SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo.

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

SGX393 inhibits the CML mutant Bcr-AblT315I and preempts in vitro resistance when combined with nilotinib or dasatinib.

Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl(T315I), however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl(T315I), with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl(T315I). These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

Effect of experimental pH on the in vitro permeability in intact rabbit intestines and Caco-2 monolayer.

The effect of experimental (apical) pH on absorptive permeability (Pe) was investigated in animal intestinal tissues and Caco-2 cell monolayers to examine whether the introduction of physiological pH such as 6.5 relates to the better prediction of animal intestinal Pe. Transport studies were conducted in a 24-well transwell for Caco-2 and diffusion chambers for rabbit intestinal permeability. Twenty-four test compounds were chosen (seven acidic, seven basic, eight neutral, and two zwitterionic) and Pe was measured at a 100microM donor concentration with two apical pHs, Krebs Bicarbonate Ringer's buffer (pH 7.4) and 4-morpholineethanesulfonic acid (MES) buffer (pH 6.5). Samples were collected over a 90-min interval and analyzed by LC/UV, LC/MS, or LSC. Upon the apical pH change from 7.4 to 6.5, Caco-2 Pe of acidic and basic compounds changed significantly, whereas rabbit intestinal Pe did not change possibly by the presence of mucous layer. When the intestinal Pe was correlated with pH 6.5 or 7.4 Caco-2 Pe, the correlation of pH 6.5 duodenum and jejunum Pe with pH 6.5 Caco-2 Pe was very poor. However, pH 7.4 Caco-2 Pe correlated relatively well with pH 6.5 duodenum and jejunum Pe and pH 7.4 ileum and colon Pe. The results suggested that pH 7.4 Caco-2 Pe is a good qualitative predictor for physiological intestinal permeability from duodenum to colon.

Modulation of nonspecific binding in ultrafiltration protein binding studies.

PURPOSE: The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units. METHODS: UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 microM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37 degrees C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC. RESULTS: Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pre-treatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED. CONCLUSIONS: The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.

Heparinase enhances collateral vessel development in the ischemic limb.

Several angiogenic factors are bound to heparin and stored in the extracellular matrix of diverse tissues; thus, the controlled release of these factors can provide a novel approach of angiogenic stimulation to ischemic tissues. The purpose of this study was to test the hypothesis that heparinase, when administered in vivo, might enhance collateral vessel development in the ischemic limb by the controlled release of angiogenic factors such as basic fibroblast growth factor (bFGF). Eleven male New Zealand White rabbits underwent ligation and excision of the common and superficial femoral arteries in the left hindlimb. In the heparinase group (n = 6), 9.7 IU of heparinase (in 3 ml saline) was injected intramuscularly to the left thigh daily for 10 days, beginning 11 days after surgery. In the control group (n = 5), inactivated heparinase (also in 3 ml saline) was administered following the same experimental protocol. Calf systolic pressure was measured in both hindlimbs and was expressed as a ratio of left to right (L/R ratio) before injections (on day 10) and after injections (on days 20, 30, and 40). Vascularization was quantified by comparing the number of vessels along a line drawn across the mid-thigh on angiograms taken 4 seconds after contrast injection at day 40 when the study was terminated. The intramuscular injection of heparinase improves perfusion to the ischemic limb through the process of enhanced collateral vessel development. The angiogenic effect of heparinase on the ischemic tissue may result from release of endogenous angiogenic factors, such as bFGF.