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Development of Novel Triazolo-

Thiadiazoles from Heterogeneous

ldquoGreenrdquo Catalysis as Protein

Tyrosine Phosphatase 1B InhibitorsC P Baburajeev1 Chakrabhavi Dhananjaya Mohan2 Hanumappa Ananda2Shobith Rangappa3 Julian E Fuchs4 Swamy Jagadish2 Kodappully Sivaraman Siveen5Arunachalam Chinnathambi6 Sulaiman Ali Alharbi6 M E Zayed6 Jingwen Zhang5Feng Li5 Gautam Sethi5 Kesturu S Girish7 Andreas Bender4 Basappa1 ampKanchugarakoppal S Rangappa2

Condensed-bicyclic triazolo-thiadiazoles were synthesized via an ecient ldquogreenrdquo catalyst strategy

and identied as eective inhibitors of PTP1B in vitro The lead compound 6-(2-benzylphenyl)-3-

phenyl-[124]triazolo[3][134]thiadiazole (BPTT) was most eective against human hepatoma cells

inhibits cell invasion and decreases neovasculature in HUVEC and also tumor volume in EAT mouse

models This report describes an experimentally unidentied class of condensed-bicyclic triazolo-

thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds

Protein tyrosine phosphorylation plays a pivotal role in various growth actors signaling to induce cellprolieration differentiation and survival1 Protein tyrosine kinases (PKs) and protein tyrosine phos-phatases (PPs) are the two counteracting proteins which regulate tyrosine phosphorylation PKs phos-phorylate specific tyrosine residues in their substrate and PPs dephosphorylate them 23 Dysregulationin the activities o PKs and PPs have been entangled with tumorigenesis Recent findings suggestthe involvement o PPs in potentiating the action o PKs resulting in the neoplastic transormation 4PP1B is an ubiquitously expressed non-transmembrane phosphatase which belongs to the PP super-amily and the implication o PP1B in the dephosphorylation o Src (Y530) is well-demonstrated inprogression o oncogenesis in breast cancer cells5 More recently involvement o PP1B in regulationo multiple signaling cascades to promote tumor progression and survival is elucidated in various can-cers6ndash8 Several PP1B small molecule inhibitors have been designed and proved to induce their effectat sub-micro molar concentration but ailed to possess increased bioavailability due to reduced cellpermeability and hydrophobicity 9 Tereore PP1B has been emerged as a promising next generation

therapeutic target to design novel effective easily bioavailable drugs to fight against cancer10

Hereinwe adopted the synthetic strategy that could generate libraries o biologically active condensed-bicyclic

1Laboratory of Chemical Biology Department of Chemistry Bangalore University Palace Road Bangalore

560001 India 2Department of Studies in Chemistry University of Mysore Manasagangotri Mysore-570006

India 3Frontier Research Center for Post-genome Science and Technology Hokkaido University Sapporo

0600808 Japan 4Centre for Molecular Informatics Department of Chemistry University of Cambridge Lenseld

Road CB2 1EW Cambridge United Kingdom 5Department of Pharmacology Yong Loo Lin School of Medicine

National University of Singapore-117597 Singapore 6Department of Botany and Microbiology College of

Science King Saudi University Riyadh -11451 Kingdom of Saudi Arabia 7Department of Studies in Biochemistry

University of Mysore Manasagangotri Mysore-570006 India These authors contributed equally to this work

Correspondence and requests for materials should be addressed to B (email salundibasappagmailcom) or

KSR (email rangappaksgmailcom)

Received 15 January 2015

Accepted 18 August 2015

Published 21 September 2015

OPEN

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triazolo-thiadiazoles (CB) which are identified as inhibitors o PP1B via structure-based virtualscreening o over 2 million compounds o diverse scaffold in order make them orally available drugswith desirable physicochemical properties

ResultsChemistry Te synthesis o CB libraries entails the union o two nuclei which accelerated thedevelopment o biologically interesting molecules in chemical biology and drug discovery Various drugsincluding riazolam Ribavarin erconazole Itraconazole Etizolam Fluconazole Alprazolam Furacylinand Ceazoline possess triazoles and thiadiazoles as the backbone and have been built on these hetero-

cyclic nuclei Presence o= N-C-S moiety could be the possible reason or the broad spectrum o phar-macological effects o triazole-thiadiazoles1112 On the other hand effective synthesis o CB scaffoldrequires highly hazardous phosphorous oxy chloride and overnight stirring at reflux temperature Wepreviously reported the synthesis o CB scaffold using phosphorous oxy chloride as cyclizing agent13Initially we carried out the microwave-assisted one pot synthesis o 4-amino-5-phenyl-4H-124-triazole-3-thiol using methyl benzoate hydrazine hydrate and carbon disulphides In the next step we ocusedto synthesize CB libraries by using heterogeneous solid-acid catalysis which becomes ideal in greenchemistry due to its decreased reactor and plant correction problems reusability environmentally benignsaving o energy and more simple and convenient procedure 1415 Further we employed the applicationo recyclable solid-acid catalyst called glycerol-based carbon-SO3H to prepare the novel 36-disubstitut-ed(124)-triazolo(34-b)(134) thiadiazole derivatives Further the glycerol-based sulonic acid unction-alized carbon catalyst system was used to synthesize the libraries o CBs under optimized reactionconditions (Fig 1A) Te effect o solvents or the synthesis o CB was also studied (Supplementaryable 1) Te experiments were perormed to study the recyclability o the catalyst system and showed that

