A novel mode of protein kinase inhibition exploiting hydrophobic motifs of autoinhibited kinases: discovery of ATP-independent inhibitors of fibroblast growth factor receptor.

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.

ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

A novel platform for accelerated pharmacodynamic profiling for lead optimization of anticancer drug candidates.

Oncology drug discovery is, by definition, a target-rich enterprise. High-throughput screening (HTS) laboratories have supported a wide array of molecularly targeted and chemical genomic approaches for anticancer lead generation, and the number of hits emerging from such campaigns has increased dramatically. Although automation of HTS processes has eliminated primary screening as a bottleneck, the demands on secondary screening in appropriate cell-based assays have increased concomitantly with the numbers of hits delivered to therapeutic area laboratories. The authors describe herein the implementation of a novel platform using off-the-shelf solutions that have allowed them to efficiently characterize hundreds of HTS hits using a palette of Western blot-based pharmacodynamic assays. The platform employs a combination of a flatbed bufferless SDS-PAGE system, a dry ultra-rapid electroblotting apparatus, and a highly sensitive and quantitative infrared imaging system. Cumulatively, this platform has significantly reduced the cycle time for HTS hit evaluation. In addition, the routine use of this platform has resulted in higher quality data that have allowed the development of structure-activity databases that have tangibly improved lead optimization. The authors describe in detail the application of this platform, designated the Accelerated Pharmaco-Dynamic Profiler (APDP), to the annotation of inhibitors of 2 attractive oncology targets, BRAF kinase and Hsp90.

Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway.

TGF-beta can signal by means of Smad transcription factors, which are quintessential tumor suppressors that inhibit cell proliferation, and by means of Smad-independent mechanisms, which have been implicated in tumor progression. Although Smad mutations disable this tumor-suppressive pathway in certain cancers, breast cancer cells frequently evade the cytostatic action of TGF-beta while retaining Smad function. Through immunohistochemical analysis of human breast cancer bone metastases and functional imaging of the Smad pathway in a mouse xenograft model, we provide evidence for active Smad signaling in human and mouse bone-metastatic lesions. Genetic depletion experiments further demonstrate that Smad4 contributes to the formation of osteolytic bone metastases and is essential for the induction of IL-11, a gene implicated in bone metastasis in this mouse model system. Activator protein-1 is a key participant in Smad-dependent transcriptional activation of IL-11 and its overexpression in bone-metastatic cells. Our findings provide functional evidence for a switch of the Smad pathway, from tumor-suppressor to prometastatic, in the development of breast cancer bone metastasis.

Dual induction of apoptosis and senescence in cancer cells by Chk2 activation: checkpoint activation as a strategy against cancer.

The human checkpoint kinase 2 (Chk2) plays a central role in regulation of the cellular response to DNA damage, resulting in cell cycle arrest, DNA repair, or apoptosis depending on severity of DNA damage and the cellular context. Chk2 inhibitors are being developed as sensitizers for chemotherapeutic agents. In contrast, here we report that direct activation of Chk2 alone (without chemotherapeutic agents) led to potent inhibition of cancer cell proliferation. In the absence of de novo DNA damage, checkpoint activation was achieved by increased Chk2 expression, as evidenced by its phosphorylation at Thr68, resulting in senescence and apoptosis of cancer cells (DLD1 and HeLa). The Chk2-induced apoptosis was p53 independent and was mediated by caspase activation triggered by loss of mitochondrial potential. The Chk2-induced senescence was also p53 independent and was associated with induction of p21. These results suggest that direct activation of checkpoint kinases may be a novel approach for cancer therapy.

A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells.

Genome-wide transcriptional profiling of human epithelial cells revealed that repression of Id inhibitors of differentiation (Id1, Id2, and Id3) is a general feature of the TGFbeta cytostatic program. Opposite responses of Id1 to TGFbeta and the related factor BMP are dictated by the specific ability of the TGFbeta mediator, Smad3, to activate expression of stress response factor ATF3 and then recruit this factor to the Id1 promoter. Thus, a Smad3-mediated primary gene response, ATF3 induction, enables Smad3 to participate in an ATF3-mediated, secondary gene response. As a common target of TGFbeta/Smad signals and stress signals via p38 kinase, ATF3 additionally serves to channel synergy between these pathways in the response of epithelial cells to stress and injury.

E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression.

Smad3 is a direct mediator of transcriptional activation by the TGFbeta receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. We defined how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners.