Production of phosphorylated and unphosphorylated c-Met: Residue 956 through 1391 (the catalytic domain) of human c-Met was amplified with an N-terminal 6X-HIS tag using the polymerase chain reaction and human liver cDNA. The resulting fragment was cloned into pENTR (Invitrogen, CA), sequence confirmed and transferred to pDEST8 using the Invitrogen Gateway System. Baculovirus was generated with the cMET/pDEST8 clone following the general procedures of the Bac-to-Bac Baculovirus Expression Systems (Invitrogen, CA). E.coli DH10Bac cells were transformed with the cMET/pDEST8 clone and Bacmid DNA was prepared for transfection into Sf21 insect-cells. Conditioned medium containing baculovirus particles was collected from the transfected cells and used for additional infections of Sf21 cells for amplification of the virus. Production of unphosphorylated c-Met was achieved by expressing full length protein tyrosine phosphatase 1B (PTB1B) using an equivalent procedure.

The phosphorylated form of the human c-Met catalytic domain was optimized for expression in Sf21 cells grown in Expression Systems ESF 921 serum-free medium. Cells were infected with baculovirus at a multiplicity of infection (MOI) of 5 and harvested 48 hours post-infection. Unphosphorylated human c-Met (catalytic domain) was produced by baculovirus coinfected with human full-length PTP-1B tyrosine phosphatase at a MOI ratio of 1:1 in Sf21 cells for 48 hours. The degree or lack of phosphorylation was examined by western analysis using both anti-phospho c-Met (pY1234/pY1235, the activation loop phosphorylation sites) and anti-phosphotyrosine antibodies on both the cell lysate and purified protein. The cells were suspended in cold lysis buffer (100 mM potassium phosphate, pH 7.5/250 mM NaCl/5% glycerol/5 mM BME/1% Triton) and lysed by dounce homogenization. The His-tagged c-Met protein was bound to Ni-NTA sepharose, washed with buffer (100 mM potassium phosphate, pH 7.5/250 mM NaCl/5% glycerol/5 mM BME) containing 10 mM and 40 mM imidazole with 100- and 20-column volumes, respectively. His-tagged c-Met was eluted with 10-column volumes of 300 mM imidazole in the same buffer. Eluted protein was dialyzed against the same buffer, concentrated and reloaded on a Talon column. The column was washed with 60- and 10-column volumes of 10 mM and 25 mM imidazole, respectively. c-Met was eluted with 10-column volumes of 150 mM imidazole.

For the western blotting analysis, unphosphorylated and phosphorylated c-Met samples were resolved on SDS-12% PAGE gel and transferred to PVDF membrane (Invitrogen, CA). Phosphorylation was detected using anti-phosphotyrosine or anti-phospho c-Met (pY1234/pY 1235) (Cell Signal, MA) antibodies using standard protocols.

The assay buffer contained 50 mM Tris-HCl, 10 mM MgCl₂, 100 mM NaCl, 0.1 mg/ml BSA, 5mM DTT, pH 7.8. For HTS 0.8 μL of 5 mM of the test compounds dissolved in DMSO were dotted on 384-well plates. DMSO titration suggested that the maximum tolerated concentration of the solvent is 4%. To measure IC₅₀s the compound plate was prepared by 3-fold and 11-point serial dilutions. 0.8 µL of the compound in DMSO was transferred from the compound plate to the assay plate. The final concentration of DMSO was 2%. Solutions of 8 nM unphosphorylated c-Met or 0.5 nM phosphorylated c-Met were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 μM in assay buffer containing 400 μM ATP (unphosphorylated c-Met) or 160 uM ATP (phosphorylated c-Met). A 20 μL volume of enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate and then 20 μL/well of substrate solution to initiate the reaction. The plate was protected from light and incubated at 25 °C for 90 min. The reaction was stopped by adding 20 μL of a solution containing 45 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.4 mg/ml BSA, 200 nM SA-APC and 3 nM EU-Py20. The plate was incubated for 15-30 min at room temperature and HTRF (homogenous time resolved fluorescence) was measured on a Perkin Elmer Fusion α-FP instrument. The HTRF program settings used were as follows: Primary excitation filter 330/30, Primary window: 200 uSec, Primary delay: 50 uSec, Number of flashes: 15, Well read time: 2000 µSec, Secondary excitation filter: 330/30, Secondary window: 400 µSec, Secondary delay: 400 µSec, Primary, Secondary delay: 400 µSec, Primary emission filter: EM 665/10, Secondary emission filter EM 620/10. Percentage of inhibition was calculated for each concentration and IC₅₀ value was generated from curve fitting with GraphPad Prism 3 software. The K_m values for ATP were measured in the presence of 1 μ M substrate peptide.

We produced the catalytic domain of c-Met using the baculovirus expression system. Because c-Met has been shown to be a substrate for protein tyrosine phosphatase 1B (PTP1B), unphosphorylated and phosphorylated c-Met was generated in the presence and absence of coexpressed PTP1B, respectively [5Wang W, Marimuthu A, Tsai J, et al. Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase Proc Natl Acad Sci 2006; 103: 3563-68.]. Phosphorylation of c-Met was monitored by western analysis using both anti-phospho c-Met (pY1234/pY1235, the activation loop phosphorylation sites) and anti-phosphotyrosine antibodies. Expression of c-Met in the absence of PTP1B produced a kinase that had significant phosphorylation of the activation loop while c-Met expressed in the presence of PTP1B produced no detectable phosphorylation either in the activation loop or elsewhere in the kinase domain (Fig. 1). Given the 100-fold higher protein load for the unphosphorylated form and lack of detectable phosporylation in that form by western analysis, we conservatively estimate that the level of phosporylation must be less than 0.1%. Similar results have been reported elsewhere using Escherichia coli as an expression host [5Wang W, Marimuthu A, Tsai J, et al. Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase Proc Natl Acad Sci 2006; 103: 3563-68.].

Fig. (1) Western blot analysis of total phosphorylation of Met. Samples of 5 µg and 50 ng of unphosphorylated (right band) and phosphorylated (left band) c-Met, respectively were resolved by SDS-PAGE under reducing conditions (upper panel). Phosphorylation content was determined using pan anti-phosphotyrosine antibodies (middle panel). Phosphorylation of Y1234 and Y1235 was detected by anti-phospho c-Met (pY1234/pY 1235 (lower panel).

Steady state kinetic analysis was undertaken for each form of the enzyme. The phosphorylated and unphosphorylated forms of c-Met show K_m values for ATP of 60 ± 12 and 200 ± 38 μ M, respectively. Relative K_cat values were determined to be 1.0 and 0.065 for phosphorylated and unphosphorylated forms, respectively (Table 1). While traditionally unphosphophoryalted forms of kinases were assumed to be enzymatically inactive several other unphosphorylated kinases have also be reported to be catalytically active [10Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural mechanism for STI-571 inhibition of Abelson tyrosine Kinase Science 2000; 289: 1938-42.-12Giaiwala KS, Wu JC, Christensen J, et al. Kit kinase Mutants show unique mechanisms of drug resistance to Imatinib and Sunitinib in gastrointestinal stromal tumor patients Proc Natl Acade Sci 2009; 106: 1542-47.]. The activity of unphosphorlayted kinases may represent a basal signaling level in cellular environments. The two forms of c-Met are clearly kinetically distinguishable as reflected by effects on both K_cat and K_m in steady state kinetic analysis.

Table 1

Kinetic Parameters for Phosphorylated and Unphosphorylated c-Met

While it is theoretically possible that unphosphorylated c-Met undergoes autophosphorylation under the assay conditions, this seems unlikely for several reasons. 1) No phosphorylation was detected by western analysis after incubating this form of the enzyme under assay conditions (data not shown) 2) product formation was linear with time (see Fig. 2). Since the phosphorylated enzyme has higher catalytic activity, c-Met phosphorylation during the kinase assay would have resulted in upward curvature for product formation. Timofeevski et al. noted increase in K_cat over time under conditions where autophosphorylation can occur [13Sergei L Timofeevski, Michele A McTigue, Kevin Ryan, et al. Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors Biochemistry 2009; 48(23): 5339-49.]. No such change is observed under our reaction conditions. 3) As will be shown below, the two forms of the enzyme have reproducible and different IC₅₀ values for a series of inhibitors (Figs. 3A & D). 4) The low concentrations of the kinase in the assay make trans-autophosphorylation unlikely. Timofeevski et al. noted strong c-Met concentration dependence for autophosphorylation [13Sergei L Timofeevski, Michele A McTigue, Kevin Ryan, et al. Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors Biochemistry 2009; 48(23): 5339-49.] and performed autophosphorylation reactions at 10 uM enzyme (in the presence of 4 mM ATP at 4 °C which can overall lead to ~2-fold decrease in the catalytic activity compared to 200 µM ATP at 25 ºC). Their c-Met concentrations are 2500-fold higher than in the enzyme assays conducted here.

Fig. (2) Time course of the phsophorylation of the peptide substrate by unphosphorylated Met. 4 nM of unphosphorylated c-Met was incubated with 1 µM of peptide substrate Biotin- EQEDEPEGDYFEWLE-amide and 200 µM ATP. The reaction was carried out at 25 °C in 50 mM Tris-HCl, 10 mM MgCl2 ,100 mM NaCl, 0.1 mg/ml BSA, 5mM DTT, pH 7.8. Points are duplicates and the data were fit to a line.

Fig. (3) Comparison of dose-dependent inhibition of phosphorylated and unphosphorylated c-Met by small molecule inhibitors. 4 nM of the unphosphorylated was incubated with 1 µM of peptide substrate, 200 µM ATP and small molecule inhibitors (filled triangles) for 90 minutes at 25 °C. 0.25 nM of the phosphorylated c-Met was mixed with1 µM of peptide substrate 80 µM ATP and small molecule inhibitors (open squares) for 90 minutes at 25 °C. Hit 7 is from Table 2 and Hit 3 is from Table 3. Points are duplicates normalized to 100% for the uninhibited kinase. Data were fit to a hyperbola on logarithmic scale.