there was a significant reduction in the yield o the product afer the third run (Supplementary able 2)Newly prepared novel CBs are shown in Supplementary able 3 We also proposed the plausiblemechanism or the use o green-catalyst or the first time (Supplementary Scheme 1)

Pharmacology Identification o potent antiprolierative agent in CBs In order to know the bio-activity o CBs we investigated the cytotoxic effect o newly synthesized compounds on HepG2 cellsas described previously 1617 Among the panel o new triazolo-thiadiazole based compounds (6-(2-benzylphenyl)-3-phenyl-[124]triazolo[34ndashb][134]thiadiazole) (BP) which possess benzyl phenyl sub-stitutions on the triazolo-thiadiazole ring was presented as the most active antiprolierative agent againsthepatoma (HepG2) cells with an IC50 o 3micro M We considered the lead compound BP or urtherstudies

BPTT causes arrest of HepG2 cells in SubG1 phase During apoptosis activation o caspase acti- vated DNAse (CAD) leads to disintegration o genomic DNA into oligomeric ragments Te cells with

lesser DNA content leads to the ormation o hypodiploid cells and in turn accumulation o cells inSubG1 phase18 We urther investigated the effect o BP on cell cycle distribution o HepG2 cells asdescribed previously 1819 Hepatoma cells were treated with different concentrations o BP (0ndash20 micro M)and evaluated or pattern o cell cycle distribution using propidium iodide We ound the arrest o HepG2cells in SubG1 phase in the dose dependent manner with maximum activity at 20 micro M (Fig 1B) Tisresult suggests that BP was capable o inducing substantial apoptosis in HepG2 cells

BPTT inhibits the protein expression of PARP Bcl-2 Survivin Caspase-3 and Cyclin D1 inHepG2 cells Based on the observation that BP intereres with cell cycle distribution to induce itsantiprolierative effect we profiled the expression o cell cycle regulator (cyclin D1) apoptotic markers(caspase-3 and PARP) and antiapoptotic (Bcl-2 and Survivin) proteins We observed the downregula-tion o aoresaid proteins in a time dependent manner and complete inhibition o these protein expres-sion at 72 h (Fig 2A)

PTP1B inhibitory activity Several studies have showed that CBrsquos are known to inhibit the cata-lytic activity o PP1B Among more than 2 million o diverse structures tested in vitro CB was theonly scaffold identified as the non-structural inhibitor or PP1B20 Tereore we considered the leadcompound BP to evaluate or PP1B inhibitory activity We estimated the PP1B enzyme activity bydetermining the ree phosphate released rom the PP1B substrate in the presence and absence o BPusing PP1B inhibitor screening assay kit ( Abcam USA) Te compound BP inhibited the catalyticactivity o PP1B by 45 and 38 at the concentration o 30 micro M and 20micro M respectively (Fig 1C) Teinhibition o catalytic activity o PP1B by BP was significantly higher compared to the previouslyreported structures20

BPTT modulates the phosphorylation of STAT3 in HepG2 cells PP1B have been reported todownregulate phosphorylation o SA3 (Y705) and On the other hand PP1B inhibitors upregulatethe phosphorylation o SA321 Based on this observation we next evaluated the effect o BP onthe phosphorylation o SA3 at Y705 Western blotting analysis showed that treatment o HepG2 cellswith BP initially resulted in a decreased SA3 activation up to 60 minutes Tereafer a gradual

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increase in SA3 phosphorylation was noted in a time dependent manner up to 8 h ( Fig 2B) On theother hand PP1B have also been demonstrated to interere with VEGF-induced phosphorylation oVEGFR2 (Y1175)22 Hence next we examined the effect o BP on VEGF-stimulated phosphorylationo VEGFR2 in HUVEC On treatment with BP we observed only a marginal increase in the phos-phorylation o VEGFR2 (data not shown)

In silico interaction of BPTT with the phosphatase domain of the human PTP1B Furtherin silico docking was perormed to rationalize and compare the molecular interactions o the newly

