Development of Novel Triazolo-Thiadiazoles from Heterogeneous “Green” Catalysis as Protein Tyrosine Phosphatase 1B Inhibitors

Condensed-bicyclic triazolo-thiadiazoles were synthesized via an efficient “green” catalyst strategy and identified as effective inhibitors of PTP1B in vitro. The lead compound, 6-(2-benzylphenyl)-3-phenyl-[1,2,4]triazolo[3][1,3,4]thiadiazole (BPTT) was most effective 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 unidentified class of condensed-bicyclic triazolo-thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds.

Scientific RepoRts | 5:14195 | DOi: 10.1038/srep14195 triazolo-thiadiazoles (CBTT), which are identified as inhibitors of PTP1B via structure-based virtual screening of over 2 million compounds of diverse scaffold in order make them orally available drugs with desirable physicochemical properties.

Results
Chemistry. The synthesis of CBTT libraries entails the union of two nuclei, which accelerated the development of biologically interesting molecules in chemical biology and drug discovery. Various drugs including Triazolam, Ribavarin, Terconazole, Itraconazole, Etizolam, Fluconazole, Alprazolam, Furacylin and Cefazoline possess triazoles and thiadiazoles as the backbone and have been built on these heterocyclic nuclei. Presence of = N-C-S moiety could be the possible reason for the broad spectrum of pharmacological effects of triazole-thiadiazoles 11,12 . On the other hand, effective synthesis of CBTT scaffold requires highly hazardous phosphorous oxy chloride and overnight stirring at reflux temperature. We previously reported the synthesis of CBTT scaffold using phosphorous oxy chloride as cyclizing agent 13 . Initially, we carried out the microwave-assisted one pot synthesis of 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiol using methyl benzoate, hydrazine hydrate and carbon disulphides. In the next step, we focused to synthesize CBTT libraries by using heterogeneous solid-acid catalysis, which becomes ideal in green chemistry due to its decreased reactor and plant correction problems, reusability, environmentally benign, saving of energy and more simple and convenient procedure 14,15 . Further, we employed the application of recyclable solid-acid catalyst, called glycerol-based carbon-SO 3 H to prepare the novel 3,6-disubstituted(1,2,4)-triazolo (3,4-b) (1,3,4) thiadiazole derivatives. Further, the glycerol-based sulfonic acid functionalized carbon catalyst system was used to synthesize the libraries of CBTTs under optimized reaction conditions (Fig. 1A). The effect of solvents for the synthesis of CBTT was also studied (Supplementary Table 1). The experiments were performed to study the recyclability of the catalyst system and showed that there was a significant reduction in the yield of the product after the third run (Supplementary Table 2). Newly prepared novel CBTTs are shown in Supplementary Table 3. We also proposed the plausible mechanism for the use of green-catalyst for the first time (Supplementary Scheme 1).
BPTT causes arrest of HepG2 cells in SubG1 phase. During apoptosis, activation of caspase activated DNAse (CAD) leads to disintegration of genomic DNA into oligomeric fragments. The cells with lesser DNA content leads to the formation of hypodiploid cells and in turn accumulation of cells in SubG1 phase 18 . We further investigated the effect of BPTT on cell cycle distribution of HepG2 cells as described previously 18,19 . Hepatoma cells were treated with different concentrations of BPTT (0-20 μ M) and evaluated for pattern of cell cycle distribution using propidium iodide. We found the arrest of HepG2 cells in SubG1 phase in the dose dependent manner with maximum activity at 20 μ M (Fig. 1B). This result suggests that BPTT was capable of inducing substantial apoptosis in HepG2 cells.
BPTT inhibits the protein expression of PARP, Bcl-2, Survivin, Caspase-3 and Cyclin D1 in HepG2 cells. Based on the observation that BPTT interferes with cell cycle distribution to induce its antiproliferative effect, we profiled the expression of cell cycle regulator (cyclin D1), apoptotic markers (caspase-3, and PARP) and antiapoptotic (Bcl-2, and Survivin) proteins. We observed the downregulation of aforesaid proteins in a time dependent manner and complete inhibition of these protein expression at 72 h ( Fig. 2A).

