Echistatin is a potent inhibitor of bone resorption in culture

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.     

Interleukin-4 is a potent mitogen for capillary endothelium

Interleukin-4 (IL-4) is a mitogen for both microvascular (human adrenal capillary, HACE) and large vessel (human umbilical vein, HUVEC) endothelial cells. Comparison of growth promotion by IL-4 to that by the potent endothelial mitogen fibroblast growth factor (FGF) showed the activity of IL-4 on HACE cells to be strong (50% of that with FGF) but on HUVEC's weak (12% of that with FGF). Growth stimulation was characterised by both 3H-thymidine incorporation and by cell number, and was maximal at 1 nM IL-4. The presence of IL-4 receptors on HACE cells and HUVEC's was confirmed by specific binding of radioiodinated IL-4. Scatchard analysis confirmed a single high affinity binding receptor on both HACE cells (Kd = 80 pM, 358 receptors/cell) and HUVEC's (Kd = 88 pM, 2,580 receptors/cell). Potent activity on capillary as opposed to large vessel endothelium places IL-4 in a unique position amongst endothelial mitogens.     

Dihydroxybergamottin caproate as a potent and stable CYP3A4 inhibitor

We investigated the inhibitory activity of the furanocoumarin derivatives from grapefruit juice to the drug metabolizing enzyme, cytochrome P450 (CYP) 3A4. Although two known furanocoumarin dimers GF-I-1 (1) and GF-I-4 (2) showed potent CYP3A4 inhibition with IC50 value of 0.07 microM, a semi-synthetic dihydroxybergamottin caproate (11), which was more stable and more simple than the dimers, exhibited comparable activity against CYP3A4.     

Identifying 6,7,4'-trihydroxyisoflavone as a potent tyrosinase inhibitor

A known biotransformed compound, 6,7,4'-trihydroxyisoflavone, was identified as a potent tyrosinase inhibitor. It inhibited mushroom tyrosinase with an IC50 value of 9.2 microM, which is six times the anti-tyrosinase activity of kojic acid (IC50 = 54.4 microM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated 6,7,4'-trihydroxyisoflavone to be a competitive inhibitor of tyrosinase when L-tyrosine was used as a substrate. Its biosynthesis precursors and analogs, including glycitein, daidzein, and genistein, showed little anti-tyrosinase activity. The results suggest that hydroxyl groups at the C-6 and C-7 positions of the isoflavone skeleton might play an important role in the expression of tyrosinase inhibitory activity.     

4-Hydroxy-oxyphenbutazone is a potent inhibitor of cytokine production

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.     

Quinolinium salt as a potent inhibitor of lymphocyte apoptosis

The synthesis of several quinolinium salts and related compounds and their ability to inhibit glucocorticoid-induced apoptosis in murine thymocytes are described. Interestingly, 1-[2-methoxyimino-2-(4-pyrrolidin-1-yl-phenyl)ethyl]quinolinium bromide (11) showed a potent protective effect with an EC(50) of 0.013 microM, which was at least 300-fold more potent than the reference compound pifithrin-alpha.     

Follistatin as a potent regulator of bone metabolism

Follistatin is a monomeric glycoprotein, distributed in a wide range of tissues. Recent work has demonstrated that this protein is a pluripotential molecule that has no structural similarity but is functionally associated with members of the transforming growth factor (TGF)-β superfamily, which indicates its wide range of action. Members of the TGF-β superfamily, especially activins and bone morphogenetic proteins are involved in bone metabolism. They play an important role in bone physiology, influencing bone growth, turnover, bone formation and cartilage induction. As follistatin is considered to be the antagonist of the TGF-β superfamily members, it plays an important role in bone metabolism and development.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Gnetol as a potent tyrosinase inhibitor from genus Gnetum

Gnetol (2,3',5',6-tetrahydroxy-trans-stilbene), a naturally occurring compound particularly found in the genus Gnetum, had a strong inhibitory effect on murine tyrosinase activity. Gnetol (IC50, 4.5 microM) was stronger than kojic acid (IC50, 139 microM) as a standard inhibitor for murine tyrosinase activity. Moreover, gnetol significantly suppressed, melanin biosynthesis in murine B16 melanoma cells.     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

2-hydroxy-4-isopropylbenzaldehyde, a potent partial tyrosinase inhibitor

Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It partially inhibits the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an IC(50) of 2.3 microM. The inhibition kinetics analyzed by Dixon plots found that chamaecin is a mixed type inhibitor. This inhibition may come in part from its ability to form a Schiff base with a primary amino group in the enzyme.     

Suppression of Interleukin-11–mediated bone resorption by cyclooxygenases inhibitors

We previously found that human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo. These cancer cells produced interleukin-11 (IL-11) by themselves and stimulated its production from osteoblasts. Interleukin-11 could increase the number of osteoclasts and raise the calcium concentration in the medium of neonatal murine calvaria organ culture, indicating bone resorption in vitro. Therefore, IL-11 could play an important role in the promotion of osteolysis at the site of bone metastasis. In the present study, we used the calvaria culture system to try to clarify the mechanisms of IL-11–mediated bone resorption. The murine calvaria expressed both the specificity-determining α subunit and the signal–transducing β subunit (gp130) of the IL-11 receptor. When IL-11 was added to the calvaria culture, the concentrations of prostaglandin E₂ (PGE₂) was elevated. Pretreatment of calvaria with cyclooxygenases inhibitors (e.g., indomethacin, NS-398, and dexamethasone) suppressed the production of PGE₂ and the bone resorption induced by IL-11. Addition of exogenous PGE₂ overcame the inhibitory effect of cyclooxygenases inhibitors and promoted bone resorption. These results indicate that IL-11 promotes bone resorption through a PGE₂ synthesis–dependent mechanism and that cyclooxygenases inhibitors could be interesting drugs to suppress IL-11–mediated osteolytic bone metastasis of cancer cells. J. Cell. Physiol. 175:247–254, 1998. © 1998 Wiley-Liss, Inc.     

4-Hydroxytamoxifen is a potent inhibitor of the mitochondrial permeability transition

The effects of 4-hydroxytamoxifen (OHTAM), the major active metabolite of the antiestrogen tamoxifen used in the breast cancer therapy, were studied on the mitochondrial permeability transition (MPT) and bioenergetic functions of mitochondria to evaluate the mechanisms underlying the cell death and toxic effects. The MPT was induced in vitro by incubating rat liver mitochondria with 1 mM inorganic phosphate plus Ca2+ and with tert-butyl hydroperoxide. OHTAM provides protection against the Ca2+-induced mitochondrial swelling, depolarization of the mitochondrial membrane potential (deltapsi), loss of electrophoretic Ca2+ uptake capacity and uncoupling of respiration, similarly to cyclosporine A. The concentrations of OHTAM used do not significantly affect deltapsi, respiratory control and adenosine diphosphate/oxygen ratios and induce repolarization and Ca2+ re-uptake, suggesting that such inhibitory effects of OHTAM were due to the prevention of the MPT induction and not due to the inhibition of the mitochondrial Ca2+ uniporter. Since the MPT induction has been linked to an oxidized shift in the mitochondrial redox state and/or increase in the generation of reactive oxygen species, the MPT prevention by OHTAM may be related to its high antioxidant capacity.     

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of TAE684 as a potent LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.     

Syntheses of (+/-)-shinflavanone and its structural analogues as potent inhibitors of bone resorption pits formation.

The first total syntheses of (+/-)-shinflavanone and its structural analogues were achieved. (+/-)-Shinflavanone, appears to be a strong inhibitor of bone resorption pits formation by osteoclast-like cell induced by 1alpha, 25-dihydroxy vitamine D3 (IC50 = 0.70 microg/mL).     

2-hydroxyethylhydrazine as a potent inhibitor of phospholipid methylation in yeast

The effect of 2-hydroxyethylhydrazine on the phosphatidylethanolamine methylation pathway in yeast was studied. 2-Hydroxyethylhydrazine inhibited the growth of cells. The concentration required for 50% inhibition was 66 microM. The growth rate decreased by 2-hydroxyethylhydrazine was restored by the addition of a low concentration of choline. Incorporation of radioactivity from L-[3-14C]serine, L-[methyl-14C]methionine and S-adenosyl-L-[methyl-14C]methionine into phosphatidylcholine was markedly reduced by 2-hydroxyethylhydrazine. The restoration of growth by choline was not due to the reversal of the inhibition, but to the formation of phosphatidylcholine via the CDPcholine pathway. Thus, the site of action of 2-hydroxyethylhydrazine in vivo was the phosphatidylethanolamine methylation pathway. Experiments with methylation mutants indicated that all three steps of methylation were sensitive to 2-hydroxyethylhydrazine. 2-Hydroxyethylhydrazine was shown to inhibit the methyltransferase after it had become chemically or metabolically transformed in cells. 2-Hydroxyethylhydrazine-resistant mutants were obtained and were found to have a defect in choline transport activity. Genetic data indicated that the uptake of 2-hydroxyethylhydrazine into cells is mediated by the choline transport system.     

Discovery of a potent, non-triketone type inhibitor of 4-hydroxyphenylpyruvate dioxygenase

3-Cyclopropanecarbonyloxy-2-cyclohexen-1-one has been found to be a new, potent, low molecular weight non-triketone type inhibitor of 4-hydroxyphenylpyruvate dioxygenase with IC50 value of 30 nM. Preliminary studies suggest that the two carbonyl groups present in the compound are crucial for the inhibition activity.