Figure 1 (A) Schematic representation or the synthesis o CBs (B) BP causes arrest o HepG2 cells

in SubG1 phase Hepatoma cells treated with BP (0ndash20micro M) displayed the arrest o HepG2 cells in SubG1

phase o cell cycle (C) Inhibition o the catalytic activity o PP1B by BP was shown PP1B activity wasmeasured by determining the ree phosphate released rom the PP1B substrate with BP Optical density

was read at 620 nm and readings were converted to nmol o PO42minus Each column represents the means o

two determinations plt 005

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synthesized CB libraries with the reported structures towards PP1B As Park et al successullyemployed computational techniques to study interactions o CBrsquos with PP1B20 we aimed at a similardescription o protein-ligand interactions based on an X-ray structure o the phosphatase domain o

the human PP1B (PDB 2FH7) We prepared the structure or docking in MOE using protonate3D(Molecular operating environment) and removed two deeply buried water molecules resolved in thecrystal structure to allow a binding mode similar to the predictions o Park et al (waters 75 and 132)Computational docking studies predict the series o CBs to occupy the active site pocket o PP1Bsimilar to predictions o Park et al (Fig 2C) Te binding poses o CBs show major shape overlapand position aromatic rings in similar positions Te thiadiazole shows hydrogen bonding to the proteinbackbone whilst other ragments orm cation-pi interactions with Arg-1595 and pi-pi interactions withyr-1422 respectively In summary we ound that the newly synthesized compounds could serve aslead-structures that targets PP1B

BPTT mitigates VEGF-induced HUVEC capillary-like structure formation and viability in

vitro Sanggenon C is a phytochemical known to have inhibitory activity against PP1B and angiogen-esis23ndash25 Tereore we evaluated the effect o BP on angiogenesis using in vitro capillary tube ormationassay which represents a simple reliable and powerul model or studying inhibitors o angiogenesis26We examined the effect o BP on tubulogenesis in HUVECs in the presence and absence o VEGF

Figure 2 (A) BP inhibits the protein expression o PARP Bcl-2 Survivin Caspase-3 and Cyclin D1

in HepG2 cells HepG2 cells were treated with BP afer which whole-cell extracts were prepared andprotein was resolved on SDS-PAGE gel electrotranserred onto nitrocellulose membranes and probed orinterested antibodies and the gels in this figure are cropped ull length gels are presented in supplementaryfigure S1 (B) BP modulates the phosphorylation o SA3 at tyrosine-705 in time dependent manner

and has no effect on the protein expression o PP1B and the gels in this figure are cropped ull lengthgels are presented in supplementary figure S1 (C) Molecular interaction studies o BP towards PP1BPredicted binding modes o a thiadiazole derivative o Park et al11 (lef) and BP (right) are highly similar

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as described previously 27 When HUVECs were cultured on Matrigel they spontaneously orm threedimensional capillary-like tubular structures In presence o VEGF HUVECs orm robust tubular-likestructures when seeded on growth actorndashreduced two-dimensional Matrigel and BP treatment sub-stantially decreased the continuity and number o HUVEC capillary-like structures (Fig 3A)

BPTT suppresses VEGF-induced microvessel formation ex vivo Further the anti-angiogenicpotential o BP was demonstrated using the rat thoracic aortic ring assay an ex vivo angiogenesismodel28 Te serum-ree three-dimensional rat aortic model closely resembles the complexities o invivo angiogenesis rom endothelial activation to pericyte acquisition and remodeling26 We observedthe significant sprouting o microvessels on VEGF stimulation leading to the ormation o a networko vessels around the aortic rings reatment o BP significantly inhibited VEGF-induced sproutingo microvessels (Fig 3B) Te results o the capillary tube ormation and rat aortic assays significantlysupport the multiaceted role o BP in antiangiogenesis

BPTT suppresses CXCL12 induced migration of HepG2 cells PP1B regulates the breast can-cer cell invasion by modulating invadopodia dynamics29 and various studies have demonstrated therole o PP1B in cancer cell invasion30 In order to determine the efficacy o BP against invasion oHepG2 cells we perormed in vitro invasion assay using Bio-Coat Matrigel invasion assay system (BDBiosciences San Jose CA USA) as described earlier31 In this assay system we used CXCL12 as aninducer and addition o CXCL12 was ound to augment the invasive potential o HepG2 cells On treat-ment with BP we observed significant reduction in the motility o cells that could invade the Matrigelcoated polycarbonate membrane thereby indicating that BP substantially intereres with invasion o

HepG2 cells (Fig 3C)

In vivo Ehrlich Ascites Tumor model Given the relevance with the results o ex vivo experimentswe also evaluated the in vivo antiangiogenic potential o BP via intraperitoneal administration in anEhrlich ascites tumor model as described earlier3233 It was ound that BP at the concentration o10 mgkg induced significant decrease o body weight tumor volume ( Fig 4AB) and peritoneal angi-ogenesis (Fig 5A) compared with the DMSO-treated controls Te unpaired ANOVA t test showed astatistically significant difference in tumor growth between the BP-treated and control groups Weurther measured microvessel density in peritoneum o experimental animals using H amp E stainingand ound the reduction o angiogenesis by 4468 and 4681 in BP and topotecan treated grouprespectively (Fig 5B)

DiscussionIn the present study we report the novel bioactive triazolo-thiadiazole derivative that targets PP1B anddemonstrated the mechanism o action in vitro and in vivo PP1B is an abundantly expressed proteintyrosine phosphatase associated with endoplasmic reticulum and it has been linked with regulation oseveral cellular events including cell growth differentiation and transormation5 It was identified thatPP1B negatively regulates tyrosine phosphorylation o several upstream signaling proteins includingc-Src JAK2 VEGFR2 and insulin receptors2934 Tough SHP1 SHP2 and HCPPA are known to inter-act with c-Src JAK2 and VEGFR2 their role in physiological conditions remain unclear 35ndash37 Te mainchallenge in targeting PP1B relies in selective inhibition due to high degree o homology among PPs 9Te prior idea about the CB being an effective scaffold against PP1B we identified and evaluatedlead structure BP against PP1B enzyme activity Further we validated the effect o BP on SA3and VEGFR2 in HepG2 cells and HUVECs respectively Initially up to 90 mins we observed the decreasein SA3 phosphorylation this is unexpected because PP1B dephosphorylates JAK2 in turn down-regulates SA3 phosphorylation However we observed gradual increase in SA3 phosphorylation(Y705) afer 90 mins o incubation with BP We did not observe change in the protein expression oPP1B indicating that BP directly intereres with the catalytic activity and had no effect on its protein

expression PP1B negatively regulates autophosphorylation o VEGFR2 via binding to the cytoplas-mic domain o VEGFR2 in endothelial cells35 We tried to analyze phospho-VEGFR2 in BP treatedHUVECs and observed only a marginal increase in the phosphorylation (data not shown) SanggenonC a phytochemical isolated rom Chinese medicinal plant Morus mongolica has been ound to exhibitinhibitory activity against PP1B and also demonstrated to possess antiangiogenic properties via inhibi-tion o hypoxia-inducible actor-1α BP appears to have similar inhibitory effect against PP1B andangiogenesis and needs to be explored urther or its potential against human diseases

ConclusionHerein or the first time we report the use o heterogeneous ldquogreenrdquo catalyst to prepare the noveltriazolo-thiadiazoles We have depicted target o lead structure as PP1B and experimentally demon-strated the same Moreover this report describes an experimentally unidentified class o small moleculestargeting PP1B Further derivatization o the lead structure may result in drug-seeds with enhanced

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Figure 3 (A) In vitro anti-angiogenic activity o BP using HUVEC In presence o VEGF HUVECs ormtubular structures on the Matrigel and in the presence o BP substantially decreased the continuity andnumber o HUVEC capillary-like structures (B) Inhibitory activity o BP on rat-aortic ring ormation by

fibro-adipose tissue o Sprague-Dawley rats Te treatment o BP significantly inhibited VEGF-inducedsprouting o microvessels (C) In vitro anti-invasive activity o BP using HepG2 cells In this assay systemwe used CXCL12 as an inducer o invasion o HepG2 cells Te treatment with HepG2 cells reduced the

motility o cells that could invade Matrigel Data are the representatives o three independent experimentsplt 005

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PP1B inhibitory activity Tis finding opens an avenue or the development o novel triazolo-thiadiazoles

based small molecules as therapeutic agents that target PP1B in human diseases

MethodsSynthesis Te synthesis and physical amp chemical characterization and spectral data on CB wereprovided as a supplementary file

MTT assay Te anti-prolierative effect o new compounds against HepG2 cells was determined bythe M dye uptake method as described previously 38 Briefly the cells (25times 104ml) were incubated intriplicate in a 96-well plate in the presence or absence o different concentrations o BP in a final vol-ume o 02 ml or indicated time intervals at 37 degC Tereafer 20 micro l M solution (5 mgml in PBS) wasadded to each well Afer 2 h incubation at 37 degC 01 ml lysis buffer (20 SDS 50 dimethyl-ormamide)was added incubation was continued overnight at 37 degC and then the optical density (OD) at 570 nmwas measured by ecan plate reader

Flow cytometric analysis Te effect o BP on cell cycle o HepG2 cells was perormed asdescribed previously 18 o determine the effect o BP on the cell cycle cells were treated with BPat indicated doses up to 48 h Tereafer cells were washed fixed with 70 ethanol and incubated or30 min at 37 degC with 01 RNase A in PBS Cells were then washed again resuspended and stained inPBS containing 25micro gml propidium iodide (PI) or 30 min at room temperature Cell distribution acrossthe cell cycle was analyzed with a BD FACSVerse flow cytometer

