Heparanase Inhibitory Activity Research Articles

PG545 Prevents Osteoarthritis Development by Regulating PI3K/AKT/mTOR Signaling and Activating Chondrocyte Autophagy

Introduction: Osteoarthritis (OA) is a degenerative disease common in the elderly and is characterized by joint pain, swelling, and restricted movement. In recent years, heparanase has been reported to play an important role in the development of osteoarthritic cartilage. PG545 is a heparan sulfate mimetic with heparanase inhibitory activity. In this study, the therapeutic effects and possible mechanisms of PG545 were investigated in a chondrocyte injury model induced by interleukin-1β (IL -1β). Methods: Following treatment with PG545 or the autophagy inhibitor 3-methyladenine (3-MA), chondrocyte viability was detected using Cell Counting Kit-8 and fluorescein diacetate/propidium iodide double staining. The apoptosis rate of chondrocytes was determined by flow cytometry. Expression of light chain 3 and P62 was monitored by immunofluorescence labeling. Western blot, lentivirus infection with red fluorescent protein and green fluorescent protein, and quantitative real-time polymerase chain reaction were used to determine the expression levels of chondrocyte markers, apoptosis-related factors, autophagy proteins, and key proteins of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. The expression and activity of stress-specific enzymes such as malondialdehyde, superoxide dismutase, and catalase (CAT) were investigated. Chondrocytes with ATG5 knockdown were used to investigate the relationship between the therapeutic effect of PG545 and autophagy. The therapeutic effect of PG545 was verified in vivo. Results: PG545 had a significant protective effect on chondrocytes by reducing oxidative stress, apoptosis, and degradation of chondrocytes and increasing chondrocyte proliferation. PG545 was effective in inducing autophagy in IL-1β-treated cells, while 3-MA attenuated the effect. The PI3K/Akt/mTOR pathway may be involved in the promotion of autophagy and OA treatment by PG545. Conclusion: PG545 was able to restore impaired autophagy and autophagic flux via the PI3K/Akt/mTOR pathway, thereby delaying the progression of OA, suggesting that PG545 may be a novel therapeutic approach for OA.

Chemoenzymatic Synthesis of D‐Glucaro‐δ‐lactam Containing Oligosaccharides as Putative Heparanase Inhibitors

AbstractHeparanase is an endo‐β‐glycosidase that cleaves heparan sulfate chains at the level of the glycosidic bond of selected glucuronic acid residues. Heparin fragments, that can compete with heparan sulfate for binding to heparanase, have been reported as weak heparanase inhibitors. To reinforce their inhibitory activity, a known β‐glucuronidase inhibitor has been introduced at their reducing end to directly interfere with the active site of the enzyme. We report here the synthesis of such oligosaccharides, displaying D‐glucaro‐δ‐lactam at their reducing end. Thanks to the trichloroacetimidate glycosylation method, we could devise experimental conditions allowing O‐glycosylation of the lactam ring, and we thus chemically prepared trisaccharides that were further elongated and sulfated using enzymatic methods. Heparanase inhibitory activity was not significantly improved by the presence of the D‐glucaro‐δ‐lactam ring.

Structure and heparanase inhibitory activity of a new glycosaminoglycan from the slug Limacus flavus

A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was −OH (˜80%) or –OSO3− (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 μM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.

Identification of Novel Class of Triazolo-Thiadiazoles as Potent Inhibitors of Human Heparanase and their Anticancer Activity

BackgroundExpression 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.MethodsIn 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.ResultsAmong 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.ConclusionsOverall, we identified biologically active heparanase inhibitor which could serve as a lead structure in developing compounds that target heparanase in cancer.

ChemInform Abstract: Synthetic Route to a Benzooxazole Derivative with Heparanase Inhibitory Activity.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthetic Route to a Benzooxazole Derivative with Heparanase Inhibitory Activity

The synthesis of a benzooxazol-5-yl acetic acid derivative (9) with strong heparanase and angiogenesis inhibitory activity, and thus possible commercial interest, is described in detail.