PTP1B inhibitory activity.
Several studies have showed that CBTT's are known to inhibit the catalytic activity of PTP1B. Among more than 2 million of diverse structures tested in vitro, CBTT was the only scaffold identified as the non-structural inhibitor for PTP1B 20 . Therefore, we considered the lead compound BPTT to evaluate for PTP1B inhibitory activity. We estimated the PTP1B enzyme activity by determining the free phosphate released from the PTP1B substrate in the presence and absence of BPTT using PTP1B inhibitor screening assay kit (Abcam, USA). The compound BPTT inhibited the catalytic activity of PTP1B by 45% and 38% at the concentration of 30 μ M and 20 μ M respectively (Fig. 1C). The inhibition of catalytic activity of PTP1B by BPTT was significantly higher compared to the previously reported structures 20 .
BPTT modulates the phosphorylation of STAT3 in HepG2 cells. PTP1B have been reported to downregulate phosphorylation of STAT3 (Y705) and On the other hand, PTP1B inhibitors upregulate the phosphorylation of STAT3 21 . Based on this observation, we next evaluated the effect of BPTT on the phosphorylation of STAT3 at Y705. Western blotting analysis showed that treatment of HepG2 cells with BPTT initially resulted in a decreased STAT3 activation up to 60 minutes. Thereafter, a gradual increase in STAT3 phosphorylation was noted in a time dependent manner up to 8 h (Fig. 2B). On the other hand, PTP1B have also been demonstrated to interfere with VEGF-induced phosphorylation of VEGFR2 (Y1175) 22 . Hence, next, we examined the effect of BPTT on VEGF-stimulated phosphorylation of VEGFR2 in HUVEC. On treatment with BPTT, we observed only a marginal increase in the phosphorylation of VEGFR2 (data not shown).
In silico interaction of BPTT with the phosphatase domain of the human PTP1B. Further, in silico docking was performed to rationalize and compare the molecular interactions of the newly Tyr-1422 respectively. In summary, we found that the newly synthesized compounds could serve as lead-structures that targets PTP1B.
BPTT mitigates VEGF-induced HUVEC capillary-like structure formation and viability in vitro. Sanggenon C is a phytochemical known to have inhibitory activity against PTP1B and angiogenesis [23][24][25] . Therefore, we evaluated the effect of BPTT on angiogenesis using in vitro capillary tube formation assay which represents a simple, reliable and powerful model for studying inhibitors of angiogenesis 26 . We examined the effect of BPTT on tubulogenesis in HUVECs in the presence and absence of VEGF in HepG2 cells. HepG2 cells were treated with BPTT, after which whole-cell extracts were prepared, and protein was resolved on SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for interested antibodies and the gels in this figure are cropped, full length gels are presented in supplementary figure S1. (B) BPTT modulates the phosphorylation of STAT3 at tyrosine-705 in time dependent manner and has no effect on the protein expression of PTP1B and the gels in this figure are cropped, full length gels are presented in supplementary figure S1. (C) Molecular interaction studies of BPTT towards PTP1B; Predicted binding modes of a thiadiazole derivative of Park et al. 11 (left) and BPTT (right) are highly similar.
Scientific RepoRts | 5:14195 | DOi: 10.1038/srep14195 as described previously 27 . When HUVECs were cultured on Matrigel, they spontaneously form three dimensional capillary-like tubular structures. In presence of VEGF, HUVECs form robust tubular-like structures when seeded on growth factor-reduced two-dimensional Matrigel and BPTT treatment substantially decreased the continuity and number of HUVEC capillary-like structures (Fig. 3A).

BPTT suppresses VEGF-induced microvessel formation ex vivo. Further, the anti-angiogenic
potential of BPTT was demonstrated using the rat thoracic aortic ring assay, an ex vivo angiogenesis model 28 . The serum-free three-dimensional rat aortic model closely resembles the complexities of in vivo angiogenesis from endothelial activation to pericyte acquisition and remodeling 26 . We observed the significant sprouting of microvessels on VEGF stimulation, leading to the formation of a network of vessels around the aortic rings. Treatment of BPTT significantly inhibited VEGF-induced sprouting of microvessels (Fig. 3B). The results of the capillary tube formation and rat aortic assays significantly support the multifaceted role of BPTT in antiangiogenesis.
BPTT suppresses CXCL12 induced migration of HepG2 cells. PTP1B regulates the breast cancer cell invasion by modulating invadopodia dynamics 29 and various studies have demonstrated the role of PTP1B in cancer cell invasion 30 . In order to determine the efficacy of BPTT against invasion of HepG2 cells, we performed in vitro invasion assay using Bio-Coat Matrigel invasion assay system (BD Biosciences, San Jose, CA, USA), as described earlier 31 . In this assay system, we used CXCL12 as an inducer and addition of CXCL12 was found to augment the invasive potential of HepG2 cells. On treatment with BPTT, we observed significant reduction in the motility of cells that could invade the Matrigel coated polycarbonate membrane, thereby indicating that BPTT substantially interferes with invasion of HepG2 cells (Fig. 3C).

