, D., Zhu, X., Cui, C., Dong, M., Jiang, H., Li, Z., Liu, Z., Zhu, W., andWang, J. G. (2013) Discovery of novel acetohydroxyacid synthase inhibi-tors as active agents against Mycobacterium tuberculosis by virtual screen-ing and bioassay. J. Chem. Inf. Model 53, 343–353

49. Hirabayashi, Y., and Furuya, S. (2008) Roles of L-serine and sphingolipidsynthesis in brain development and neuronal survival. Prog. Lipid Res. 47,188 –203

50. Kitabatake, M., So, M. W., Tumbula, D. L., and Soll, D. (2000) Cysteine



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

biosynthesis pathway in the archaeon Methanosarcina barkeri encoded byacquired bacterial genes? J. Bacteriol. 182, 143–145

51. Largen, M., and Belser, W. L. (1975) Tryptophan biosynthetic pathway inthe Enterobacteriaceae: some physical properties of the enzymes. J. Bac-teriol. 121, 239 –249

52. Pizer, L. I. (1965) Glycine synthesis and metabolism in Escherichia coli. J.Bacteriol. 89, 1145–1150

53. Snyder, S. H., and Kim, P. M. (2000) D-Amino acids as putative neu-rotransmitters: focus on D-serine. Neurochem. Res. 25, 553–560

54. Ulane, R., and Ogur, M. (1972) Genetic and physiological control of serineand glycine biosynthesis in Saccharomyces. J. Bacteriol. 109, 34 – 43

55. Jung, S. K., Ko, Y., Yu, K. R., Kim, J. H., Lee, J. Y., Chae, C. H., Ji, S., Kim,C. H., Lee, H. K., Choi, E. B., Kim, B. Y., Erikson, R. L., Chung, S. J., and Kim,S. J. (2012) Identification of 3-acyl-2-phenylamino-1,4-dihydroquinolin-

4-one derivatives as inhibitors of the phosphatase SerB653 in Porphy-romonas gingivalis, implicated in periodontitis. Bioorg. Med. Chem. Lett.22, 2084 –2088

56. Durrant, J. D., Urbaniak, M. D., Ferguson, M. A., and McCammon, J. A. (2010)Computer-aided identification of Trypanosoma brucei uridine diphosphategalactose 4�-epimerase inhibitors: toward the development of novel therapiesfor African sleeping sickness. J. Med. Chem. 53, 5025–5032

57. Possemato, R., Marks, K. M., Shaul, Y. D., Pacold, M. E., Kim, D., Birsoy, K.,Sethumadhavan, S., Woo, H. K., Jang, H. G., Jha, A. K., Chen, W. W.,Barrett, F. G., Stransky, N., Tsun, Z. Y., Cowley, G. S., Barretina, J., Kalaany,N. Y., Hsu, P. P., Ottina, K., Chan, A. M., Yuan, B., Garraway, L. A., Root,D. E., Mino-Kenudson, M., Brachtel, E. F., Driggers, E. M., and Sabatini,D. M. (2011) Functional genomics reveal that the serine synthesis pathwayis essential in breast cancer. Nature 476, 346 –350



at Cam

bridge University L

ibrary on Novem


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis...

Documents

Transcript of High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis...