Quantitative high-throughput cell-based assays for inhibitors of ROCK kinases.

The serine/threonine kinases ROCK1 and ROCK2 are direct targets of activated rho GTPases, and aberrant rho/ROCK signaling has been implicated in a number of human diseases. We have developed novel methods for high-throughput assays of ROCK inhibitors that provide for quantitative evaluation of the ability of small molecules to inhibit the function of ROCK kinases in intact cells. Conditions for extraction of known phosphorylated substrates of ROCK were identified, and the involvement of ROCK in phosphorylation of these substrates was evaluated using small interfering RNA (siRNA). Of the potential substrates tested, MYPT1 was identified as a substrate whose phosphorylation was reduced markedly in the combined absence of ROCK1 and ROCK2 proteins, and ELISA methods were developed to allow quantitative measurement of the degree of phosphorylation of MYPT1 at residue T853 in cells grown in 96-well plates. These methods are amenable to high-throughput assays for identification of ROCK inhibitors within libraries of small molecules and can be used to compare compound potencies to prioritize compounds of interest for additional evaluation. These methods should be useful in drug discovery efforts directed toward identifying potent ROCK inhibitors for potential treatment of cancer, hypertension, or other diseases involving rho/ROCK signaling.

OSI-930: a novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models.

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.

ROCK1 and ROCK2 are required for non-small cell lung cancer anchorage-independent growth and invasion.

Evidence is emerging that the closely related ROCK1 and ROCK2 serine/threonine kinases support the invasive and metastatic growth of a spectrum of human cancer types. Therefore, inhibitors of ROCK are under preclinical development. However, a key step in their development involves the identification of genetic biomarkers that will predict ROCK inhibitor antitumor activity. One identified mechanism for ROCK activation in cancer involves the loss of function of the DLC1 tumor suppressor gene, which encodes a GTPase activating protein (RhoGAP) for the RhoA and RhoC small GTPases. DLC-1 loss may lead to hyperactivation of RhoA/C and its downstream effectors, the ROCK kinases. We therefore determined whether loss of DLC-1 protein expression identifies non-small cell lung carcinoma (NSCLC) cell lines whose growth and invasion phenotypes are sensitive to ROCK inhibition. We identified and characterized a novel small molecule pharmacologic inhibitor of ROCK and additionally applied genetic approaches to impair ROCK1 and/or ROCK2 activity, and we determined that although NSCLC anchorage-dependent growth was ROCK-independent, both anchorage-independent growth and Matrigel invasion were ROCK-dependent. However, loss of DLC-1 expression did not correlate with ROCK activation or with OXA-06 sensitivity. Unexpectedly, suppression of ROCK1 or ROCK2 expression alone was sufficient to impair anchorage-independent growth, supporting their nonoverlapping roles in oncogenesis. Mechanistically, the block in anchorage-independent growth was associated with accumulation of cells in the G(0)-G(1) phase of the cell cycle, but not increased anoikis. We conclude that ROCK may be a useful therapeutic target for NSCLC.