sGC ( Kd = 0.6-1.1 μM ); Hypoxia-inducible factor-1alpha (HIF-1alpha/HIF-1α)

YC-1 is a soluble guanylyl cyclase (sGC) allosteric activator. YC-1 sensitizes the enzyme to its gaseous activators, carbon monoxide or nitric oxide, and raises the enzyme's catalytic rate. While YC-1 by itself only activates the enzyme by a factor of ten, it increases the activation of sGC that is dependent on CO and NO, which can stimulate the highly purified enzyme by a factor of several hundred to several thousand [1]. In vitro, it suppresses HIF-1 activity, vascular contraction, and platelet aggregation. YC-1 entirely prevents HIF-1α expression at the post-transcriptional level, which in turn suppresses HIF-1 transcription factor activity in hepatoma cells cultured in hypoxic environments. This indicates that YC-1's effects are probably related to the oxygen-sensing pathway rather than the activation of soluble guanylyl cyclase[2].

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YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme. [1]

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sGC with Ferric Heme Is Activated by YC-1. [1]
Next, we investigated the effect of ODQ-dependent oxidation on the activity of sGC in nonreducing conditions. As expected, the basal sGC activity was not affected by ODQ (Fig. 2, dashed line), whereas the NO-dependent activation of sGC was inhibited in a concentration-dependent manner by ODQ (Fig. 2, dotted line). However, YC-1-dependent activation of sGC was only partially attenuated by ODQ treatment and reached a plateau at about 1–3 μM ODQ. Even at concentrations of 10 μM ODQ sGC enzyme was still activated by 100 μM YC-1, although only at 30–40% of nontreated levels (Fig. 2, solid line). At 10 μM ODQ the sGC heme was more than 90% oxidized, as suggested by spectral studies and nonresponsiveness to NO. The differences in the inhibitory effect of ODQ on NO- and YC-1-stimulated sGC suggest that YC-1 activation of sGC has two components. One component is heme-dependent, whereas another is heme-independent.

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Heme-Deficient Mutant His-105 → Cys Is Activated by YC-1. [1]
To confirm the conclusion that YC-1 activation only partially depends on the heme prosthetic group, we generated a heme-deficient sGC enzyme. We substituted the heme-coordinating His-105 of the β subunit with a cysteine residue and coexpressed this mutant β subunit with the hexahistidine-tagged α subunit. As expected, the purified α1β1Cys-105 enzyme was heme-deficient and lacked the characteristic Soret band at 431 nm (Fig. 3). The mutant enzyme was not activated by SNP (Fig. 4B), whereas the wild-type enzyme showed a significant increase in cGMP production upon NO treatment (Fig. 4A). Analysis of the YC-1 effect on the mutant enzyme indicated that treatment with 100 μM YC-1 resulted in a 3-fold activation of the α1β1Cys-105 enzyme (Fig. 4B). In comparison, the wild-type enzyme showed a 7-fold activation under similar conditions (Fig. 4A). This YC-1 activation of the α1β1Cys-105 enzyme supports our conclusions that YC-1 binding and sGC activation can occur in the absence of a heme group. However, activation of the heme-deficient mutant enzyme was about 30% of the wild-type heme-containing enzyme. YC-1 activation of the mutant enzyme was not enhanced with SNP addition (Fig. 4B), supporting the evidence of heme deficiency.

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Effect of YC-1 on the HIF-1-Mediated Expressions of Hypoxia-Inducible Genes [1]
Previously, we found that YC-1 treatment inhibits HIF-1α protein expression and decreases the mRNA levels of erythropoietin and VEGF in Hep3B cells cultured under hypoxic conditions. To investigate the inhibitory effect of YC-1 on HIF-1-mediated hypoxic responses, Hep3B cells were treated with YC-1 under hypoxic conditions. The HIF-1α protein level increased in cells cultured under these conditions for 4 hours without YC-1 but underwent a dose-dependent decrease in cells cultured with YC-1 (Fig. 1, A). The expression of several HIF-1-regulated genes (VEGF, aldolase A, and enolase 1) showed a dose-dependent decrease in cells cultured with YC-1 for 16 hours, whereas the expression of β-actin mRNA was not affected (Fig. 1, B). The HIF-1α mRNA level was also relatively unchanged in cells cultured with YC-1, suggesting that YC-1-mediated decrease in HIF-1α protein expression occurs at a post-transcriptional level.

