Virtual screening, identification and experimental testing of novel inhibitors of PBEF1/Visfatin/NMPRTase for glioma therapy.

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by  Chandra, Nagasuma

Chandraetal.JournalofClinicalBioinformatics2011,1:5 JOURNAL OF
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CLINICAL BIOINFORMATICS
RESEARCH Open Access
Virtual screening, identification and experimental
testing of novel inhibitors of PBEF1/Visfatin/
NMPRTase for glioma therapy
Nagasuma Chandra1*, Raghu Bhagavat1, Eshita Sharma1, P Sreekanthreddy2, Kumaravel Somasundaram2*
Abstract
Background: Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes nicotinamide phosphoribosyltransferase
(NMPRTase), which catalyses the rate limiting step in the salvage pathway of NAD+ metabolism in mammalian
cells. PBEF1 transcript and protein levels have been shown to be elevated in glioblastoma and a chemical inhibitor
of NMPRTase has been shown to specifically inhibit cancer cells.
Methods: Virtual screening using docking was used to screen a library of more than 13,000 chemical compounds.
A shortlisted set of compounds were tested for their inhibition activity in vitro by an NMPRTase enzyme assay.
Further, the ability of the compounds to inhibit glioma cell proliferation was carried out.
Results: Virtual screening resulted in short listing of 34 possible ligands, of which six were tested experimentally,
using the NMPRTase enzyme inhibition assay and further with the glioma cell viability assays. Of these, two
compounds were found to be significantly efficacious in inhibiting the conversion of nicotinamide to NAD+, and
out of which, one compound, 3-amino-2-benzyl-7-nitro-4-(2-quinolyl-)-1,2-dihydroisoquinolin-1-one, was found to
inhibit the growth of a PBEF1 over expressing glioma derived cell line U87 as well.
Conclusions: Thus, a novel inhibitor has been identified through a structure based drug discovery approach and is
further supported by experimental evidence.
Background addition to its role as a redox cofactor, is also used as a
Gliomasareprimarymalignanttumors,originatinginthe substrate in several biochemical reactions including
brain,andaccountfor80%ofadultprimarybraintumors. mono-andpoly-ADPribosylation(ARTandPARP cata-
Theprognosisforpatientswithglioblastomamultiforme, lyzed), protein deacetylation and ADP-ribose cyclization
avirulentvarietyofthediseaseisratherpoor,withamed- [3].NMPRTasecatalyzestheconversionoffreenicotina-
ian survival of less than one year [1]. Several molecular midetonicotinamidemononucleotide(NMN),whichisa
andbiochemicalabnormalitiessuchasspecificchromoso- keystepinthesalvagepathwayofNAD+.Expressionlevels
malaberrations, upregulationofepidermalgrowthfactor of NMPRTase (also known as visfatin/Pre B-cell enhan-
receptor(EGFR), lossofphosphateandtensinhomology cingfactor1(PBEF1)),wasfoundtobeupregulatedincol-
(PTEN), havebeenclearlyassociatedwithgliomas. Some orectal cancers [4], suggesting that NMPRTase may be
pathwaysassociatedwithhighergradegliomasareupregu- crucial for maintaining cellular NAD+ levels in tumors.
lationofPDGFRA(plateletderivedgrowthfactorreceptor Microarrayanalysesofgliomacells(gradeIItoIV)versus
a), CDK4 (cyclin dependent kinase 4) and down-regula- normal brainglial cellshas identified differentialexpres-
tion of retinoblastoma (RB1) [1]. NAD+ biosynthesis has sionofNMPRTaseingliomawith2-5foldupregulationin
been shown to be activated in cancers [2]. NAD+, in glioma cells, depending on the grade of the tumor
(increasedexpressionofNMPRTasewithgreaterprogres-
sion ofthe disease, GradeIV>Grade III>Grade II)[5].
*Correspondence:[email protected];[email protected]
1BioinformaticsCentre,IndianInstituteofScience,Bangalore560012,India Observation of increased rates of NAD+ metabolism in
2MicrobiologyandCellBiology,IndianInstituteofScience,Bangalore glioma, suggests that the cancer cells may be critically
560012,India
dependent upon metabolites produced in the pathway,
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Chandraetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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and presents a possible strategy to counter the disease, a. Selection of ligand library and preparation of ligands
throughtheinhibitionofkeyenzymesinthepathway. and protein
The crystal structures of free NMPRTase, NMPRTase Virtualscreeningwasperformedtoidentifypossiblelead
bound to NMN, and NMPRTase bound to the inhibitor compounds from the Maybridge HitFinder™ database.