In vivo Ehrlich Ascites Tumor model. Given the relevance with the results of ex vivo experiments,
we also evaluated the in vivo antiangiogenic potential of BPTT via intraperitoneal administration in an Ehrlich ascites tumor model as described earlier 32,33 . It was found that BPTT at the concentration of 10 mg/kg induced significant decrease of body weight, tumor volume (Fig. 4A,B) and peritoneal angiogenesis (Fig. 5A) compared with the DMSO-treated controls. The unpaired ANOVA t test showed a statistically significant difference in tumor growth between the BPTT-treated and control groups. We further measured microvessel density in peritoneum of experimental animals using H & E staining and found the reduction of angiogenesis by 44.68% and 46.81% in BPTT and topotecan treated group respectively (Fig. 5B).

Discussion
In the present study, we report the novel, bioactive triazolo-thiadiazole derivative that targets PTP1B and demonstrated the mechanism of action in vitro and in vivo. PTP1B is an abundantly expressed protein tyrosine phosphatase associated with endoplasmic reticulum and it has been linked with regulation of several cellular events including cell growth, differentiation and transformation 5 . It was identified that PTP1B negatively regulates tyrosine phosphorylation of several upstream signaling proteins including c-Src, JAK2, VEGFR2 and insulin receptors 29,34 . Though SHP1, SHP2 and HCPTPA are known to interact with c-Src, JAK2 and VEGFR2, their role in physiological conditions remain unclear [35][36][37] . The main challenge in targeting PTP1B relies in selective inhibition due to high degree of homology among PTPs 9 . The prior idea about the CBTT being an effective scaffold against PTP1B, we identified and evaluated lead structure BPTT against PTP1B enzyme activity. Further, we validated the effect of BPTT on STAT3 and VEGFR2 in HepG2 cells and HUVECs respectively. Initially up to 90 mins, we observed the decrease in STAT3 phosphorylation, this is unexpected because PTP1B dephosphorylates JAK2 in turn downregulates STAT3 phosphorylation. However, we observed gradual increase in STAT3 phosphorylation (Y705) after 90 mins of incubation with BPTT. We did not observe change in the protein expression of PTP1B indicating that BPTT directly interferes with the catalytic activity and had no effect on its protein expression. PTP1B negatively regulates autophosphorylation of VEGFR2 via binding to the cytoplasmic domain of VEGFR2 in endothelial cells 35 . We tried to analyze phospho-VEGFR2 in BPTT treated HUVECs and observed only a marginal increase in the phosphorylation (data not shown). Sanggenon C, a phytochemical isolated from Chinese medicinal plant Morus mongolica has been found to exhibit inhibitory activity against PTP1B and also demonstrated to possess antiangiogenic properties via inhibition of hypoxia-inducible factor-1α . BPTT appears to have similar inhibitory effect against PTP1B and angiogenesis and needs to be explored further for its potential against human diseases.