Synthesis and heparanase inhibitory activity of sulfated mannooligosaccharides related to the antiangiogenic agent PI-88

A stepwise synthetic route to the mannooligosaccharides from the neutral fraction of Pichia holstii phosphomannan hydrolysate, including a tetrasaccharylamine component, was developed using only two or three readily available d-mannose building blocks. These compounds were sulfonated to give the corresponding sulfated oligosaccharides which are closely related to the constituents of the anticancer agent PI-88. The synthetic approach is well suited to the preparation of analogues as demonstrated by the synthesis of a series of (1 → 3)-linked mannooligosaccharides. The inhibitory activity of the sulfated oligosaccharides against heparanase was determined using a Microcon ultrafiltration assay. The tetra- and pentasaccharides were potent competitive inhibitors of heparanase ( K i = 200–280 nM) whilst the shorter di- and trisaccharides were partial competitive inhibitors and did not completely inhibit the enzyme even at very high concentrations.

Antimetastatic Activities of Modified Heparins: Selectin Inhibition by Heparin Attenuates Metastasis

Heparin, which is traditionally used as an anticoagulant but has a variety of additional biological activities, was shown in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity.

1-[4-(1 H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1 H-benzoimidazol-2-yl)-phenyl]-urea derivatives as small molecule heparanase inhibitors

A novel class of 1-[4-(1 H-benzoimidazol-2-yl)-phenyl]-3-[4-(1 H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1 H-benzoimidazol-2-yl)-phenyl]-urea ( 7a) and 1,3-bis-[4-(5,6-dimethyl-1 H-benzoimidazol-2-yl)-phenyl]-urea ( 7d), which displayed good heparanase inhibitory activity (IC 50 0.075–0.27 μM). Compound 7a showed good efficacy in a B16 metastasis model.

N-(4-{[4-(1 H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamide derivatives as small molecule heparanase inhibitors

A novel class of N-(4-{[4-(1 H-benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamides are described as inhibitors of the endo-β-glucuronidase heparanase. Among them are N-(4-{[4-(1 H-benzoimidazol-2-yl)-phenylamino]-methyl}-phenyl)-3-bromo-4-methoxy-benzamide ( 15h), and N-(4-{[5-(1 H-benzoimidazol-2-yl)-pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benzamide ( 23) which displayed good heparanase inhibitory activity (IC 50 0.23–0.29 μM), with the latter showing oral exposure in mice.

2,3-Dihydro-1,3-dioxo-1H-isoindole-5-carboxylic acid derivatives: a novel class of small molecule heparanase inhibitors

A novel class of 2,3-dihydro-1,3-dioxo-1H-isoindole-5-carboxylic acids are described as inhibitors of the endo-β-glucuronidase heparanase. Several of the compounds, for example, 2-[4-propylamino-5-[5-(4-chloro)phenyl-benzoxazol-2-yl]phenyl]-2,3-dihydro-1,3-dioxo-1H-isoindole-5-carboxylic acid (9c), display potent heparanase inhibitory activity (IC50 200–500 nM) and have high selectivity (>100-fold) over human β-glucuronidase. They also show anti-angiogenic effects. Such compounds should serve as useful biological tools and may provide a basis for the design of novel therapeutic agents.

2,3-Dihydro-1,3-dioxo-1 H-isoindole-5-carboxylic acid derivatives: a novel class of small molecule heparanase inhibitors

A novel class of 2,3-dihydro-1,3-dioxo-1 H-isoindole-5-carboxylic acids are described as inhibitors of the endo-β-glucuronidase heparanase. Several of the compounds, for example, 2-[4-propylamino-5-[5-(4-chloro)phenyl-benzoxazol-2-yl]phenyl]-2,3-dihydro-1,3-dioxo-1 H-isoindole-5-carboxylic acid ( 9c), display potent heparanase inhibitory activity (IC 50 200–500 nM) and have high selectivity (>100-fold) over human β-glucuronidase. They also show anti-angiogenic effects. Such compounds should serve as useful biological tools and may provide a basis for the design of novel therapeutic agents.

Exploitation of heparanase inhibitors from microbial metabolites using an efficient visual screening system.

In this paper we describe the establishment of an efficient visual method for screening heparanase inhibitors, and we present the results of screening 10,000 microbial culture broths. Heparanase-overexpressing stable clones of the human hepatocellular carcinoma HepG2 cells were established and used as an enzyme source. Digestion of heparan sulfate (HS) was detected using novel HS-containing tablets or SDS-polyacrylamide gel electrophoresis. This method was able to find suramin, a known heparanase inhibitor, from a library of typical enzyme inhibitors. By screening 10,000 culture broths of microorganisms (actinomycetes, fungi, and bacteria) an actinomycete strain, RK99-A234, was found to have heparanase inhibitory activity. RK-682 was identified in the fermentation broth as a heparanase inhibitor, IC50 = 17 microM.