FK866 [6], have been recently reported. FK866 is a TheMaybridgeHitFinder™setsarestructuralrepresen-
potent small-molecule inhibitor of human NMPRTase, tatives of large non-redundant chemical libraries. This
and the consequent reduction in NAD+ levels can cause collectionincludes14,400compoundsthatrepresentthe
apoptosis of tumor cells while having minimal effect on drug-likediversityoftheMaybridgeScreeningCollection
normal cells [7]. FK866 also turns out to be the only (~56,000 compounds). All the screening compounds fit
promising inhibitor known, for the enzyme. The struc- Lipinskiguidelinesfordrug-likeness;partitioncoefficient,
tures provide a basis for understanding substrate specifi- ClogP ≤ 5, H-bond donors ≤ 5, H-bond acceptors ≤ 10,
city, mechanism of enzyme action, and hence provide a molecularweight≤500[12].TheMaybridgeHitFinder™
framework for design of novel NMPRTase inhibitors. set was obtained from http://www.maybridge.com. The
An extremely useful step in the rational design of ligandfileswerepreparedfordockingusingSchrodinger
inhibitors is to utilize the three dimensional structural Ligprep software [13]. In addition to the generation of
information of the target protein and identify a possible energy minimized 3D structures, Schrodinger Ligprep
lead compound from large libraries of compounds. The wasalso usedforadditionofhydrogensanddesaltingof
most efficient method to do this, of course would be to metal ions. The main objective of using LigPrep was to
carry out virtual screening. In the recent years several obtain low energy 3D structures of the set of ligands in
docking algorithms have been developed which are the library, for use in further computational studies.
being used for virtual screening of potential ligands to a OPLS_2005force field wasutilized to optimize the geo-
given protein at the three dimensional level [8,9]. We metry and minimize the energy. Force field parameters
recently developed a parallel version of a popular dock- wereassignedtotheligandatomsusingdefaulttreatment
ing algorithm - AutoDock [10] and have implemented for possible tautomers, and ionization at a selected pH
this on an IBM Bluegene supercomputer [11], rendering range (7 ± 2 by default), and ring conformations (1 ring
the docking approach amenable for high throughput vir- conformer by default).The Ligparse module was used
tual screening. Here we report virtual screening of a duringLigprepandtheligandswithfollowingproperties
large library of compounds and short listing of six can- were removed from the set: molecular weight less than
didate molecules that are likely to bind to NMPRTase. 200, number of neutral acceptor groups greater than 10
These compounds were subsequently tested experimen- and number of neutral donor groups greater than 5. A
tally for their ability to (a) inhibit the conversion of total of 13214 ligands were selected and retained out of
nicotinamide to NAD+ by NMPRTase and (b) inhibit thetotal14,400originalligands.
efficiently the growth of a PBEF1 over expressing glio- b. Docking
blastoma derived cell line U87. Based on these studies, a The co-ordinates of the human NMPRTase (2GVG-
promising lead compound has been identified. complex with the reaction product nicotinamide mono-
nucleotide; 2GVJ- complex with an inhibitor, n-[4-(1-
Methods benzoylpiperidin-4-yl)butyl]-3-pyridin-3-ylpropanamide-
Reagents and cell lines FK866 were obtained from Protein Data Bank (PDB)
Cancer cell lines U373, U138, LN229, U343, U87, U251 [14]. The protein file was prepared for docking by
and LN18 (all glioma derived cell lines), SW480 (colon removal of water molecules, addition of polar hydrogens,
carcinoma), HaCat (immortalized human keratinocytes) removal of ligand and phosphate groups in active site,
and HBL100 (immortalized human breast epithelial and addition of Kollman charges [15]. The macromole-
cells) cells were cultured in Dulbecco’s Modified Eagle cule was treated to be completely rigid for all docking
Medium (DMEM) respectively with 10% Fetal bovine studies to reduce the extensive computational costs. A
serum, penicillin and streptomycin at 37 °C in a humidi- grid box encompassing both the NMN and FK866 sites
fied atmosphere with 5% CO . The six best docked (86×60×50; 0.375 Å spacing) was constructed and used
2
compounds were purchased from Maybridge, Thermo for all the docking runs. Our definition of the site as
Fisher Scientific, UK. C14-Nicotinamide (Specific activ- input for the docking program encompasses the phos-
ity 55 mCi/mmol) was purchased from American Radio phate site completely, since the grid box with dimen-
labeled Chemicals, USA. sions 80 × 60 × 50 points with a default spacing of
0.375 Å, is sufficiently large to encompass the entire
Virtual screening binding pocket and any nearby minor sites such as that
Different steps involved in virtual screening are briefly of the phosphate site. Thus, the search space for gener-
described below: ating ligand map files using Autogrid, is big enough to
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encompass the PO4 site, and is not neglected. Docking visualization tool Pymol [19] was used to generate the
parameter files were prepared for each ligand using the images of the docked complexes.
following parameters: ga_pop_size 150; ga_num_evals
2500000; ga_num_generations 500; ga_run 100 and RNA isolation and RT-qPCR
rmstol 1.0. The Maybridge HitFinder™ dataset was RNA isolation and RT-qPCR were carried out as
docked using the parallel version of AutoDock 3, avail- described before [5]. Total RNA was extracted from
able in the laboratory using 256 processors on an IBM cancer cell lines by using the TRI reagent (Sigma). The
cluster. This process greatly reduced the computational RNA samples were quantified by measuring the absor-
cost and time involved in virtual screening of the large bance using a spectrophotometer and visualized on a
dataset (~13214). Clustering was performed based on MOPS formaldehyde gel for quality assurance. The rela-
the similarity in binding modes and affinities in the run tive quantification of the expression levels of selected
cycles. The size of the clusters refer to the total number genes was carried out using a two-step strategy: In the
of conformations of the ligand that bind in the same first step, cDNA was generated from RNA derived from
orientation within the specified RMSD threshold (1 Å different tissue samples using a cDNA archive kit (ABI
was used in our study) and binding with the same PRISM); subsequently, real-time quantitative PCR was
energy. Thus, a cluster is defined as a unit of such simi- carried out in an ABI PRISM 7900 (Applied Biosystems)
lar confirmations. More the number in each cluster bet- sequence detection system with the cDNA as template
ter is the accuracy and confidence of the predicted pose using PBEF1 specific primer set and a Dynamo kit con-
of the ligand molecule. The Ligand Protein Contacts taining SYBR green dye (Finnzymes). All measurements
(LPC) [16] was used for obtaining the interactions of were made in triplicates. The genes GARS (glycyl-tRNA
docked ligand atoms with the macromolecule, hydrogen synthetase), AGPAT1 (1-acylglycerol-3-phosphate O-
bonding, van der Waals contacts and the solvent acces- acyltransferase 1), ATP5G1 [ATP synthase, H+ trans-
sible surface area. porting, mitochondrial F0 complex, subunit C1 (subunit
c. Short listing of potential leads 9)], and RPL35A (ribosomal protein L35a) were used as
The docking log files (.dlg) were parsed using in-house internal controls because their expression levels were
perl scripts to scan the clustering histograms, and iden- found to be unaltered in microarray experiments. The
tify ligands that have docked poses with binding energy fold change (log2 ratio) in PBEF1 gene expression was
lower than the cut-off criteria and cluster size greater calculated over its mean expression in normal brain
than the defined cut-off (See Table 1). The cut-off samples obtained from previously published results [5].
values were obtained from docking the known inhibitor Delta-delta CT method was used for the calculation of
FK866, and product NMN, to the receptor, and retriev- ratios. Sequences of reverse transcription-PCR primers
ing the docking energy and cluster size values for poses and conditions used will be provided on request.
that have least deviation from the crystal pose (RMSD <
1.0). As a newer version of AutoDock became available Western blot analysis
during the course of this work, the docking exercise was Western blot analysis was performed as described pre-
repeated for the short listed compounds with the same viously [5] with rabbit polyclonal antibody against GST-
parameters using AutoDock4 (version 4.0.1). PBEF1 raised in the laboratory using a standard immu-
Energy minimization nization protocol and antitubulin antibody.
Minimization of the docked ligands in the best ranked
poses was done using CNS software suite [17]. Conju- NMPRTase assay
gate gradient method was used for minimization with The measurement of NMPRTase activity was carried out
flexibility allowed only for those atoms within the 6 Å as described before [7]. To prepare cytoplasmic extract,
radius of every atom of the ligand for 150 runs. The as source of NMPRTase, we collected logarithmically
topology and parameter files for the compounds were growing U87 glioblastoma cells by centrifugation and
obtained from XPLO-2D software [18]. Molecular washed three times with Ca2+ Mg2+ free PBS. The cells
pellet (2-3 × 107 cells) was suspended and lysed in 1 mL
of 0.01 M NaH PO (pH 7.4) by one round of freezing
2 4
and slow thawing. The clear supernatant was recovered
Table 1Docking resultsofcontrol compounds
on ice after centrifugation at 23,000 × g at 0°C for 90
minutes. 70 mL of 1% protamine sulfate was added per
Biochemicalenergyof AD4docked RMSDfrom Sizeof
ml of supernatant and incubated for 15 min on ice, fol-
binding(kcal/mol) (kcal/mol) crystalpose cluster
lowed by centrifugation at 23,000 × g at 0°C for 30 min-
NMN - -8.02 0.87 35
utes. The final supernatant was stored in small aliquots
FK866 -9.25 -8.63 0.77 16
at -80°C. The NMPRTase activity was determined in 0.5
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mL of reaction solution consisting of 5 mM MgCl , the hydrophobic stacking, was used as a criterion for fil-
2
2 mM ATP, 0.5 mM phosphoribosyl PPI, 0.1 mM tering docked ligands, subsequent to the selection on
14[C]-nicotinamide (specific activity: 50 mCi/mmol; the basis of binding energy and cluster size. The unique-
American Radio labeled Chemicals, Inc.) and 50 mM ness of the binding site has been studied by comparing
Tris (pH 8.8) at 37°C. The reaction was started by add- NMPRTase with closely related NAPRT and QPRT
ing 100 μL of cell extract and stopped after 1 hr with enzymes, present in the same biochemical pathway
excess of cold nicotinamide and heating (2 min, 105°C). [6]. A multiple sequence alignment of the human
The precipitate was removed by centrifugation at 2500 NMPRTase with the other two proteins showed that
× g at 4°C for 10 min and the supernatant was stored at they have diverged in terms of sequences considerably,
-20°C. The 14[C]-labeled components in the cell but adopt the same fold. Yet, analysis of their binding
extracts were separated and identified using thin-layer sites using PocketMatch [21], indicates that considerable
chromatography (cellulose/1 M ammonium sulphate: difference exists at the binding site level, suggesting that
ethanol (3:7)). The chromatograms were run, exposed to design of specific inhibitors can be achieved. It has also
imaging plates (Fuji) and read using Phosphorimager been reported earlier that FK866, a potent inhibitor of
(Fuji). The 14[C]-labeled NAD+ was quantified using NMPRTase does not inhibit NAPRT, consistent with
Alpha Innotec software. this observation [6].
MTT assay Identification and analysis of potential compounds
MTT assay was carried out as described previously [20]. As a control study, the enzyme’s reaction product NMN
A total of 1.5 × 103 cells/well were plated in a 96-well as well as the known inhibitor, FK866 were docked to
plate. After 24 h of plating, the cells were treated with the protein, an exercise which resulted in reproducing
indicated amounts of compounds. A measure of 20 μL the crystal structure poses for both compounds. Table 1
(5 mg/mL) of MTT was added to each well 48 hrs after lists the interaction energies computed for the docked
the addition of the compounds. MTT is a tetrazolium NMN and FK866 as well as the deviations from the
salt that is converted by living cells into purple forma- crystallographic observed poses. The energy values com-
zan crystals. The medium was removed from the wells puted for these reference compounds were used as
3 hrs after MTT addition and 200 μL of DMSO was reference values for identifying possible ligands from the
added to dissolve the formazan crystals, and then the large compound library. All those compounds which
absorbance was measured at 550 nm in an ELISA exhibited interaction energies above this threshold or in
reader. other words indicated binding weaker than the reference
compounds were eliminated from the list for further
Results analysis. The result of the virtual screening of the data-
Description of the binding site set is summarized in Table 2.
The crystal structures of NMPRTase, in complex with For selection of potential ligands, analysis of ligand
the known inhibitor FK866, and reaction product NMN, protein contacts for top ranking poses of every ligand
reveal that the active enzyme exists as a dimer. The was carried out and the interactions of docked com-
structures of the two complexes were very similar to pounds were visualized. Interactions conserved with
each other and no significant conformational changes NMN and FK866 binding were calculated and compared
were observed upon ligand binding [6], making it mean- with that of the short listed compounds. The best poses
ingful to compare the binding modes of the two ligands. were identified using the following criteria in the given
The catalytic centre is present at the interface of the order of preference i) lowest binding energy in the lar-
two chains. There are two active sites per dimer and gest sized cluster ii) number of hydrogen bonds with the
residues from both the chains are involved in the inter- active site residues and iii) conservation of interactions
actions with the product NMN at each site [6]. The with those from NMN/FK866 binding. Preference is
binding pockets of the two ligands overlap partly with given to the largest sized least binding energy cluster
each other, together extending into a single large pocket and then examined to verify if one or more hydrogen
resembling a long tunnel, with NMN binding at one bonds are conserved between the natural substrate
end and FK866 spanning till the other end. Some resi- NMN, or the previously known inhibitor FK866. This
dues are common to both, indicating the overlap in was to ensure that the ligands shortlisted were actually
their binding poses. An essential feature of both the docking into the binding site of interest. All the 34
binding poses is the presence of hydrophobic stacking in shortlisted from an initial list of 13214 compounds pass
which an aromatic group in the ligand is sandwiched these criteria and is shown in Table 2. The top six in
between F193 of one subunit and Y18 of another subu- this list that were readily purchasable were considered
nit. The conservation of these interactions, especially for further studies which are shown in Table 3. The
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Table 2Virtual Screening results and the two control compounds, FK866 and NMN are
#ligands Energy #ligands Cluster Potential shown in Figure 2.
cut-off above sizecut- ligands
cut-off off
Inhibition of NMPRTase activity by selected lead
Maybridge 13214 <-8.5 34 >15 6 compounds
HitFinder™ kcal/
We then tested whether the selected lead compounds
mol
could inhibit NMPRTase activity. We measured the abil-
Summaryofdockingresults;(1)Dockingofreferencecompoundsto
NMPRTasereproducingthecrystalposes;(2)Anoverviewofresultsofvirtual ity of NMPRTase to convert 14[C]-nicotinamide to 14
screeningtothesameproteinmolecule. [C]-NAD+. We had earlier shown that most of the glio-
blastoma tissues have elevated levels of transcript
docking results for all the six compounds showed that and protein of PBEF1/NMPRTase [5]. To prepare
docked poses passing the defined thresholds were found NMPRTase enzyme, we first tested a panel of glioma
to cluster into two main groups, corresponding to the derived cell lines for NMPRTase transcript and protein
binding modes of NMN and FK866 respectively, with levels. We found that, out of seven glioma cell lines
docking energies nearly comparable between the modes tested, two cell lines, U87 and U138, had substantially
for each ligand. The binding sites for both possible high levels of PBEF1 transcripts in comparison to nor-
modes are shown in Figure 1 and Additional file 1, mal brain samples (Figure 3A). Western blotting analysis
Figure S1, whereas the list of interactions at the site for also corroborated above results that U87 and U138 had
both possible modes are indicated in Table 3, and illu- relatively higher levels of PBEF1/NMPRTase protein
strated in Figure 1. 2D-structures of the six compounds levels (Figure 3B).
Table 3Binding Free Energyforthe six identified compounds andthe control compounds
Comp Compoundname BindingFree Number Residuesinthebindingpockets
no. energy(kcal/ inclusters
mol)
1 Dipotassium9-oxo-9H-fluorene-2,7- -8.56 54 F ,R ,G ,R ,G ,V ,G Y 1,T 1,R 1,D 1
193 196 197 311 353 356 384, 18 391 392 393
disulfonate (ModeI)
-7.98 32 Y ,H ,F ,D ,V ,A ,A ,S ,I ,R ,I ,Y 1
188 191 193 219 242 244 245 275 309 311 351 18
(ModeII)
2 Ethyl-5-amino-6-cyano-7-(2-furyl)-4-oxo-3- -9.45 44 F ,R ,D ,E ,H ,R ,D ,G ,D ,G ,V ,
193 196 219 246 247 311 313 353 354 355 356
phenyl-3,4-dihydro-1-phthalazinecarboxylate (ModeI) G ,S ,G ,G ,G D 1,Y 1,E 1,K 1,K 1
381 382 383 384 385, 16 18 149 415 423
-8.12 14 Y ,H ,G ,Y ,S ,V ,P ,P ,I ,R ,V ,
188 191 217 240 241 242 273 307 309 349 350
(ModeII) I ,I ,A ,Q 1
351 378 379 92
3 1,[3,5-Di(2H-1,2,3-benzotriazol-2-yl)-2,4- -9.55 18 F ,R ,R ,G ,D ,G ,V ,G ,S ,G ,G
193 196 311 353 354 355 356 381 382 383 385,
dihydroxyphenyl]ethan-1-one (ModeI) D 1,Y 1,R 1,R 1,D 1,N 1
16 18 40 392 393 396
-8.69 15 Y ,K ,H ,F ,G ,D ,Y ,S ,V ,A ,S ,
188 189 191 193 217 219 240 241 242 244 275
(ModeII) I ,R ,R ,V ,I ,E ,N ,I ,A ,Y 1
309 311 349 350 351 376 377 378 379 18
4 7a-methyl-2,4,5-triphenyl-7,7a- -8.56 40 F ,R ,H ,G ,D ,G ,V ,G ,S ,G ,G ,
193 196 247 353 354 355 356 381 382 383 384
dihydrocyclopenta[b]pyran-7-one (ModeI) G Y 1,R 1,R 1,S 1,K 1,K 1
385, 18 40 392 398 415 423
-8.23 27 Y ,K ,H ,Y ,S ,V ,P ,I ,R ,V ,E ,I ,A
188 189 191 240 241 242 307 309 349 350 376 378 379
(ModeII)
5 3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2- -10.54 38 F ,R ,D ,A ,H ,R ,D ,G ,D ,G ,V
193 196 219 244 247 311 313 353 354 355 356,
dihydroisoquinolin-1-one (ModeI) Y 1,E 1,R 1,S 1,F 1,K 1,K 1
18 149 392 398 399 415 423
-9.84 16 G ,Y ,K ,H ,Y ,S ,V ,S ,I ,R ,I ,
185 188 189 191 240 241 242 275 309 349 351
(ModeII) E ,A
376 379
6 N-[(5-methyl-2-phenyl-2H-1,2,3-triazol-4-yl) -9.86 16 F ,R ,D ,V ,A ,S ,G ,D ,G ,V ,G ,
193 196 219 242 244 275 353 354 355 356 381
methyl]-2H-chromene-3-carboxamide (ModeI) S ,G ,G ,G D 1,Y 1
382 383 384 385, 16 18
-9.64 10 Y ,H ,F ,D ,Y ,S ,V ,A ,S ,I ,I ,
188 191 193 219 240 241 242 244 275 309 351
(ModeII) Y 1,H 1,Q 1
18 90 92
7 CONTROL -8.63 16 F ,G ,Y ,R ,G ,D ,R ,D ,G V ,G ,G
193 194 195 196 197 219 311 313 353, 356 384 385
n-[4-(1-benzoylpiperidin-4-yl)butyl]-3-pyridin- D 1,Y 1,R 1,D 1
16 18 392 393
3-ylpropanamide(FK866)
8 CONTROL -8.02 27 F ,R ,G ,D ,G ,V ,D ,G ,S ,G ,G ,
193 196 353 354 355 356 357 381 382 383 384
G ,K ,R 1,D 1
385 389 392 393
Nicotinamideribosemonophosphate(NMN)
BindingenergyforsixcompoundsandcontrolcompoundsNMN,andFK866alongwithresiduesineachsite.Thesubscriptreferstotheresiduenumber.
Residuesinvolvedinhydrogenbondinteractionsareshowninboldface.
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a b
c d
e
f
Figure1CrystalposesoftheControlcompoundsanddockedposesoftheidentifiedcompounds.Crystallographicallyobservedbinding
modesoftheknownandnewligandsinNMPRTase;(a)NMN,(b)FK866,anddockedbindingmodesofcompounds4and5.(c)and(e)panels
indicatethefirstbindingmodesofcompounds5and4respectivelywhilepanels(d)and(f)indicatethesecondbindingmodesofcompounds
5and4respectively.Theligandsareinballandstickrepresentationandcoloredbystandardatomtypes;theAchainresiduesofthesiteare
showninredandCchainresiduesareinblue,inallthepanels.
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1) 2) 3)
4) 5) 6)
7) 8)
Figure22D- structuresof compounds.2D-structuresofthesixcompoundsalongwith thecontrolcompoundsFK866andNMN;panels
numbered1to6indicatethe2D-structuresforcompounds1-6,andpanelsnumbered7and8-forFK866andNMNrespectively.
We chose U87 cells as the source of NMPRTase. The compoundstoinhibitthegrowthofagliomaderivedcell
NMPRTase enzyme extract from U87 converted the 14 line U87, which haselevatedlevelsofPBEF1. FK866, the
[C] labeled nicotinamide to NAD+ (Figure 3C lane 1). knownNMPRTaseinhibitor,inhibitedthegrowthofU87
FK866, the known inhibitor of NMPRTase inhibited this cells efficiently with an IC of 170 μM (Figure 5 and
50
reaction efficiently (Figure 3C compare lane 2 with 1). Table 4). Of the six selected lead compounds, we found
As expected, the extract from U251 cells, which had thatonlycompounds1and5inhibitedthegrowthofU87
very low levels of PBEF1 transcript and protein, did not cellswithanIC of335and325μMrespectively(Figure
50
convert 14[C] labeled nicotinamide to NAD+ (Figure 3C 5 and Table 4). Since compound 1 did not inhibit
lane 3). We then tested the ability of six selected lead NMPRTase activity(Figure 4),itmightutilizea different
compounds to inhibit NMPRTase activity. We found mechanism to inhibit the growth of U87 cells. However,
that, of the six compounds tested, compounds 4 and 5 compound 5 inhibited NMPRTase activity as well as the
inhibited NMPRTase activity (Figure 4A and 4B) signifi- growthofU87cells.Furthertoconfirmthatinhibitionof
cantly. Compound 5 was found to be more potent in U87cellgrowthbyFK866andcompound5isbecauseof
NMPRTase inhibition (Figure 4A and 4B compare lanes theirabilitytoinhibitNMPRTase, we testedtheeffect of
23-26 with lanes 1 and 2). these two compounds on the growth U251 cells which
doesnotexpressNMPRTase.Asexpected,neitherFK866
Inhibition of growth PBEF1 over expressing glioblastoma nor compound 5 inhibited U251 cells, while adriamycin
cell line U87 inhibited very efficiently (data notshown).We thuscon-
TocorrelatetheNMPRTaseinhibitionpropertywithcell cludethatcompound5isapotentinhibitorofNMPRTase
growth inhibition, we then tested the ability of these andcancercellgrowth.
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A B
5 373 87 251 138 N229 343
4 PBEF1/NMPRtase U U U U L U
1 2 3 4 5 6
3
2
o PBEF1
ati
r 1
2
g
o
L 0
-1 TUBULIN
-2
3 7 9 8 3 1 8 0 T 0
37 U8 22 13 34 25 N1 48 Ca 10
U LN U U U L SW Ha HBL
Glioma
cell lines
C
C14 Nicotinamide
C14NAD
1 2 3 4
U87 + + - -
U251 - - + +
FK866 (5μM) - + - +
Figure3NMPRTasetranscriptandproteinlevelsingliomacellsandNMPRTaseenzymeassay.A.Log2-transformedgeneexpression
ratiosobtainedfromreal-timequantitativePCRanalysisareplottedforPBEF1.Eachbarrepresentsadataderivedfromtheindicatedcellline.In
eachsample,foldchangeingeneexpressioniscalculatedoveritsmeanexpressioninnormalbrainsamples.B.Equalamountsoftotalprotein
lysatesfromindicatedcelllinesweresubjectedtowesternblottingtodetectlevelsofPBEF1andTubulinproteins.C.NMPRTaseassaywas
carriedoutasdescribedinthemethodssectionwithextractsobtainedfromU87orU251cellseitherwithorwithouttheFK866.