Western Blotting Western blot analysis was perormed as previously described39 Briefly BPtreated MDA-MB-231whole-cell extracts were lysed in lysis buffer (20 mM ris pH 74) 250 mM NaCl2 mM EDA (pH 80) 01 riton X-100 001 mgml aprotinin 0005 mgml leupeptin 04 mM PMSFand 4 mM NaVO4) Lysates were then spun at 14000 rpm or 10 min to remove insoluble material andprotein concentration was quantified Tereafer proteins were resolved on SDS gel Afer electropho-resis the proteins were electrotranserred to a nitrocellulose membrane blocked with 5 nonat milkand probed with various antibodies overnight at 4 degC Te blot was washed exposed to HRP-conjugated

secondary antibodies or 1 h and finally examined by chemiluminescence (ECL GE Healthcare)

PTP1B inhibitory activity PP1B activity was measured by determining the ree phosphate releasedrom the PP1B substrate with a PP1B Inhibitor Screening Assay kit rom Abcam 2xPP1B substratesolutions (120micro M) 2xPP1B Enzyme solutions (2 ngwell) and BP solutions (0ndash30micro M) were pre-pared and added to the 96 wells plate as described in the kit instructions Afer incubations at 30 degC or30 mins reactions were then terminated by addition o 25 micro l o Red Reagent and optical density is readat 620 nm Te readings were converted to nmol o PO4

2minus with a phosphate standard curve

Molecular Modeling In silico docking was carried out using MOE Te coordinates o the thiadiazolemoiety kindly provided by Park et al were chosen as grid centre or docking with deault parametersin MOE Predicted binding poses were visualized using Pymol

Invasion assay Te in vitro invasion assay was perormed using Bio-Coat Matrigel invasion assaysystem (BD Biosciences San Jose CA USA) according to the manuacturerrsquos instructions 1times 105

Figure 4 (A) Relative body weight o EA bearing mice treated with vehicle alone BP and topotecan(B) Relative ascites fluid in EA bearing mice treated with vehicle alone BP and topotecan Data are

represented as meanplusmn SE plt 005

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7SCIENTIFIC REPORTS | 514195 | DOI 101038srep14195

PP1B inhibitory activity Tis finding opens an avenue or the development o novel triazolo-thiadiazoles

based small molecules as therapeutic agents that target PP1B in human diseases

MethodsSynthesis Te synthesis and physical amp chemical characterization and spectral data on CB wereprovided as a supplementary file

MTT assay Te anti-prolierative effect o new compounds against HepG2 cells was determined bythe M dye uptake method as described previously 38 Briefly the cells (25times 104ml) were incubated intriplicate in a 96-well plate in the presence or absence o different concentrations o BP in a final vol-ume o 02 ml or indicated time intervals at 37 degC Tereafer 20 micro l M solution (5 mgml in PBS) wasadded to each well Afer 2 h incubation at 37 degC 01 ml lysis buffer (20 SDS 50 dimethyl-ormamide)was added incubation was continued overnight at 37 degC and then the optical density (OD) at 570 nmwas measured by ecan plate reader

Flow cytometric analysis Te effect o BP on cell cycle o HepG2 cells was perormed asdescribed previously 18 o determine the effect o BP on the cell cycle cells were treated with BPat indicated doses up to 48 h Tereafer cells were washed fixed with 70 ethanol and incubated or30 min at 37 degC with 01 RNase A in PBS Cells were then washed again resuspended and stained inPBS containing 25micro gml propidium iodide (PI) or 30 min at room temperature Cell distribution acrossthe cell cycle was analyzed with a BD FACSVerse flow cytometer

Western Blotting Western blot analysis was perormed as previously described39 Briefly BPtreated MDA-MB-231whole-cell extracts were lysed in lysis buffer (20 mM ris pH 74) 250 mM NaCl2 mM EDA (pH 80) 01 riton X-100 001 mgml aprotinin 0005 mgml leupeptin 04 mM PMSFand 4 mM NaVO4) Lysates were then spun at 14000 rpm or 10 min to remove insoluble material andprotein concentration was quantified Tereafer proteins were resolved on SDS gel Afer electropho-resis the proteins were electrotranserred to a nitrocellulose membrane blocked with 5 nonat milkand probed with various antibodies overnight at 4 degC Te blot was washed exposed to HRP-conjugated

secondary antibodies or 1 h and finally examined by chemiluminescence (ECL GE Healthcare)

PTP1B inhibitory activity PP1B activity was measured by determining the ree phosphate releasedrom the PP1B substrate with a PP1B Inhibitor Screening Assay kit rom Abcam 2xPP1B substratesolutions (120micro M) 2xPP1B Enzyme solutions (2 ngwell) and BP solutions (0ndash30micro M) were pre-pared and added to the 96 wells plate as described in the kit instructions Afer incubations at 30 degC or30 mins reactions were then terminated by addition o 25 micro l o Red Reagent and optical density is readat 620 nm Te readings were converted to nmol o PO4

2minus with a phosphate standard curve

Molecular Modeling In silico docking was carried out using MOE Te coordinates o the thiadiazolemoiety kindly provided by Park et al were chosen as grid centre or docking with deault parametersin MOE Predicted binding poses were visualized using Pymol

Invasion assay Te in vitro invasion assay was perormed using Bio-Coat Matrigel invasion assaysystem (BD Biosciences San Jose CA USA) according to the manuacturerrsquos instructions 1times 105

Figure 4 (A) Relative body weight o EA bearing mice treated with vehicle alone BP and topotecan(B) Relative ascites fluid in EA bearing mice treated with vehicle alone BP and topotecan Data are

represented as meanplusmn SE plt 005

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HepG2 cells were suspended in serum-ree DMEM and seeded into the Matrigel transwell chambersconsisting o polycarbonate membranes with 8micro m pores Afer pre-incubation with or without BP or8 h the transwell chambers were then placed into appropriate wells o a 24-well plate in which either thebasal medium only or basal medium containing CXCL12 had been added Afer incubation the uppersuraces o the ranswell chambers were wiped with cotton swabs and the invading cells were fixed andstained with crystal violet solution Te invading cells were then counted in five randomly selected areasunder microscopic observation

Capillary-like tube formation assay ube ormation was assessed as described previously 27 BrieflyHUVECs were pretreated with various dilutions o BP or 12 h and then seeded onto the Matrigellayer in 24-well plates at a density o 5times 104 cells in Medium 200 with or without VEGF Afer 6 h tubu-lar structure o endothelial cells was photographed using an inverted microscope Tree independentexperiments were perormed

Rat aortic ring assay Rat aortic ring assay was perormed as described previously 28 In brie aortasisolated rom Sprague-Dawley rats were cleaned o fibro adipose tissue and colateral vessels and cutinto approximately 1 mm long rings Te aortic rings were randomized into Growth Factor Reduced

Figure 5 (A) In vivo anti-angiogenic activity o BP in the peritoneal cavity o EA implanted mouse

model (B) Analysis o angiogenesis (MVD) in the peritoneum o EA implanted mouse using H amp E staining

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Matrigel-coated wells and urther sealed with a 100micro l overlay o Matrigel Medium 200 containing withand without VEGF along with different dilutions o BP was added to the wells and incubated at37 degC5 CO2 or 6 days At the end o incubation the microvessel sprouting ormed were fixed andphotographed using a Nikon inverted microscope (magnification 100X) wo independent experimentswere perormed

In vivo tumor model studies 6ndash8 weeks old emale Swiss albino mice were kept in a 12 h light and12 h dark cycle and ed with standard chow ormula and reverse osmosis water and were acclimatizedor 1 week and were used or establishing Ehrlich ascites tumor (EA) model or urther experiments as

described earlier40 Animals were obtained and maintained in the animal house Department o Studiesin Zoology Manasagangotri Mysuru India Te animal experiment was reviewed and approved by theInstitutional Animal Ethical Committee University o Mysore Mysuru Animal handling and experi-ments were carried out in accordance with the guidelines o the Committee or the Purpose o Controland Supervision o Experiments on Animals (CPCSEA) Experimental animals were injected with 5times 106

viable EA cells intraperitoneally and weight o the animals were recorded daily up to 11th day to analyzethe tumor growth o investigate the in vivo efficacy o BP we injected 10 mgkg body weight o BPinto the peritoneum o the EA bearing mice daily rom the 5th day o transplantation Te animals weresacrificed on 12th day and ascites fluid was collected by making an incision in the abdomen Te numbero microvessels in a hotspot adjacent to tumor mass was analysed using H amp E staining to determine theeffect o BP on angiogenesis Te peritoneal cavity o DMSO-treated topotecan treated and BPtreated animals were photographed

Statistical analysis Te mean values are expressedplusmn SE or control and experimental samples andeach experiment is repeated a minimum o two times Statistical analysis was perormed by one-wayANOVA Plt 005 was considered statistically significant (GraphPad Prism version 60 GraphPad Sofware)

References1 Hubbard S 983122 amp ill J H Protein tyrosine 983147inase structure and unction Annual review o biochemistry 69 373ndash398 doi

101146annurevbiochem691373 (2000)2 den Hertog J Protein-tyrosine phosphatases in development Mechanisms o Development 85 3ndash14 httpdxdoiorg101016

S0925-4773(99)00089-1 (1999)3 Pagano M A ibaldi E Gringeri E amp Brunati A M yrosine phosphorylation and liver regeneration A glance at intracellular

transducers IUBMB Lie 64 27ndash35 doi 101002iub576 (2012)4 Jiang Z X amp Zhang Z Y argeting PPs with small molecule inhibitors in cancer treatment Cancer metastasis reviews 27

263ndash272 doi 101007s10555-008-9113-3 (2008)5 Bjorge J D Pang A amp Fujita D J Identification o protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity

capable o dephosphorylating and activating c-Src in several human breast cancer cell lines Te Journal o biological chemistry 275 41439ndash41446 doi 101074jbcM004852200 (2000)

6 Bentires-Alj M amp Neel B G Protein-tyrosine phosphatase 1B is required or HE9831222Neu-induced breast cancer Cancer research 67 2420ndash2424 doi 1011580008-5472can-06-4610 (2007)