To assess whether the decreased VEGF mRNA levels affected levels of VEGF protein secreted into the medium, we measured VEGF protein levels in Hep3B cell-conditioned medium. After 24 hours, the VEGF protein level in medium from cells cultured under hypoxic conditions (mean = 1208 pg/mL, 95% CI = 1112 to 1304 pg/mL; P<.001 versus normoxic conditions) was more than twice that from cells cultured under normoxic conditions (mean = 559 pg/mL, 95% CI = 392 to 726 pg/mL) (Fig. 1, C). Compared with the VEGF protein level in medium from untreated cells grown under hypoxic conditions, the VEGF protein level in medium from cells cultured with YC-1 was reduced in a dose-dependent manner (P<.001) (Fig. 1, C).

We next examined whether the effects of YC-1 were specific to Hep3B cells by assessing the expression of HIF-1α protein and VEGF mRNA in other tumor cell lines (NCI-H87, SiHa, SK-N-MC, and Caki-1) cultured under hypoxic conditions in the absence or presence of YC-1. HIF-1α protein and VEGF mRNA were induced in all cell lines cultured under hypoxic conditions in the absence of YC-1 (Fig. 2). The levels of HIF-1α protein and VEGF mRNA were dose-dependently reduced in cells cultured under hypoxic conditions in the presence of YC-1 (Fig. 2). These results confirm that YC-1 inhibits the HIF-1-mediated induction of hypoxia-inducible genes, regardless of the tumor cell type.

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Splenic lymphocytes incubated with YC-1 in vitro had cytolytic activity against NK-cell sensitive YAC-1 cells that was comparable with that from splenic lymphocytes incubated without YC-1 (Fig. 7, A). Moreover, splenic lymphocytes from mice treated with YC-1 for 2 weeks had cytolytic activity that was comparable with that from vehicle-treated mice [2].

When YC-1 is given to experimental animals, mean arterial pressure drops and platelet-rich thrombosis is inhibited, both of which are correlated with elevated cGMP levels [1]. YC-1 efficiently stops tumor growth in mice that are tumor-bearing. While YC-1's anti-platelet aggregation effect does not seem to affect tumor growth, the inhibition of HIF-1 activity in tumors from mice treated with the drug is linked to blocked angiogenesis and an inhibition of tumor growth[2].

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Effects of YC-1 on Tumor Growth In Vivo [2]
Because of the observed in vitro effects of YC-1, we investigated whether YC-1 inhibits angiogenesis in solid tumors by suppressing the activity of HIF-1 and whether YC-1 inhibits tumor growth in vivo. Mice injected with human tumor cells were treated daily with YC-1 for 2 weeks. Tumors in YC-1-treated mice were visibly smaller than those in vehicle-treated mice (Fig. 3, A). The change in tumor size was measured and plotted as average tumor size versus time (Fig. 3, B). Tumor growth was minimal in mice treated with YC-1 the day after the tumor cells were injected (the last day of the experiment: mean = 422 mm3, 95% CI = 283 to 561 mm3; P<.001 versus vehicle-treated group, mean = 1082 mm3, 95% CI = 880 to 1284 mm3) and was halted in mice treated with YC-1 after the tumors had become established (mean = 126 mm3, 95% CI = 97 to 155 mm3; P<.001 versus vehicle-treated group). NCI-H87 (Fig. 3, C), SiHa (Fig. 3, D), SK-N-MC (Fig. 3, E), and Caki-1 (Fig. 3, F) xenograft tumors were also statistically significantly smaller in mice treated with YC-1 than in mice treated with the vehicle (P<.01 for all comparisons). These results indicate that YC-1 effectively inhibits tumor growth in tumor-bearing mice.

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Effects of YC-1 on Angiogenesis, HIF-1α Protein, and VEGF Expression [2]
To determine the mechanism by which YC-1 inhibits tumor growth, we examined Hep3B tumors morphologically and biochemically. H&E-stained tumor sections from vehicle-treated mice revealed well-developed blood vessels containing red blood cells and several mitotic figures (Fig. 4, A). By contrast, tumor sections from YC-1-treated mice revealed frequent acinus formation without well-developed blood vessels (Fig. 4, A).

To determine whether the inhibitory effect of YC-1 on tumor growth is associated with the suppression of tumor angiogenesis, we examined the distribution of the endothelial marker CD31. Few CD31-immunopositive vessels were observed in tumor sections from YC-1-treated mice, whereas many vessels were observed in tumor sections from vehicle-treated mice (Fig. 4, B).

Because HIF-1 is important in angiogenesis, we next assessed HIF-1α expression in tumor sections from vehicle- and YC-1-treated mice (Fig. 4, C). Hep3B tumors from vehicle-treated mice showed HIF-1α protein in both the nucleus and perinuclear areas but only in relatively hypoxic regions away from blood vessels (Fig. 4, C). By contrast, tumor sections from YC-1-treated mice showed no HIF-1α-immunoreactive cells (Fig. 4, C).

We quantified the numbers of HIF-1α-positive cells and CD31-positive vessels in tumor sections from vehicle- and YC-1-treated mice (Fig. 5). Regardless of tumor cell origin, the expression of HIF-1α protein and blood vessel formation was statistically significantly lower in mice treated with YC-1 for 2 weeks than in vehicle-treated mice (P<.01 for all comparisons) (Fig. 5).

We also measured the extent of necrosis in Hep3B tumor sections stained with H&E. No statistically significant difference in the percentage of necrosis was found between tumors from vehicle-treated mice (40 lesions examined; mean = 16.3%, 95% CI = 12.2% to 20.4%) and those from YC-1-treated mice (six lesions examined; mean = 18.3%, 95% CI = 10.2% to 26.4%; P = .17). Similarly, for the other tumor types, the differences in the extent of necrosis between tumors from vehicle- and YC-1-treated mice were not statistically significant (data not shown).

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Effect of YC-1 on NK Cell Function [2]
To confirm the effects of YC-1 on HIF-1α expression in Hep3B tumors, we isolated the HIF-1α protein by immunoprecipitation and immunoblotting. HIF-1α was detected by immunoprecipitation in tumor lysates incubated with anti-HIF-1α antibody, but not in those incubated with a preimmune serum (data not shown). The level of HIF-1α protein expression was markedly lower in YC-1-treated tumors than in vehicle-treated tumors (Fig. 6, A). In addition, levels of VEGF protein and mRNA, and of aldolase and enolase mRNAs were also lower in YC-1-treated tumors than in vehicle-treated tumors (Fig. 6, A and B). The decreased expression of VEGF, aldolase, and enolase may in turn account for the blocked angiogenesis and the growth retardation observed in YC-1-treated tumors.

PDGF is another vasoactive factor that, like VEGF, promotes angiogenesis and growth in solid tumors. PDGF is stored in the α-granules of platelets, and its secretion is stimulated by platelet aggregation. Because YC-1 inhibits platelet aggregation, it is possible that some of the antiangiogenic effects of YC-1 are mediated by reduced levels of PDGF in tumors, although such an effect has not before been reported. Thus, to test the possibility that YC-1 inhibits PDGF-induced angiogenesis in tumors, we examined the levels of PDGF-A and PDGF-B protein in Hep3B tumors (Fig. 6, A). No substantial differences in the levels of PDGF-A or PDGF-B were observed in tumors from vehicle- and YC-1-treated mice. This result suggests that the anti-platelet aggregation effect of YC-1 does not appear to affect tumor growth.

