The innate immune signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies. (Cancer Res 2006; 66(17): 8715-21)

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between (1)H and (13)C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure-activity relationship (SAR) studies led to 16 and 18, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (16) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis.