The signaling pathway of the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is important for cell growth, survival, and motility and is functionally linked to the signaling pathway of VEGF, which is widely recognized as a key effector in angiogenesis and cancer progression. Dysregulation of the MET/VEGF axis is found in a number of human malignancies and has been associated with tumorigenesis. Cabozantinib (XL184) is a small-molecule kinase inhibitor with potent activity toward MET and VEGF receptor 2 (VEGFR2), as well as a number of other receptor tyrosine kinases that have also been implicated in tumor pathobiology, including RET, KIT, AXL, and FLT3. Treatment with cabozantinib inhibited MET and VEGFR2 phosphorylation in vitro and in tumor models in vivo and led to significant reductions in cell invasion in vitro. In mouse models, cabozantinib dramatically altered tumor pathology, resulting in decreased tumor and endothelial cell proliferation coupled with increased apoptosis and dose-dependent inhibition of tumor growth in breast, lung, and glioma tumor models. Importantly, treatment with cabozantinib did not increase lung tumor burden in an experimental model of metastasis, which has been observed with inhibitors of VEGF signaling that do not target MET. Collectively, these data suggest that cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling.

The Met receptor tyrosine kinase and its ligand,

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models, leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973, a novel selective MEK inhibitor, and GDC-0941, a class I PI3K inhibitor, are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here, we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice, we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis, including Bcl-2 family proapoptotic regulators. Therefore, these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing. Cancer Res; 72(1); 210-9. Ó2011 AACR.

Growth and differentiation factor (GDF) 11 is a member of the transforming growth factor β superfamily recently identified as a potential therapeutic for age‐related cardiac and skeletal muscle decrements, despite high homology to myostatin (Mstn), a potent negative regulator of muscle mass. Though several reports have refuted these data, the in vivo effects of GDF11 on skeletal muscle mass have not been addressed. Using in vitro myoblast culture assays, we first demonstrate that GDF11 and Mstn have similar activities/potencies on activating p‐SMAD2/3 and induce comparable levels of differentiated myotube atrophy. We further demonstrate that adeno‐associated virus‐mediated systemic overexpression of GDF11 in C57BL/6 mice results in substantial atrophy of skeletal and cardiac muscle, inducing a cachexic phenotype not seen in mice expressing similar levels of Mstn. Greater cardiac expression of Tgfbr1 may explain this GDF11‐specific cardiac phenotype. These data indicate that bioactive GDF11 at supraphysiological levels cause wasting of both skeletal and cardiac muscle. Rather than a therapeutic agent, GDF11 should be viewed as a potential deleterious biomarker in muscle wasting diseases.

The ERK/MAP kinase cascade is a key mechanism subject to dysregulation in cancer and is constitutively activated or highly upregulated in many tumor types. Mutations associated with upstream pathway components RAS and Raf occur frequently and contribute to the oncogenic phenotype through activation of MEK and then ERK. Inhibitors of MEK have been shown to effectively block upregulated ERK/MAPK signaling in a range of cancer cell lines and have further demonstrated early evidence of efficacy in the clinic for the treatment of cancer. Guided by structural insight, a strategy aimed at the identification of an optimal diphenylamine-based MEK inhibitor with an improved metabolism and safety profile versus PD-0325901 led to the discovery of development candidate 1-({3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl}carbonyl)-3-[(2S)-piperidin-2-yl]azetidin-3-ol (XL518, GDC-0973) (1). XL518 exhibits robust in vitro and in vivo potency and efficacy in preclinical models with sustained duration of action and is currently in early stage clinical trials. KEYWORDS: MAPK pathway, MEK, cancer, kinase inhibitor, XL518, GDC-0973 T he MAPK cascade, or mitogen-activated protein kinase signal transduction pathway, is a mechanism commonly subject to dysregulation in cancer, and constitutive or highly upregulated signaling is a frequent hallmark of oncogenic transformation and progression. Controlled by activation of RAS at the cell surface interior, the subsequent stimulation of Raf and then MEK and ERK serves to regulate a range of key intracellular effectors associated with cell proliferation, 1 invasion, 2 angiogenesis, 3 and apoptotic resistance. 4 Mutated RAS is associated with almost one-third of human cancers, and a majority of malignant melanomas and papillary thyroid cancers harbor B-Raf mutations. 5,6 Inhibitors of MEK have demonstrated efficacy against malignant tumors characterized by mutations in either RAS or Raf in preclinical models, and early development candidates including GSK1120212, In some tumors, activation of both the RAS driven ERK/ MAPK cascade and the PI3K-Akt pathway is observed, resulting in degenerate and convergent oncogenic signals, and upregulation or constitutive PI3K pathway activation is associated with resistance to MEK inhibitor single agent treatment.10 Several combination approaches using inhibitors of mTor, PI3K, Akt, and Raf have more recently been validated in preclinical models and are being pursued clinically. Herein, the discovery of XL518 (GDC-0973) (1), a potent and selective MEK inhibitor, is described. XL518 is currently in early stage clinical testing as both a single agent and in combination with the class I PI3K inhibitor GDC-0941. 11Our goal at the outset was the identification of a potent and selective MEK inhibitor with sustained duration of efficacy suitable for qd dosing and an optimized safety profile relative to clinical precursors. The diphenylamine series disclosed by the Pfizer/Warner Lambert groups served as a starting point for our effort. A key aspect tha...

ABSTRACT:The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO 2 , -CN, and CH 3 CO-), 3S-C 8 -, and 3′-dephospho-3S-C 8 CoA. These analogues lack a C-H and cannot undergo R, -dehydrogenation. Instead they deprotonate at RC-H at pH g 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pK a s of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO 2 ), ≈14.5 (-CN), and ≈14.6 (CH 3 CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo RC-H deprotonation. While the extent of deprotonation varies, the anionic products form charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (K d ) of p-NO 2 -phenylacetyl-CoA (4NPA-CoA), 3S-C 8 -CoA, and 3′-dephospho-3S-C 8 CoA, four pK a s at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pK a ≈5, ligand (L-H) in the MCADH∼ligand complex; (b) pK a ≈6, Glu376-COOH in uncomplexed MCADH; (c) pK a ≈7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK ≈8, Glu99-COOH in the MCADH∼4NPA-CoA complex. The pK a ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by g1 pK unit in that with 3S-C 8 CoA and further in the presence of C 8 -CoA, the best substrate. The RC-H pK a s of 4NPA-CoA, of 3S-C 8 -CoA, and of 3′-dephospho-3S-C 8 CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pK a values are therefore lowered by 8 to g11 pH units (50 to g 65 kJ mol -1 ) and are close to the pK a of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2′-OH of FAD and Glu376-NH. It is concluded that the pK a shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, RC-H abstraction is fast (k app ≈55 s -1 at pH 7.5 and 25°C, deuterium isotope effect ≈1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pK a s of ≈5 and ≈7.3, which correspond to those determined in static experiments.Acyl-CoA dehydrogenases catalyze the R, -dehydrogenation of fatty acid acyl-CoA conjugates to their corresponding enoyl-CoA products; the redox equivalents formed in this reaction are transferred to electron transferring flavoprotein and further to the respiratory chain (1, 2). A peculiarity of the R, -dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase ...

Background: A limited number of approved therapeutic options are available to metastatic medullary thyroid cancer (MTC) patients, and the response to conventional chemotherapy and/or radiotherapy strategies is inadequate. Sporadic and inherited mutations in the tyrosine kinase RET result in oncogenic activation that is associated with the pathogenesis of MTC. Cabozantinib is a potent inhibitor of MET, RET, and vascular endothelial factor receptor 2 (VEGFR2), as well as other tyrosine kinases that have been implicated in tumor development and progression. The object of this study was to determine the in vitro biochemical and cellular inhibitory profile of cabozantinib against RET, and in vivo antitumor efficacy using a xenograft model of MTC. Methods: Cabozantinib was evaluated in biochemical and cell-based assays that determined the potency of the compound against wild type and activating mutant forms of RET. Additionally, the pharmacodynamic modulation of RET and MET and in vivo antitumor activity of cabozantinib was examined in a MTC tumor model following subchronic oral administration. Results: In biochemical assays, cabozantinib inhibited multiple forms of oncogenic RET kinase activity, including M918T and Y791F mutants. Additionally, it inhibited proliferation of TT tumor cells that harbor a C634W activating mutation of RET that is most often associated with MEN2A and familial MTC. In these same cells grown as xenograft tumors in nude mice, oral administration of cabozantinib resulted in dose-dependent tumor growth inhibition that correlated with a reduction in circulating plasma calcitonin levels. Moreover, immunohistochemical analyses of tumors revealed that cabozantinib reduced levels of phosphorylated MET and RET, and decreased tumor cellularity, proliferation, and vascularization. Conclusions: Cabozantinib is a potent inhibitor of RET and prevalent mutationally activated forms of RET known to be associated with MTC, and effectively inhibits the growth of a MTC tumor cell model in vitro and in vivo.

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