ZM336372

ZM336372 is a potent inhibitor of C-Raf with IC50 value of 70 nM [1]. It is also reported that ZM336372 inhibits SAPK2b/p38β2 with IC50 value of 2 μM.

C-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases and plays an important role in the ERK1/2 pathway. It has been reported that abnormal expression of C-Raf correlates with human diseases and also involves in a variety of cancers.

ZM336372 is a potent C-Raf inhibitor and has a different selectivity with the reported C-Raf inhibitor PD98059. When tested with primary neurons, ZM3363372 treatment decreased low potassium-induced apoptosis percent by inhibiting C-Raf activation [2]. In tumor spheroids pretreated with H2O2 and ZM336372 for 24 hours totally abolished the ROS-induced eNOS up-regulation via mediating ERK1/2 signaling pathway [3].

References:
1.  Hall-Jackson, C.A., et al., Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol, 1999. 6(8): p. 559-68.
2.  Burgess, S. and V. Echeverria, Raf inhibitors as therapeutic agents against neurodegenerative diseases. CNS Neurol Disord Drug Targets, 2010. 9(1): p. 120-7.
3.  Wartenberg, M., et al., Reactive oxygen species-mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids. Int J Cancer, 2003. 104(3): p. 274-82.

ZM336372, a Raf-1 activator, causes suppression of proliferation in a human hepatocellular carcinoma cell line.[Pubmed:18299943]

J Gastrointest Surg. 2008 May;12(5):852-7.

Hepatocellular carcinoma has been described to exhibit characteristics similar to that of neuroendocrine tumors (NETs). This includes similar anti-neoplastic responses to extracellular signal-regulated kinase (ERK) activation. NET cells and HepG2 cells have both shown growth inhibition with ERK activation. ZM336372, a Raf-1 activating agent, has been shown to cause growth inhibition and suppression of hormone secretion in a neuroendocrine cell line. Here we examine treatment of the HepG2 cell line with ZM336732 to determine if a similar anti-proliferative response will be obtained. HepG2 cells were treated with ZM336372 or solvent (dimethyl sulfoxide). The resulting effect on the proliferation was measured using the 3,4-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Western blot analysis was performed to examine the activation of the Raf-1/mitogen-activated protein kinase kinase/ERK pathway, chromogranin A production, and p21CIP1 level. Growth inhibition was observed with ZM336372 in a dose-dependent fashion. Minimal baseline phosphorylation of ERK 1/2 was observed; however, activation was observed after treatment with ZM336372. Chromogranin A secretion was suppressed due to treatment with ZM336372. A dose-dependent up-regulation of p21CIP1 was observed in response to ZM336372 treatment. ZM336372 causes growth inhibition, suppression of hormone secretion, and up-regulation of cell cycle inhibitors in a human hepatocellular carcinoma cell line, similar to that previously seen in NETs.

Neuroendocrine tumor cell growth inhibition by ZM336372 through alterations in multiple signaling pathways.[Pubmed:18063082]

Surgery. 2007 Dec;142(6):959-64; discussion 959-64.

BACKGROUND: We have shown previously that activation of the Raf-1/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling pathway by ZM336372 inhibits carcinoid cells growth. In the present study, we further characterize the molecular details of the growth inhibition by the signaling-based compound ZM336372 in neuroendocrine neoplasms (NENs). METHODS: NEN cells were treated with ZM336372 (20 to 100 mumol/L) or carrier (DMSO). Western Blot was used to determine the activation of the Raf-1/MEK/ERK, other pathways activation, and cellular bioactive hormone production. RESULTS: ZM336372 in NEN cells resulted in increasing raf-1 activation and inactivation of glycogen synthase kinase-3 beta (GSK-3beta) as measured by phosphorylation of ERK1/2 and GSK-3beta, respectively. There was no alteration in the levels of phosphorylated Akt, an important mediator of the phosphatidyl inositol 3 kinase pathway. Importantly, blocking of raf-1 pathway by U0126, a potent inhibitor, in the presence of ZM336372 did not reduce the levels of p-GSK-3beta, indicating that GSK-3beta inactivation is independent of raf-1 pathway activation. Moreover, the levels of chromogranin A and achaete-scute complex like-1 reductions were persistent even after blocking the raf-1 pathway. Treatment with ZM336372 in the presence of small interfering RNA against raf-1 resulted in an increase in Raf-1 production, suggesting that ZM336372 upregulates raf-1 at the transcriptional level. CONCLUSION: This is the first description of a novel compound ZM336372 that regulates multiple pathways in NEN cells.