To examine whether there were differences in the number of NK cells that infiltrated the tumors in vivo, we quantified the number of NK cells in tumor sections by immunostaining with anti-asialo GM1 antibody (supplemental Fig. 1, available at http://jncicancerspectrum.oupjournals.org/jnci/content/vol95/issue7/index.shtml). NK cells were observed in Hep3B tumor sections from vehicle- and YC-1-treated mice, although the difference in number was not statistically significant (vehicle-treated tumors [n = 6], mean = 8.8 per mm2, 95% CI = 6.9 to 10.7 per mm2; YC-1-treated tumors [n = 6], mean = 8.4 per mm2, 95% CI = 5.8 to 11.0 per mm2, P = .7). These results suggest that YC-1 has no effect on NK cell function.

The appearance of the CO-bound Soret absorption band is monitored while titrating CO from a saturated solution into sGC protein in order to determine CO dissociation constants. Excess dithionite is added to buffer that has been purged of Ar to prepare the Ms sGC β₁(1-380) and Bt sGC β₁(1-197) samples. CO binding experiments using a Cary 50 spectrophotometer with a modified sample holder are carried out in a 10 cm pathlength cuvette for Ms sGC-β₁(1-380) and Ms sGC-NT21. A single site saturation ligand binding model in SigmaPlot is used to plot binding data in the presence and absence of 50 μM Lificiguat (YC-1).

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sGC Activity Assay. [1]
sGC activity was measured by the formation of [32P]cGMP from [32P]GTP as described. The reaction volume of 100 μl contained 50 mM TEA (pH 7.4), 1 mM EGTA, 10 mM 3-isobutyl-1-methylxanthine, 1 mg/ml BSA, 200 μM GTP, phosphocreatine, phosphocreatine kinase, 3 mM MgCl2, 1 mM cGMP, and ≈1–2 × 105 cpm of [32P]GTP. sGC enzyme (0.5 μg per assay) is used to measure basal activity, and 0.1 μg is used for sodium nitroprusside (SNP) or YC-1 stimulation. When necessary, DTT to a final concentration of 1 mM is added. The enzyme was activated with SNP (100 μM) or YC-1 (100 μM) or inhibited by the indicated concentration of ODQ. YC-1 and ODQ stock solutions were prepared in DMSO, but the DMSO concentration in the reaction mixture did not exceed 0.1%.

The assay for measuring cell proliferation uses a Cell Counting Kit-8 (CCK-8). In a nutshell, 3×10³/well of cells are cultured in 96-well plates, incubated for 24 hours, and then treated with Lificiguat (YC-1) or Sorafenib. Each well receives an addition of CCK-8 reagent following a 72-hour treatment. After 2.5 hours of incubation at 37°C, the absorbance is measured at 450 nm using an automated ELISA plate reader. Utilizing Microsoft Excel software, any synergistic effects arising from the combination of the compounds are measured in order to calculate the combination index values (CI>1: antagonistic effect, CI=1: additive effect, and CT<1: synergistic effect).

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Conditioned Media and VEGF Enzyme-Linked Immunosorbent Assay [2]
Hep3B cells were plated in a six-well plate at a density of 1 × 105 cells/well in α-modified Eagle medium supplemented with 10% heat-inactivated FBS and incubated overnight. Cells were treated with YC-1 (0.01–10 μM) or vehicle (DMSO) for 5 minutes and were then subjected to normoxia or hypoxia for 24 hours. VEGF levels in the conditioned media were quantified by using the Quantikine human VEGF Immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s recommended protocol. The VEGF concentrations were quantified by comparison with a series of VEGF standard samples included in the assay kit.

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NK Cell Activity [2]
Splenic lymphocytes from 12 nude mice were used to determine the effect of YC-1 on NK cell activity in vitro and in vivo. Individual spleens were homogenized in PBS by passing tissues through steel mesh using a plunger and centrifuged over a Ficoll-Paque (Amersham Biosciences) gradient at 400g at room temperature for 30 minutes to isolate the lymphocyte population. The lymphocytes were removed and washed three times in PBS. NK cell activity in the total lymphocyte population was assessed using a 4-hour 51Cr-release assay with NK-sensitive YAC-1 cells as the target cell population. The YAC-1 cells were labeled with sodium chromate (Na251CrO4) at 0.25 mCi/mL for 1.5 hours at 37 °C in a humidified atmosphere containing 5% CO2, as described.

To examine the in vitro effect of YC-1 on NK cell activity, splenic lymphocytes (6.25 × 104 to 5 × 105) were incubated with YC-1 (0.1–10 μM) or DMSO for 24 hours and then incubated at the indicated effector : target cell ratio with 1 × 104 51Cr-labeled YAC-1 cells in 96-well round-bottom plates at 37 °C in a humidified atmosphere containing 5% CO2. After 4 hours, the plates were centrifuged at 200g at 4 °C for 10 minutes, and 100-μL samples of medium were removed and counted for 1 minute in a gamma counter. Splenic lymphocytes taken from a single mouse were used in each experiment. Each assay was repeated three times, and the average value is the result from one experiment. Results are expressed as the mean of the average values from four separate experiments and 95% confidence intervals (CIs).

To examine the in vivo effect of YC-1 on NK cell activity, mice received a daily intraperitoneal injection of DMSO (n = 4) or of YC-1 (30 μg/g; n = 4) for 2 weeks. Splenic lymphocytes were isolated from each mouse and tested immediately for NK cell activity. The spontaneous release of 51Cr from YAC-1 cells was usually lower than 10% of the total 51Cr loaded. NK cell activity was calculated as follows: (experimental release minus spontaneous release)/(total release minus spontaneous release) × 100. Each assay was repeated three times, and the average value is the result from one experiment. Results are expressed as the mean of four separate experiments and 95% CIs.

YC-1 was resuspended in dimethyl sulfoxide (DMSO) at a stock concentration of 120 mg/mL, and stored at –30 °C.

Mice: The nu/nu mice used are fifty-eight females that are four weeks old. The mammary fat pads of naked mice are inoculated with MDA-MB-468 breast cancer cells (5×10⁶ cells/mouse) suspended in 0.1 mL of Matrigel solution (50% v/v Matrigel in PBS). The tumor-bearing mice are randomly assigned to treatment groups after the tumor masses reach 100 mm³. These groups receive varying doses of Lificiguat (YC-1)/YC-1-S. A YC-1 injection (30 or 60 mg/kg) or a YC-1-S oral administration is given to the mice. Tumor volume (mm³) is computed using the formula length×(width)²×0.5. Tumor size and mouse body weight are measured once every three days. Tumor nodules are removed and weighed when the experiments are over and the mice are put down. Western blotting is used to examine tumor samples.
Rats: Male Wistar albino rats, ages 4 months (200-250 g) and 24 months (550-600 g), are utilized. Before use, ligiguat (YC-1) is prepared and administered intraperitoneally (i.p.) in a dose of 0.1 mL per 100 g of body weight. For two weeks, each rat was given 1 mg/kg/day of ligiguat (YC-1). Rats that are 4 months and 24 months old (n = 10, for each group) are given DMSO. All outcomes are measured, and doses are chosen to validate the chosen doses on locomotor activity.

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Xenografts of Human Tumors [2]
Male nude (BALB/cAnNCrj–nu/nu) mice were use. Eighty mice aged 7–8 weeks were injected with tumor cells for the xenograft experiments. Sixty-nine mice bearing tumors were used for the experiments; the other 11 mice were excluded because of technical problems associated with the injection or because of lack of tumor growth. Viable Hep3B cells (5 × 106) were injected subcutaneously into the flanks of 25 of the 69 mice. The mice were immediately randomly assigned to one of three groups. The first group (n = 12) was a control group and received the vehicle (DMSO). The second group (n = 7) received daily intraperitoneal injections of YC-1 (30 μg/g) beginning the day after the injection of Hep3B cells and continuing for 2 weeks. The third group (n = 6) received daily intraperitoneal injections of YC-1 (30 μg/g) for 2 weeks after the Hep3B tumors measured 100–150 mm3, after approximately 40 days.
NCI-H87, SiHa, SK-N-MC, or Caki-1 tumor cells (5 × 106) were injected subcutaneously into the flanks of the other 44 mice. Of the mice in each group, 13, 10, 10, and 11, respectively, developed tumors. The tumor-bearing mice in each group were randomly assigned to either a control group or an experimental group. After the tumors reached an approximate volume of 100–150 mm3, the mice in the experimental group received daily intraperitoneal injections of YC-1 (30 μg/g) for 2 weeks. The mice in the control groups received daily intraperitoneal injections of DMSO.