Antimetastatic effects associated with anticoagulant properties of heparin and chemically modified heparin species in a mouse mammary tumor model

Fibrin deposits surrounding circulating tumor cells may protect them from mechanical trauma and destruction by the host immune system, and may facilitate microvascular entrapment required for metastasis. We report that heparin inhibited the clotting of plasma induced by mouse mammary carcinoma cells in a dose-dependent manner and blocked the production of experimental lung metastasis when administered i.p. at the time of intravenous injection of tumor cells. On the other hand, O/N-desulfated N-acetylated heparin did not exhibit anticoagulant properties and had no effects on metastasis formation. Similarly, reduction of metastasis was not seen with N-desulfated N-acetylated heparin, another chemically modified heparin which does not inhibit blood coagulation but has heparanase inhibitory activity. Our data demonstrate the existence of a strong association between antimetastatic and anticoagulant properties of heparin species in the present experimental system.

Suramin. A potent inhibitor of melanoma heparanase and invasion

Suramin, a polysulfonated naphthylurea, has anti-reverse transcriptase and anti-proliferative activities and inhibits the binding of various growth factors to their cell surface receptors. This drug is used in the treatment of acquired immunodeficiency syndrome and several types of cancers. Increased levels of circulating glycosaminoglycans have been observed in suramin-treated cancer patients, suggesting that it may inhibit glycosaminoglycan catabolism. Melanoma-derived heparanase, a heparan sulfate-specific endo-beta-D-glucuronidase that plays an important role in metastatic melanoma cell invasion through basement membranes, is inhibited by suramin in a dose-dependent manner: 100% inhibition was observed at a concentration of approximately 100 microM. Structurally related polysulfonated compounds, such as trypan blue and Evans blue, had lower heparanase inhibitory activities: the concentrations required for 50% heparanase inhibition (ID50) were 310-320 microM and six times higher than for suramin (ID50 = 46 microM). Oversulfated heparin tetrasaccharide, whose average molecular size is similar to suramin, had also much lower heparanase inhibitory activity than suramin. The inhibition constants (Ki) for suramin and oversulfated heparin tetrasaccharide were 48 and 290 microM, respectively. Suramin had a remarkable inhibitory activity against B16 melanoma cell invasion through reconstituted basement membranes (ID50 less than 10 microM). The inhibitory effects of suramin on melanoma heparanase and cell invasion appeared to be completely independent of its antiproliferative activity, because significant effects on melanoma cell growth were not observed at the concentrations of suramin used in this study. The results suggest that the antimetastatic effects of suramin may be due to its antiinvasive rather than antiproliferative activities.

Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

Inhibition of Heparanase-Mediated Degradation of Extracellular Matrix Heparan Sulfate by Non-anticoagulant Heparin Species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

Chemically modified heparins as inhibitors of heparan sulfate specific endo-beta-glucuronidase (heparanase) of metastatic melanoma cells.

To determine the significance of the heparan sulfate (HS) degradative endo-beta-glucuronidase (heparanase) in tumor invasion and metastasis and to develop possible antimetastatic agents, we synthesized specific inhibitors of this enzyme. We previously found that heparanase activity correlates with the lung colonization abilities of murine B16 melanoma cells and is inhibited by heparin [Nakajima, M., Irimura, T., Di Ferrante, N., & Nicolson, G. L. (1984) J. Biol. Chem. 259, 2283-2290]. In this study, heparin was chemically modified in order to determine which portions of its structure are responsible for heparanase inhibitory activity and to obtain heparanase inhibitors that have minimal additional biological effects, such as anticoagulation. N-Sulfate groups and O-sulfate in heparin were removed separately, and the resultant free amino groups were acetylated or resulfated. Heparin was also reduced at the carboxyl groups of uronic acid. The heparanase inhibitory activities of these heparin derivatives were examined by high-speed gel-permeation chromatography and by the use of radioactive HS immobilized on agarose beads. The results indicated that although N-sulfate and O-sulfate groups on glucosamine residues, and carboxyl groups on uronic acid residues, are important for heparanase inhibition, they are not essential for full activity. When highly metastatic B16-BL6 melanoma cells were incubated with N-acetylated N-desulfated heparin, N-resulfated N- and O-desulfated heparin, or carboxyl-reduced heparin and injected intravenously to syngenic C57BL/6 mice, significant reductions in the numbers of experimental melanoma lung metastases occurred.