7 Julien S G et al Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis andprotects rom lung metastasis Nat Genet 39 338ndash346 doi 101038ng1963 (2007)

8 He 983122 J Yu Z H Zhang 983122 Y amp Zhang Z Y Protein tyrosine phosphatases as potential therapeutic targets Acta pharmacologicaSinica 35 1227ndash1246 doi 101038aps201480 (2014)

9 Zhang S amp Zhang Z Y PP1B as a drug target recent developments in PP1B inhibitor discovery Drug discovery today 12 373ndash381 doi 101016jdrudis200703011 (2007)

10 Bialy L amp Waldmann H Inhibitors o protein tyrosine phosphatases next-generation drugs Angewandte Chemie (Internationaled in English) 44 3814ndash3839 doi 101002anie200461517 (2005)

11 983115ini G D 983122obins 983122 983115 amp Avery L Synthesis and antitumor activity o ribavirin imidates A new acile synthesis o ribavirinamidine (1-beta-D-ribouranosyl-124-triazole-3-carboxamidine hydrochloride) Journal o medicinal chemistry 32 1447ndash1449(1989)

12 Nishida M Matsubara Mura983147awa Mine Y amp Yo983147ota Y Ceazolin a new semisynthetic cephalosporin antibiotic II Invitro and in vivo antimicrobial activity Te Journal o antibiotics 23 137ndash148 (1970)

13 Swamy S N et al Synthesis o pharmaceutically important condensed heterocyclic 46-disubstituted-124-triazolo-134-thiadiazole derivatives as antimicrobials European journal o medicinal chemistry 41 531ndash538 doi 101016jejmech200512009(2006)

14 Collis A E amp Horvath I Heterogenization o homogeneous catalytic systems Catalysis Science amp echnology 1 912ndash919 (2011)15 Li Z et al Silica-supported aluminum chloride A recyclable and reusable catalyst or one-pot three-component Mannich-type

reactions Journal o Molecular Catalysis A Chemical 272 132ndash135 (2007)16 Bharath983147umar H et al Synthesis biological evaluation and in silico and in vitro mode-o-action analysis o novel

dihydropyrimidones targeting PPA983122-[gamma] 983122SC Advances 4 45143ndash45146 doi 101039C4983122A08713E (2014)17 983115eerthy H 983115 et al Novel synthetic biscoumarins target tumor necrosis actor-alpha in hepatocellular carcinoma in vitro and in

vivo Te Journal o biological chemistry 289 31879ndash31890 doi 101074jbcM114593855 (2014)18 Mohan C D et al Development o a novel azaspirane that targets the Janus 983147inase-signal transducer and activator o transcription

(SA) pathway in hepatocellular carcinoma in vitro and in vivo Te Journal o biological chemistry 289 34296ndash34307 doi101074jbcM114601104 (2014)

19 Srivastava M et al Sapodilla plum (Achras sapota) induces apoptosis in cancer cell lines and inhibits tumor progression in miceScientific reports 4 6147 doi 101038srep06147 (2014)

20 Par983147 H Chien P N amp 983122yu S E Discovery o potent inhibitors o receptor protein tyrosine phosphatase sigma through thestructure-based virtual screening Bioorganic amp medicinal chemistry letters 22 6333ndash6337 doi 101016jbmcl201208081 (2012)

21 Zabolotny J M et al PP1B regulates leptin signal transduction in vivo Developmental cell 2 489ndash495 (2002)22 Na983147ayama M amp Berger P Coordination o VEGF receptor traffic983147ing and signaling by coreceptors Experimental cell research

319 1340ndash1347 (2013)

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1 0SCIENTIFIC REPORTS | 514195 | DOI 101038srep14195

23 Lim 983115 Edible medicinal and non-medicinal plants Vol 1 (Springer 2012)24 Zhang E H Wang 983122 F Guo S Z amp Liu B An update on antitumor activity o naturally occurring chalcones Evidence-based

complementary and alternative medicine eCAM 2013 815621 doi 1011552013815621 (2013)25 Dat N et al Hypoxia-Inducible Factor-1 Inhibitory Benzourans and Chalcone-Derived Dielsminus Alder Adducts rom Morus

