BMC Cancer. 2017 Mar 31;17(1):235. doi: 10.1186/s12885-017-3214-8.
Identification of Novel Class of Triazolo-Thiadiazoles as Potent Inhibitors of Human Heparanase and their Anticancer Activity.
Baburajeev CP1, Mohan CD2,3, Rangappa S4, Mason DJ5, Fuchs JE5, Bender A5, Barash U6, Vlodavsky I7, Basappa8, Rangappa KS9.
Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central College Campus, Palace Road, Bangalore, 560001, India.
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, 570006, India.
Department of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore, 570006, India.
Adichunchanagiri Institute for Molecular Medicine, BG Nagara, Nagamangala Taluk, Mandya, district-571448, India.
Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK.
Cancer and Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Cancer and Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel. vlodavsk@mail.huji.ac.il.
Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central College Campus, Palace Road, Bangalore, 560001, India. salundibasappa@gmail.com.
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, 570006, India. rangappaks@yahoo.com.
Expression and activity of heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is associated with progression and poor prognosis of many cancers which makes it an attractive drug target in cancer therapeutics.
In the present work, we report the in vitro screening of a library of 150 small molecules with the scaffold bearing quinolones, oxazines, benzoxazines, isoxazoli(di)nes, pyrimidinones, quinolines, benzoxazines, and 4-thiazolidinones, thiadiazolo[3,2-a]pyrimidin-5-one, 1,2,4-triazolo-1,3,4-thiadiazoles, and azaspiranes against the enzymatic activity of human heparanase. The identified lead compounds were evaluated for their heparanase-inhibiting activity using sulfate [35S] labeled extracellular matrix (ECM) deposited by cultured endothelial cells. Further, anti-invasive efficacy of lead compound was evaluated against hepatocellular carcinoma (HepG2) and Lewis lung carcinoma (LLC) cells.
Among the 150 compounds screened, we identified 1,2,4-triazolo-1,3,4-thiadiazoles bearing compounds to possess human heparanase inhibitory activity. Further analysis revealed 2,4-Diiodo-6-(3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6yl)phenol (DTP) as the most potent inhibitor of heparanase enzymatic activity among the tested compounds. The inhibitory efficacy was demonstrated by a colorimetric assay and further validated by measuring the release of radioactive heparan sulfate degradation fragments from [35S] labeled extracellular matrix. Additionally, lead compound significantly suppressed migration and invasion of LLC and HepG2 cells with IC50 value of ~5 μM. Furthermore, molecular docking analysis revealed a favourable interaction of triazolo-thiadiazole backbone with Asn-224 and Asp-62 of the enzyme.
Overall, we identified biologically active heparanase inhibitor which could serve as a lead structure in developing compounds that target heparanase in cancer.
Anticancer activity; Heparanase inhibitors; Metastasis; triazolo-thiadiazoles
Schematic representation of new heparanase inhibitors used in this study. i) hydrazine hydrate, ethanol, MWI; CS2 and KOH, 5-6 min at 700 watt; ii) SCe (20 mol%), DMF, 10 h
Identification of Novel Class of Triazolo-Thiadiazoles as Potent Inhibitors of Human Heparanase and their Anticancer Activity
BMC Cancer. 2017;17:235.
Plausible mechanism of cyclization and synthesis of title compounds
Synthetic scheme for the preparation of N-amino-triazole-amides. i) HOBt/EDC, DMF, RT, 2 h. R1 = 3a, 3c
a Screening of compounds for inhibition of heparanase enzymatic activity applying the Fondaparinux heparanase assay. PC, positive control = N-(4-{[4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamide []. b Lead molecules which exhibited inhibitory activity against human heparanase were validated using a semi-quantitative assay that measures release of radioactive heparan sulfate fragments from an insoluble extracellular matrix as described in 'Methods' section. Briefly, sulfate [35S] labeled ECM was incubated (6 h, 37 °C, pH 6.0) with recombinant human heparanase (200 ng/mL) in the absence and presence of 10 μg/mL of the test compounds. Sulfate labeled material released into the incubation medium was subjected to gel filtration on Sepharose 6B. Compound DTP effectively inhibited the cleavage and release of heparan sulfate degradation fragments
Heparanase expression and activity in various hepatocellular and lung carcinoma cell lines. Mouse Lewis lung carcinoma (LLC), human lung carcinoma (HCC827 = HCC), and human hepatocellular carcinoma (HepG2, Hep3B) cells maintained in culture were subjected to RT-PCR (a) and heparanase activity (b) assays, as described in '
Effect of DTP on LLC cell migration and Invasion. LLC cells were plated on BD BioCoat™ chambers (BD Biosciences) and cell migration (without Matrigel coat) (a) and invasion (with Matrigel coat) (b) were measured as described in 'Methods'. The effect of lead compound DTP (1–10 μM) or heparin (100 μg/mL) on cell migration and invasion is demonstrated by representative photomicrographs (magnification: ×10) and the respective bar graphs. Data are represented as mean ± S.E. *P < 0.1; **P < 0.05. ***P < 0.01
Effect of DTP on HepG2 cell migration and Invasion. HepG2 cells were plated on BD BioCoat™ chambers (BD Biosciences) and cell migration (without Matrigel coat) (a) and invasion (with Matrigel coat) (b) were measured as described in 'Methods'. The effect of lead compound DTP (1–10 μM) or heparin (100 μg/mL) on cell migration and invasion is demonstrated by representative photomicrographs (magnification: ×5) and the respective bar graphs. Data are represented as mean ± S.E. *P < 0.05
Selected docked poses for active compounds DTP, HTP and ITP (a, b and c, respectively), showing similar active site interaction modes. DTP and ITP are shown to interact with both Asn-224 and Asp-62 via the triazolo-thiadiazole backbone, and HTP is shown to interact with Asn-224 and the active site acid-base Glu-343
Antineoplastic Agents/chemistry
Antineoplastic Agents/pharmacology*
Enzyme Inhibitors/chemistry
Enzyme Inhibitors/pharmacology*
Glucuronidase/antagonists & inhibitors*
Neoplasms/drug therapy
Neoplasms/enzymology*
Thiadiazoles/chemistry*
Triazoles/chemistry*
Tumor Cells, Cultured
Thiadiazoles
Triazoles
Glucuronidase