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Tumor Histology and Immunohistochemistry for HIF-1α, CD31, and Asialo GM1 [2]
The day after the last injection of YC-1 or vehicle, the mice were killed, and the tumors were removed. The tumors were fixed with formalin and embedded in paraffin. Serial sections (6-μm thick) were cut from each paraffin block. One section was stained with hematoxylin–eosin (H&E) for histologic assessment. Other sections were immunochemically stained for HIF-1α, for the endothelial cell marker CD31, or for the NK cell marker asialo GM1. The sections were deparaffinized, rehydrated through a graded alcohol series, and heated in 10 mM sodium citrate (pH 6.0) for 5 minutes in a microwave to retrieve the antigens. Nonspecific sites were blocked with a solution containing 2.5% bovine serum albumin and 2% normal goat serum in PBS (pH 7.4) for 1 hour, and the sections were then incubated overnight at 4 °C with rabbit polyclonal anti-CD31 antibody (1 : 100 dilution in the blocking solution), rabbit polyclonal anti-asialo GM1 antibody (1 : 100 dilution in the blocking solution), or rat anti-HIF-1α antibody (1 : 100 dilution in the blocking solution), as described previously. Negative control sections were incubated with the diluent (blocking solution) in the absence of any primary antibodies. The sections were then washed and incubated with appropriate biotinylated secondary antibodies, and the avidin–biotin–horseradish peroxidase complex was used to localize the bound antibodies, with diaminobenzidine as the final chromogen. All immunostained sections were lightly counterstained with hematoxylin.

[1]. Proc Natl Acad Sci U S A . 2001 Nov 6;98(23):12938-42.

[2]. J Natl Cancer Inst . 2003 Apr 2;95(7):516-25.

Lificiguat/YC-1 is a member of the class of indazoles that is 1H-indazole which is substituted by a benzyl group at position 1 and a 5-(hydroxymethyl)-2-furyl group at position 3. It is an activator of soluble guanylate cyclase and inhibits platelet aggregation. It has a role as an antineoplastic agent, a soluble guanylate cyclase activator, an apoptosis inducer, a platelet aggregation inhibitor and a vasodilator agent. It is a member of indazoles, a member of furans and an aromatic primary alcohol.

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Our data indicate that the heme moiety is important but not necessary for YC-1-dependent activation of sGC. Previous studies of changes in Raman spectra upon YC-1 binding to a CO-sGC suggested that YC-1 binds near the heme-binding region of sGC. The partial attenuation of YC-1 activation by ODQ may indicate that there is more than one binding site for YC-1, and that at least one of these sites is affected by the oxidation of heme and resulting changes in the conformation of the heme pocket. However, there is some evidence against multiple YC-1-binding sites. Recently, a new allosteric compound, BAY41–2272, which affects the activity of sGC in a manner similar to YC-1, was described. This compound alone stimulates sGC without changing its Soret band characteristics and potentiates the effects of CO and NO in a manner similar to YC-1. Moreover, YC-1 and BAY 41–2272 share some structural similarities (Fig. 5, boxed). ODQ oxidation of the heme strongly inhibited activation of sGC by 1 μM BAY 41–2272, but an increase in sGC activity is observed at higher concentrations of BAY 41–2272 (100 μM; ref. 23). These data corroborate our findings. The crosslinking derivative of BAY 41–2272 labeled only two closely located residues 238 and 245 of the α subunit of sGC, suggesting only one binding site for BAY 41–2272. The preservation of YC-1 responsiveness of the ODQ-oxidized sGC or mutant heme-free sGC suggests that the heme moiety or heme-binding pocket has an important, but not indispensable role in the interaction of sGC with YC-1. The binding of YC-1 may be facilitated by the protoporphyrin moiety of the heme, or by the hydrophobic residues that stabilize the heme in its pocket. Interaction of YC-1 with the heme pocket might be responsible for the sensitization of sGC to nitric oxide and carbon monoxide and explain changes in the dissociation constants of these gaseous activators. Oxidation of the heme iron and associated change in the charge in proximity to the heme or the complete removal of the heme group results in some structural changes in the heme pocket. These changes would result in a decreased affinity to YC-1, but not a complete disruption of sGC/YC-1 interaction. The existence of at least one additional contact region, which is not affected by the changes in the heme pocket, may be postulated. Cys-238 and -245 of the α subunit, identified as residues interacting with BAY41–2272, could be part of such a region. Mutagenesis of these and nearby residues in the α subunit could test this hypothesis. [1]

