Wednesday, May 20, 2015

Drugs in Clinical Pipeline: ZM336372

ZM336372 [3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)amino]-4-methylphenyl]-benzamide] is a potent ATP-competitive inhibitor of Raf-1 in vitro (IC50 = 70 nM). The compound inhibited c-Raf tenfold more potently than B-Raf and did not inhibit 17 of 19 other protein kinases tested, even at 50 uM. The two exceptions were SAPK2a/p38a and SAPK2b/p38b2, both of which were inhibited by ZM 336372 with an IC50 of 2 uM under the assay conditions used [1]. The Ras/Raf-1 signalling pathway is a well-characterized system that links receptor tyrosine kinase (RTK) activation with changes in gene expression and cell behavior. Raf-1 is a serine/threonine protein kinase that phosphorylates and activates MAPK-kinase (MEK) in response to activation by Ras.

Though, ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro, paradoxically, exposure of cells to ZM 336372 induces greater than 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKKI or p42 MAPK/ERKP. 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 MKKl or p42 MAPK, or reverse the phenotype of Ras- or Raf-transformed cell lines. ZM 336372 does not by itself induce the activation of MKKl or p42 MAPK, yet removing it causes an activation of MKKl and p42 MAPK. This implies that the cell does indeed contain high specific activity c-Raf that is prevented from activating its downstream substrate MKKl because of the presence of the inhibitor. The molecular mechanism by which this feedback loop operates appears to be novel, because it is not affected by inhibitors of protein kinase C, PtdIns 3-kinase or the MAP kinase cascade, and does not involve any change in the GTP loading of Ras.

The activity of ZM 336372 is as follows:

IC50 (RAF-1 enzyme assay) = 70 nM

Common Name: ZM 336372
Synonyms:  ZM336372; ZM-336372; ZM 336372
IUPAC Name: 3-(Dimethylamino)-N-[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl] benzamide
CAS Number: 208260-29-1
SMILES: Oc1ccc(cc1)C(=O)Nc1cc(ccc1C)NC(=O)c1cccc(c1)N(C)C
Mechanism of Action: Kinase Inhibitor; RAF-1 Inhibitor
Indication: Various Cancers; Anti-tumor Therapy
Development Stage: Investigational
Company: -

The ras/Raf-1 signaling pathway has long been recognized for its importance in cancer biology. In this pathway, ras signaling often involves activation of Raf-1, a cytosolic serine/threonine kinase. Activated Raf-1 then phosphorylates mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), which then activates downstream extracellular signal–regulated kinase 1 and 2 (ERK1/2). Activating mutations in this pathway are common among adenocarcinomas of the colon, pancreas, and lung as well as squamous cell cancers. However, in neuroendocrine tumors such as small cell lung (SCLC), medullary thyroid, and carcinoid cancer, mutations causing elevated Raf-1 signaling are rarely detected in pathologic specimens. In SCLC, Raf-1 pathway activation results in phenotypic change and reduction in cellular proliferation.

ZM336372, is capable of reducing tumor cell growth as well as neuroendocrine hormones production in carcinoid cells, addressing both proliferation and palliative issues associated with carcinoid tumors. Furthermore, it is reported that treatment with ZM336372 significantly reduces chromogranin A in carcinoid tumor cells quickly and with prolonged effect. Additionally, the neuroendocrine transcription factor, hASH1, is suppressed in response to treatment with ZM336372 [2].

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. To test the role of the signalling pathway, pheochromocytoma PC-12 cells were treated with varying concentrations of ZM336372. 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. 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. 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 [3].

1. Hall-Jackson, C. A.; et. al. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol 1999, 6(8), 559-568.
2. Van Gompel, J. J.; et. al. ZM336372, a Raf-1 activator, suppresses growth and neuroendocrine hormone levels in carcinoid tumor cells. Mol Cancer Ther 2005, 4, 910.
3. Kappes, A.; et. al. ZM336372, A Raf-1 Activator, Inhibits Growth of Pheochromocytoma Cells. J Surg Res 2006, 133(1), 42-45.