Species Journal o natural products 72 39ndash43 (2008)26 Siveen 983115 S et al γ -tocotrienol inhibits angiogenesis-dependent growth o human hepatocellular carcinoma through abrogation

o A983115mO983122 pathway in an orthotopic mouse model Oncotarget 5 1897 (2014)27 Ponce M L ube ormation an in vitro matrigel angiogenesis assay Methods in molecular biology (Clifon NJ) 467 183ndash188

doi 101007978-1-59745-241-0_10 (2009)28 Nicosia 983122 F amp Ottinetti A Modulation o microvascular growth and morphogenesis by reconstituted basement membrane gel

in three-dimensional cultures o rat aorta a comparative study o angiogenesis in matrigel collagen fibrin and plasma clot In

vitro cellular amp developmental biology journal o the issue Culture Association 26 119ndash128 (1990)29 Cortesio C L et al Calpain 2 and PP1B unction in a novel pathway with Src to regulate invadopodia dynamics and breast

cancer cell invasion Te Journal o cell biology 180 957ndash971 doi 101083jcb200708048 (2008)30 Lessard L et al PP1B is an androgen receptor-regulated phosphatase that promotes the progression o prostate cancer Cancer

research 72 1529ndash1537 doi 1011580008-5472can-11-2602 (2012)31 Neelgundmath M et al Novel synthetic coumarins that targets NF-983147appaB in Hepatocellular carcinoma Bioorganic amp medicinal

chemistry letters 25 893ndash897 doi 101016jbmcl201412065 (2015)32 983122oopashree 983122 et al Novel synthetic bisbenzimidazole that targets angiogenesis in Ehrlich ascites carcinoma bearing mice

Bioorganic amp medicinal chemistry letters 25 2589ndash2593 (2015)33 Anil 983115umar C Nanjunda Swamy S Gaon983147ar S Salimath B P amp 983122angappa 983115 S N-substituted-2-butyl-5-chloro-3H-

imidazole-4-carbaldehyde derivatives as anti-tumor agents against Ehrlich ascites tumor cells in vivo Medicinal Chemistry 3 269ndash276 (2007)

34 Zhou Y amp 983122ui L Leptin signaling and leptin resistance Frontiers o medicine 7 207ndash222 doi 101007s11684-013-0263-5(2013)

35 Na983147amura Y et al 983122ole o protein tyrosine phosphatase 1B in vascular endothelial growth actor signaling and cell-cell adhesionsin endothelial cells Circulation research 102 1182ndash1191 doi 101161circresaha107167080 (2008)

36 983122ajendran P et al γ ‐ocotrienol is a novel inhibitor o constitutive and inducible SA3 signalling pathway in human

hepatocellular carcinoma potential role as an antiprolierative pro‐apoptotic and chemosensitizing agent British journal o pharmacology 163 283ndash298 (2011)

37 Sugahara 983115 Timmaiah 983115 N Bid H 983115 Houghton P J amp 983122angappa 983115 S Anti-tumor activity o a novel HS-mimetic- vascular endothelial growth actor binding small molecule PloS one 7 e39444 doi 101371journalpone0039444 (2012)

38 983115eerthy H 983115 et al Synthesis and characterization o novel 2-amino-chromene-nitriles that target Bcl-2 in acute myeloidleu983147emia cell lines PloS one 9 e107118 doi 101371journalpone0107118 (2014)

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AcknowledgementsWe are thankul to Pro Suguna Rao Department o Veterinary Pathology Veterinary College HebbalBengaluru or providing acility to carryout in vivo studies We are thankul to Dr J Shankar and Mr KS Balaji eresian College Mysore or their kind help in in vivo experiments Tis research was supportedby University Grants Commission (41-257-2012-SR) Vision Group Science and echnology Departmento Science and echnology (NO SRFLS-1422012) to B KSR thanks to Council o Scientific ampIndustrial Research (No 01 (2434) 10 EMR-II Dated 28122010) Institution o Excellence Universityo Mysore and University Grants Commission (FNo39-1062010 (SR) Dated 24-12-2010) or undingCDM thanks University o Mysore or Department o Science and echnology-Promotion o UniversityResearch and Scientific Excellence Research Associate Fellowship and AB thanks the European ResearchCommission or an ERC Starting Grant Tis work was also supported by NUHS Bench-to-Bedsidegrants to GS GS is also supported by Te Deanship o Scientific Research College o Science ResearchCentre King Saud University Kingdom o Saudi Arabia AH thanks ICMR (SRF No 45542013-PHABMS Dated 08122014) or Senior Research Fellowship

Author ContributionsCPB perormed the chemistry experiments AH CDM SJ KSS and FL perormed the biologyexperiments B and JEF perormed the computational experiments and assisted in writing themanuscript SR AC JZ MEZ KSG and SAA assisted with biology experiments GS and KSRprovided tools or research and verified the biology results and assisted in article preparation ABcontributed to in silico study design provided tools or research and verified the data and assisted inwriting the article B and KSR designed the project provided reagents and analysed the data andwrote the article All authors reviewed the manuscript

Additional InformationSupplementary information accompanies this paper at httpwwwnaturecomsrep

Competing financial interests Te authors declare no competing financial interests

How to cite this article Baburajeev C P et al Development o Novel riazolo-Tiadiazoles romHeterogeneous Green Catalysis as Protein yrosine Phosphatase 1B Inhibitors Sci Rep 5 14195 doi101038srep14195 (2015)

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Tis work is licensed under a Creative Commons Attribution 40 International License Teimages or other third party material in this article are included in the articlersquos Creative Com-

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