Conclusion
Herein, for the first time we report the use of heterogeneous "green" catalyst to prepare the novel triazolo-thiadiazoles. We have depicted target of lead structure as PTP1B and experimentally demonstrated the same. Moreover, this report describes an experimentally unidentified class of small molecules targeting PTP1B. Further derivatization of the lead structure may result in drug-seeds with enhanced MTT assay. The anti-proliferative effect of new compounds against HepG2 cells was determined by the MTT dye uptake method as described previously 38 . Briefly, the cells (2.5 × 10 4 /ml) were incubated in triplicate in a 96-well plate in the presence or absence of different concentrations of BPTT in a final volume of 0.2 ml for indicated time intervals at 37 °C. Thereafter, 20 μ l MTT solution (5 mg/ml in PBS) was added to each well. After 2 h incubation at 37 °C, 0.1 ml lysis buffer (20% SDS, 50% dimethyl-formamide) was added; incubation was continued overnight at 37 °C; and then the optical density (OD) at 570 nm was measured by Tecan plate reader.
Flow cytometric analysis. The effect of BPTT on cell cycle of HepG2 cells was performed as described previously 18 . To determine the effect of BPTT on the cell cycle, cells were treated with BPTT at indicated doses up to 48 h. Thereafter cells were washed, fixed with 70% ethanol, and incubated for 30 min at 37 °C with 0.1% RNase A in PBS. Cells were then washed again, resuspended, and stained in PBS containing 25 μ g/ml propidium iodide (PI) for 30 min at room temperature. Cell distribution across the cell cycle was analyzed with a BD FACSVerse flow cytometer.
Western Blotting. Western blot analysis was performed as previously described 39 . Briefly, BPTT treated MDA-MB-231whole-cell extracts were lysed in lysis buffer (20 mM Tris, pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.01 mg/ml aprotinin, 0.005 mg/ml leupeptin, 0.4 mM PMSF, and 4 mM NaVO 4 ). Lysates were then spun at 14,000 rpm for 10 min to remove insoluble material and protein concentration was quantified. Thereafter, proteins were resolved on SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with various antibodies overnight at 4 °C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence (ECL; GE Healthcare). PTP1B inhibitory activity. PTP1B activity was measured by determining the free phosphate released from the PTP1B substrate with a PTP1B Inhibitor Screening Assay kit from Abcam. 2xPTP1B substrate solutions (120 μ M), 2xPTP1B Enzyme solutions (2 ng/well) and BPTT solutions (0-30 μ M) were prepared and added to the 96 wells plate as described in the kit instructions. After incubations at 30 °C for 30 mins, reactions were then terminated by addition of 25 μ l of Red Reagent and optical density is read at 620 nm. The readings were converted to nmol of PO 4 2− with a phosphate standard curve.  HepG2 cells were suspended in serum-free DMEM and seeded into the Matrigel transwell chambers consisting of polycarbonate membranes with 8 μ m pores. After pre-incubation with or without BPTT for 8 h, the transwell chambers were then placed into appropriate wells of a 24-well plate, in which either the basal medium only or basal medium containing CXCL12 had been added. After incubation, the upper surfaces of the Transwell chambers were wiped with cotton swabs, and the invading cells were fixed and stained with crystal violet solution. The invading cells were then counted in five randomly selected areas under microscopic observation.

Molecular
Capillary-like tube formation assay. Tube formation was assessed as described previously 27 . Briefly, HUVECs were pretreated with various dilutions of BPTT for 12 h and then seeded onto the Matrigel layer in 24-well plates at a density of 5 × 10 4 cells in Medium 200 with or without VEGF. After 6 h, tubular structure of endothelial cells was photographed using an inverted microscope. Three independent experiments were performed.
Rat aortic ring assay. Rat aortic ring assay was performed as described previously 28 . In brief, aortas isolated from Sprague-Dawley rats were cleaned of fibro adipose tissue and colateral vessels and cut into approximately 1 mm long rings. The aortic rings were randomized into Growth Factor Reduced Matrigel-coated wells and further sealed with a 100 μ l overlay of Matrigel. Medium 200 containing with and without VEGF along with different dilutions of BPTT was added to the wells and incubated at 37 °C/5% CO 2 for 6 days. At the end of incubation, the microvessel sprouting formed were fixed and photographed using a Nikon inverted microscope (magnification, 100X). Two independent experiments were performed.
In vivo tumor model studies. [6][7][8] weeks old female Swiss albino mice were kept in a 12 h light and 12 h dark cycle and fed with standard chow formula and reverse osmosis water and were acclimatized for 1 week and were used for establishing Ehrlich ascites tumor (EAT) model for further experiments as described earlier 40 . Animals were obtained and maintained in the animal house, Department of Studies in Zoology, Manasagangotri, Mysuru, India. The animal experiment was reviewed and approved by the Institutional Animal Ethical Committee, University of Mysore, Mysuru. Animal handling and experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Experimental animals were injected with 5 × 10 6 viable EAT cells intraperitoneally, and weight of the animals were recorded daily up to 11 th day to analyze the tumor growth. To investigate the in vivo efficacy of BPTT, we injected 10 mg/kg body weight of BPTT into the peritoneum of the EAT bearing mice daily from the 5 th day of transplantation. The animals were sacrificed on 12 th day and ascites fluid was collected by making an incision in the abdomen. The number of microvessels in a hotspot adjacent to tumor mass was analysed using H & E staining to determine the effect of BPTT on angiogenesis. The peritoneal cavity of DMSO-treated, topotecan treated and BPTT treated animals were photographed.

Statistical analysis.
The mean values are expressed ± S.E. for control and experimental samples and each experiment is repeated a minimum of two times. Statistical analysis was performed by one-way ANOVA. P < 0.05 was considered statistically significant (GraphPad Prism version 6.0, GraphPad Software).