Discussion Analysis of the docked poses indicate that two bind-
Virtual screening procedure carried out resulted in ing modes are observed within the large tunnel like
short-listing of six compounds which were obtained and binding pocket encompassing sites for both the natural
tested experimentally using both the enzyme inhibition product NMN and the partly overlapping larger inhibi-
assay as well as the cell growth inhibition assay. It is tor FK866. Given that the docking algorithm uses
indeed gratifying that two of the six compounds clearly heuristics, a typical docking simulation is carried out
exhibit enzyme inhibition. This clearly demonstrates the by repeating each run about 150 times, each time
usefulness of the docking based virtual screening using a different random number as a seed. This clus-
approach used here, utilizing three dimensional struc- tering pattern obtained subsequently for each docking
tural information at atomic level detail. simulation reflects the propensity of the ligand to
Chandraetal.JournalofClinicalBioinformatics2011,1:5 Page9of12
http://www.jclinbioinformatics.com/content/1/1/5
A
C14Nicotinamide
C14NAD
U87 U87 U87 + 5 μM 10 μM 15 μM 20 μM 25 μM 5 μM 10 μM 15 μM 20 μM25 μM 5 μM 10 μM 15 μM 20 μM 25 μM 5 μM 10 μM 15 μM 20 μM 25 μM 5 μM 10 μM 15 μM 20 μM 5 μM 10 μM 15 μM 20 μM 25 μM
F
K
8
66 Com. #1 Com. #2 Com. #3 Com. #4 Com. #5 Com. #6
(5
μ
M
)
5 M
B 10 M
15 M
120 20 M
25 M
100
d
e 80
c
u
d
o 60
r
p
D 40
A
N
% 20
0
1 2 3 4 5 6 7 8 9 101112 1314151617 1819202122 23242526 2728293031
U87 ext. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
FK866 (5μM) - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Com. #1 - - + + + + + - - - - - - - - - - - - - - - - - - - - - - - -
Com. #2 - - - - - - - + + + + + - - - - - - - - - - - - - - - - - - -
Com. #3 - - - - - - - - - - - - + + + + + - - - - - - - - - - - - - -
Com. #4 - - - - - - - - - - - - - - - - - + + + + + - - - - - - - - -
Com. #5 - - - - - - - - - - - - - - - - - - - - - - + + + + - - - - -
Com. #6 - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + +
Figure4NMPRTaseinhibitorscreen.A.NMPRTaseassaywascarriedoutasdescribedinthemethodssectionwithextractsobtainedfromU87
withindicatedamountsofeitherFK866orcompound1to6.B.TheamountofC14NAD+formedintheexperimentdescribedabove(A)is
measuredandshown.Pleasenotethatcompound5and4inhibitedtheNMPRTaseactivity.
occupy the binding site in the respective modes. Figure clearly illustrates that the identified compound 5 occu-
S1a-S1f shows the binding sites of all the six com- pies the same pocket as that of FK866.
pounds. For all the six compounds tested, distinct clus- The interactions observed in the first mode include a
ters were observed indicating the two modes of stacking interaction of the aromatic ring in the ligand
binding as top ranking poses, the first overlapping in with the side chains of F193 of one subunit and Y18 of
position with that of the natural product NMN and another subunit. In the second mode, interactions with
the second overlapping substantially with that of Y188, K189, Y240, S241, V242, S275, I309, R349, I351,
FK866. Figure S1i shows the superposition of the site and E376 are commonly seen. Figure 1 shows the bind-
of FK866 (as in 2GVJ) and the site corresponding to ing sites of both modes for compound 4, 5; single
the second binding mode of compound 5, which modes of NMN (as in 2GVG) and FK866 (as in 2GVJ).
Chandraetal.JournalofClinicalBioinformatics2011,1:5 Page10of12
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Figure5Gliomacellgrowthinhibitorscreen.ViabilitywasmeasuredbyMTTassayat48hrsafteradditionindicatedcompoundstoU87cells.
Theassayswerecarriedoutintriplicatesandthemeanvalueforeachtimepointwasusedtogeneratethegraph.
Table 4IC values forthe compounds It is interesting to note that majority of these residues
50
CompoundName IC (μM) are conserved in the NMPRTase proteins from several
50
FK866 170 sources and their close sequence homologues.
Interestingly, Compound 5 docked with the lowest
Niacin >500
binding energy of all the compounds in both modes. It
Nicotinamide >500
forms more number of hydrogen bonds with the binding
Compound1 335
site residues, all of them being crucial for NMN/FK866
Compound2 >500
binding. The two modes reflect the most plausible
Compound3 >500 modes of binding for the compounds studied, and any
Compound4 >500 one of them may be more important than the other
Compound5 325 for achieving inhibition. Irrespective of that, compound
Compound6 >500 5 is seen to be a better ligand in both the poses,
further corroborated by the experimental studies. To
ListofcompoundsandtheirIC50values.