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Background: Hypoxia-inducible factor 1 alpha (HIF-1alpha), a component of HIF-1, is expressed in human tumors and renders cells able to survive and grow under hypoxic (low-oxygen) conditions. YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, an agent developed for circulatory disorders that inhibits platelet aggregation and vascular contraction, inhibits HIF-1 activity in vitro. We tested whether YC-1 inhibits HIF-1 and tumor growth in vivo.
Methods: Hep3B hepatoma, NCI-H87 stomach carcinoma, Caki-1 renal carcinoma, SiHa cervical carcinoma, and SK-N-MC neuroblastoma cells were grown as xenografts in immunodeficient mice (69 mice total). After the tumors were 100-150 mm(3), mice received daily intraperitoneal injections of vehicle or YC-1 (30 microg/g) for 2 weeks. HIF-1 alpha protein levels and vascularity in tumors were assessed by immunohistochemistry, and the expression of HIF-1-inducible genes (vascular endothelial growth factor, aldolase, and enolase) was assessed by reverse transcription-polymerase chain reaction. All statistical tests were two-sided. [2]

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In summary, we tested whether YC-1 could target HIF-1 and inhibit tumor angiogenesis in vivo. We confirmed the inhibitory effects of YC-1 on the expression of HIF-1α and on the induction of VEGF, aldolase A, and enolase 1 in cancer cells cultured under hypoxic conditions. In vivo, treatment with YC-1 halted the growth of xenograft tumors originating from Hep3B, Caki-1, NCI-H87, SiHa, and SK-N-MC cells. Tumors from YC-1-treated mice showed fewer blood vessels and lower expression of HIF-1α protein and of HIF-1-regulated genes than tumors from vehicle-treated mice. These results suggest that YC-1 is an inhibitor of HIF-1 that halts tumor growth by blocking tumor angiogenesis and tumor adaptation to hypoxia. [2]

C19H16N2O2

304.34

304.121

C, 74.98; H, 5.30; N, 9.20; O, 10.51

170632-47-0

170632-47-0

5712

White solid powder

1.2±0.1 g/cm3

522.2±50.0 °C at 760 mmHg

269.6±30.1 °C

0.0±1.4 mmHg at 25°C

1.653

3.55

1

3

4

23

386

0

O1C(C([H])([H])O[H])=C([H])C([H])=C1C1C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])N=1

OQQVFCKUDYMWGV-UHFFFAOYSA-N

InChI=1S/C19H16N2O2/c22-13-15-10-11-18(23-15)19-16-8-4-5-9-17(16)21(20-19)12-14-6-2-1-3-7-14/h1-11,22H,12-13H2

[5-(1-benzylindazol-3-yl)furan-2-yl]methanol

Lificiguat; YC1; YC 1; yc-1; Lificiguat; 170632-47-0; (5-(1-benzyl-1H-indazol-3-yl)furan-2-yl)methanol; 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole; 154453-18-6; Lificiguat [INN]; YC-1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~61 mg/mL (~200.43 mM)
Ethanol: ~31 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (8.21 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)