ZM336372 induces apoptosis associated with phosphorylation of GSK-3beta in pancreatic adenocarcinoma cell lines.[Pubmed:20031160]

J Surg Res. 2010 Jun 1;161(1):28-32.

INTRODUCTION: ZM336372 is small molecule tyrosine kinase modulator. It has been shown to inhibit glycogen synthase kinase-3beta (GSK-3beta) through phosphorylation of GSK-3beta at Ser 9. GSK-3beta has previously been shown to mediate cell survival in pancreatic cancer cells. Here we determine the effects of ZM336372 on GSK-3beta phosphorylation, apoptosis, and growth in pancreatic adenocarcinoma cell lines. METHODS: Panc-1 and MiaPaCa-2 cells were treated with ZM336372 or lithium chloride (LiCl) and compared with solvent control. The effects on proliferation for each cell line were determined using the MTT assay. Western blot analysis was performed to examine the effects of treatment on the phosphorylation of GSK-3beta. In addition, western blot was utilized to examine the cleavage of poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. RESULTS: A dose-dependent increase in phosphorylation of GSK-3beta was observed after treatment with both ZM336372 and LiCl. Growth inhibition due to treatment with ZM336372 and LiCl also occurred in a dose-dependent fashion. An increase in cleaved PARP was demonstrated after treatment with both agents, as was seen previously with GSK-3beta inhibition in pancreatic adenocarcinoma cells. CONCLUSION: This is the first description of growth inhibition and apoptosis in pancreatic cancer cells related to GSK-3beta inhibition through treatment with ZM336372.

ZM336372, a Raf-1 activator, inhibits growth of pheochromocytoma cells.[Pubmed:16603190]

J Surg Res. 2006 Jun 1;133(1):42-5.

BACKGROUND: Surgical resection is the only curative treatment for patients with pheochromocytomas, paragangliomas, and other catecholamine-producing tumors. Patients with metastatic disease can often have significant symptoms associated with catecholamine excess. Activation of the Raf-1/MEK/ERK1/2 pathway has been shown to inhibit growth and hormone production for neuroendocrine tumors (NE) such as carcinoid and medullary thyroid cancer. However, the role of the Raf-1/MEK/ERK1/2 signaling pathway in pheochromocytomas is unknown. MATERIALS AND METHODS: Pheochromocytoma PC-12 cells were treated with varying concentrations of ZM336372, a pharmacologic Raf-1 activating drug. Levels of phosphorylated ERK1/2 and the NE marker Chromogranin A (CgA) were determined by Western blot. Cellular growth was measured by MTT cell-proliferation assay. RESULTS: At baseline, PC-12 cells had very little phosphorylated ERK1/2, similar to other NE tumors. Treatment of PC-12 cells with increasing dosages of ZM336372 resulted in increased phosphorylated ERK1/2. Importantly, ZM336372 inhibited pheochromocytoma cellular proliferation. Furthermore, Raf-1 pathway activation by ZM336372 was associated with suppression of NE marker, CgA, by the tumor cells. CONCLUSIONS: These data suggest that Raf-1/MEK/ERK1/2 pathway activation may be a novel strategy to treat pheochromocytoma and other catecholamine-producing tumors. In pheochromocytoma cells, ZM336372 blocks cellular proliferation and suppresses NE vasoactive peptide production. Thus, ZM336372 may be used for both therapeutic and palliative treatment for patients with pheochromocytomas.

Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species.[Pubmed:11279018]

J Biol Chem. 2001 May 18;276(20):17420-8.

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

Paradoxical activation of Raf by a novel Raf inhibitor.[Pubmed:10421767]

Chem Biol. 1999 Aug;6(8):559-68.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.

ZM 336372 is a potent inhibitor of the protein kinase c-Raf. The IC50 value is 0.07 μM in the standard assay, which contains 0.1 mM ATP.

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