Fang-Yu Lee

YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase.

1 Our previous study demonstrated that YC‐1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC‐1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2 In human washed platelets, YC‐1 inhibited platelet aggregation and ATP released induced by U46619 (2 μm), collagen (10 μg ml−1) and thrombin (0.1 u ml−1) in a concentration‐dependent manner with IC50 values of (μm) 2.1 ± 0.3, 11.7 ± 2.1 and 59.3 ± 7.1, respectively. 3 In a 30,000 g supernatant fraction from human platelet homogenate, YC‐1 (5–100 μm) increased sGC activity in a concentration‐dependent manner. At the same concentration‐range, YC‐1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4 MY‐5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC‐1, and shifted the concentration‐anti‐aggregation curve of YC‐1 to the left. In contrast, HL‐725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti‐aggregatory effect caused by YC‐1. 5 Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC‐1, and attenuated markedly the anti‐aggregatory effect of YC‐1. The adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine (DDA) did not affect YC‐1‐induced inhibition of platelet aggregation. 6 Haemoglobin, which binds NO, prevented the activation of sGC and anti‐aggregatory effect caused by sodium nitroprusside, but did not affect YC‐1 responses. 7 These results would suggest that YC‐1 activates sGC of human platelets by a NO‐independent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.

YC-1 inhibits HIF-1 expression in prostate cancer cells: Contribution of Akt/NF-κB signaling to HIF-1α accumulation during hypoxia

Hypoxia-inducible factor 1 (HIF-1), a transcription factor that is critical for tumor adaptation to microenvironmental stimuli, represents an attractive chemotherapeutic target. YC-1 is a novel antitumor agent that inhibits HIF-1 through previously unexplained mechanisms. In the present study, YC-1 was found to prevent HIF-1α and HIF-1β accumulation in response to hypoxia or mitogen treatment in PC-3 prostate cancer cells. Neither HIF-1α protein half-life nor mRNA level was affected by YC-1. However, YC-1 was found to suppress the PI3K/Akt/mTOR/4E-BP pathway, which serves to regulate HIF-1α expression at the translational step. We demonstrated that YC-1 also inhibited hypoxia-induced activation of nuclear factor (NF)-κB, a downstream target of Akt. Two modulators of the Akt/NF-κB pathway, caffeic acid phenethyl ester and evodiamine, were observed to decrease HIF-1α expression. Additionally, overexpression of NF-κB partly reversed the ability of wortmannin to inhibit HIF-1α-dependent transcriptional activity, suggesting that NF-κB contributes to Akt-mediated HIF-1α accumulation during hypoxia. Overall, we identify a potential molecular mechanism whereby YC-1 serves to reduce HIF-1 expression.

Enhancement of learning behaviour by a potent nitric oxide-guanylate cyclase activator YC-1

Memory is one of the most fundamental mental processes, and various approaches have been used to understand the mechanisms underlying this process. Nitric oxide (NO), cGMP and protein kinase G (PKG) are involved in the modulation of synaptic plasticity in various brain regions. YC‐1, which is a benzylindazole derivative, greatly potentiated the response of soluble guanylate cyclase to NO (up to several hundreds fold). We have previously shown that YC‐1 markedly enhances long‐term potentiation in hippocampal and amygdala slices via NO‐cGMP‐PKG‐dependent pathway. We here further investigated whether YC‐1 promotes learning behaviour in Morris water maze and avoidance tests. It was found that YC‐1 shortened the escape latency in the task of water maze, increased and decreased the retention scores in passive and active avoidance task, respectively. Administration of YC‐1 30 min after foot‐shock stimulation did not significantly affect retention scores in response to passive avoidance test. Administration of scopolamine, a muscarinic antagonist, markedly impaired the memory acquisition. Pretreatment of YC‐1 inhibited the scopolamine‐induced learning deficit. The enhancement of learning behaviour by YC‐1 was antagonized by intracerebroventricular injection of NOS inhibitor L‐NAME and PKG inhibitors of KT5823 and Rp‐8‐Br‐PET‐cGMPS, indicating that NO‐cGMP‐PKG pathway is also involved in the learning enhancement action of YC‐1. YC‐1 is thus a good drug candidate for the improvement of learning and memory.

YC-1 attenuates LPS-induced proinflammatory responses and activation of nuclear factor-κB in microglia

An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC‐1, a known activator of soluble guanylyl cyclase, against LPS‐induced inflammatory responses in microglia.

YD-3, a novel inhibitor of protease-induced platelet activation

In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD‐3 [1‐benzyl‐3(ethoxycarbonylphenyl)‐indazole] were examined. YD‐3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC50=28.3 μM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet‐activating factor (PAF) or U46619. YD‐3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin‐induced fibrin formation was not affected by YD‐3, indicating YD‐3 does not inhibit the proteolytic activity of thrombin. In washed human platelets, however, YD‐3 had only mild inhibitory effect on the low concentration (0.05 u ml−1) of thrombin‐induced human platelet aggregation, and did not affect that induced by higher concentrations (0.1 u ml−1) of thrombin or SFLLRN, the protease‐activated receptor 1 (PAR1) agonist peptide. By contrast, YD‐3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC50 values of 38.1 μM and 5.7 μM, respectively. YD‐3, at 100 μM, had no effect on ristocetin‐induced glycoprotein Ib (GPIb)‐dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD‐3 on thrombin‐induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD‐3. In SFLLRN‐desensitized human platelets, high concentration of thrombin (1 u ml−1) could still elicit intracellular Ca2+ mobilization, and the rise of [Ca2+]i was prevented by either leupeptin or YD‐3. Our results suggest that YD‐3 inhibits a non‐PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.

The role of PAR4 in thrombin-induced thromboxane production in human platelets

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

Synthesis of 1-benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole derivatives as new anticancer agents

As part of our continuing search for potential anticancer drug candidates among YC-1 analogs, 1, 3-disubstituted selenolo[3,2-c]pyrazole derivatives were synthesized and evaluated for their cytotoxicity against NCI-H226 non-small cell lung cancer and A-498 renal cancer cell lines. Significant cytotoxicity was observed in 3-(5-hydroxymethyl-2-furyl) derivatives (2, 33, 36 and 37). Among them, compound 2 was found to have the most potent activity. The mode of action of compound 2 seems to differ from those of the 175 anticancer agents listed in NCIs standard database and resembles that of YC-1. Thus, we recommend that compound 2 should be developed further as new drug candidate for treatment of non-small cell lung cancer and renal cancer.

Mechanism of anti-proliferation caused by YC-1, an indazole derivative, in cultured rat A10 vascular smooth-muscle cells.

An indazole derivative, YC-1, was identified in this study to be capable of reversibly and effectively inhibiting proliferation of rat A10 vascular smooth-muscle cells (VSMCs) in vitro. YC-1 (1-100 microM) dose-dependently inhibited [3H]thymidine incorporation into DNA in rat A10 VSMCs that were synchronized by serum depletion and then restimulated by addition of 10% foetal calf serum (FCS), whereas FCS-induced [3H]thymidine incorporation into rat synchronized endothelial cells was unaffected by this agent. The dose of YC-1 required to cause inhibition of FCS-induced proliferation was similar to that necessary for the formation of cellular cyclic GMP (cGMP). Guanylate cyclase activity in soluble fractions of VSMCs was activated by YC-1 (1-100 microM), whereas cGMP-specific phosphodiesterase activity was unaffected by this compound. The anti-proliferative effect of YC-1 was mimicked by 8-bromo-cGMP, a membrane-permeable cGMP analogue, and was antagonized by KT 5823 (0.2 microM), a selective inhibitor of protein kinase G. The anti-proliferative effect of YC-1 was also antagonized by Methylene Blue (50 microM), a guanylate cyclase inhibitor, and was potentiated by 3-isobutyl-1-methylxanthine (500 microM), a phosphodiesterase inhibitor. These results verified that YC-1 is a direct soluble guanylate cyclase activator in A10 VSMCs, and the anti-proliferative effect of YC-1 is mediated by cGMP. YC-1 still inhibited FCS-induced DNA synthesis even when added 10-18 h after restimulation of the serum-deprived A10 VSMCs with 10% FCS. Flow cytometry in synchronized populations revealed an acute blockage of FCS-inducible cell-cycle progression at a point in the G1/S-phase in YC-1 (100 microM)-treated cells. The inhibition of proliferation by YC-1 was demonstrated to be independent of cell damage, as documented by several criteria of cell viability. In conclusion, YC-1 reversibly and effectively inhibited the proliferation of VSMCs, suggesting that it has potential as a therapeutic agent in the prevention of vascular diseases.

YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway.

The aim of this study was to elucidate the mechanism of YC‐1{3‐(5′‐hydroxy methyl‐2′‐furyl)‐1‐benzylindazole}‐induced human renal carcinoma cells apoptosis and to evaluate the potency of YC‐1 in models of tumour growth in mice.

CLC604 preferentially inhibits the growth of HER2-overexpressing cancer cells and sensitizes these cells to the inhibitory effect of Taxol in vitro and in vivo

HER2 has become a solicitous therapeutic target in metastatic and clinical drug-resistant cancer. Here, we evaluated whether or not 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) and its furopyrazole and thienopyrazole analogues repress the expression of the HER2 protein. Among the test compounds, (1-benzyl-3-(p-hydroxymethylphenyl)-5-methylfuro[3,2-c]pyrazol) (CLC604), an isosteric analogue of YC-1, significantly suppressed the expression of HER2, and preferentially inhibited cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Our results revealed that CLC604 reduced HER2 expression through a post-transcriptional mechanism and involvement of proteasomal activity. CLC604 disrupted the association of 90-kDa heat shock protein (Hsp90) with HER2 resulting from the inhibition of Hsp90 ATPase activity. Moreover, we found that CLC604 significantly enhanced the antitumor efficacy of clinical drugs against HER2-overexpressing tumors and efficiently reduced HER2-induced drug resistance in vitro and in vivo. These findings suggest that CLC604 should be developed further as a novel antitumor drug candidate for the treatment of drug-resistant cancer.