Table 2

IC₅₀ Values for Screening Hits (General Library)

Table 3

IC₅₀ Values for Screening Hits (Internal Kinase Inhibitors Collection)

42,000 compounds from the ChemBridge collection were screened against phosphorylated and unphosphorylated c-Met. ChemBridge is a collection of 450,000 verified, druglike, diverse, small molecule compounds. The assay performed well over the course of the screen; control wells for each plate averaged a signal/background ratio of 8.6 with Z´ (Z´ = 1- ([3 X STD _{negative control}] + [3 X STD _{positive control}])/(mean _{positive control} – mean _{negative control}) of 0.74 for the unphosphorylated and 14.5 and 0.81 for the phosphorylated form, respectively. The concentration-response curves for the positive control, SU11274, included on every screening plate, were consistent, with median minimum significant ratio (MSR) value of 1.3 for phosphorylated and 1.2 for unphophorylated [14Brian J, Eastwood BJ, Mark W, et al. The minimum significant ratio: a statistical parameter to characterize the reproducibility of potency estimates from concentration-response assays and estimation by replicate-experiment studies J Biomol Screen 2006; 11: 253-61.]. The Z´ and MSR values for both assays indicate that the screening efforts would be expected to be robust and reliable.

The compounds were tested at 100 µM. All compounds that exhibited greater than 30% inhibition against either form of the enzyme were then retested against both forms by determining their IC₅₀ values. The hits that had been shown to test positive against non-kinase targets from other HTS campaigns were eliminated as false positives (~11% of the total hits). Using a 30 µM cutoff for potency when defining a hit, the hit rate for phosphorylated c-Met was 0.09 % (38 total hits) while the hit rate for unphosphorylated c-Met is 0.13% (56 total hits). The hit rate for unphosphorylated c-Met was ~ 44% higher than for phosphorylated c-Met. While this ratio of hit rates might change if larger numbers of compounds were screened, it is clear that for the limited set tested there is a higher number of potential lead molecules generated when screening with the unphosphorylated form of the enzyme. Inspection of the relative potencies for compounds indicates that the majority of compounds are roughly equally potent on both forms of c-Met with ratios close to 1.0 (Fig. 3C). There are however, compounds that exhibit at least a 5-fold more potent IC₅₀ value for unphosphorylated c-Met relative to phosphorylated c-Met (Table 2). In fact of the 56 hits for unphosphorylated c-Met, six have greater than 5-fold specificity. These hits (ratios >5), four of which with purine moities and two of which with a novel benzyloxybenzaldehyde oxime core structure, represent potential lead molecules that would not have been identified by screening only the phosphorylated form of c-Met. On the other hand, no compound shows significantly greater potency on the phosphorylated form (ratio significantly <1.0) which accounts for the increased hit rate for the unphosphorylated form. From a structural perspective, the specific molecules must be binding to a conformation that only the unphosphorylated form can easily adopt. The nonspecific molecules, on the other hand, must bind to a conformation that is present in both forms of the enzyme. The scenario is quite plausible given the known large conformation changes that kinases undergo and that the conformation preferences are in large part determined by phosphorylation state [15Huse M, Kuriyan J. The conformational plasiticity of protein kinases Cell 2002; 107: 275-.].

We also examined two published kinase inhibitors that inhibit c-Met. Staurosporine is a well known ATP-competitive nonspecific kinase inhibitor. SU11274 is a commercially available potent c-Met inhibitor. SU11274 is a well studied specific ATP-competitive inhibitor and has been shown to be active against numerous cancer cell lines [16Sattler M, Pride YB, Ma P, et al. A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase Cancer Res 2003; 63(17): 5462-9.-18Bellon SF, Kaplan-Lefko P, Yang Y, et al. Dussault. c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations J Biol Chem 2008; 283(5): 2675-83.]. Table 2 and Figs. (3A and 3B) show the IC50 values for these two inhibitors. Staurosporine has essentially the same IC50 value for both forms of the enzyme while SU11274 is 5 nM on the unphosphorylated form and 275 nM on the phosphorylated form, a 55-fold difference in potency. Furthermore, the IC50 for SU11274 is 30 nM in SNU5 proliferation assays. SNU5 is a gastric cancer cell line in which c-Met is amplified. Given that the compound is more potent on cells than on the phosphorylated form of the enzyme, it is likely that the cellular activity is being driven by inhibition of the unphosphorylated enzyme. The discrepancy is even larger when one accounts for the high cellular ATP concentrations of approximately 1 mM (predicted IC50 values for SU11274 are 30 nM and 3331 nM for the phosphorylated and unphosphorylated forms, respectively). We have noted the same trend with a large number of compounds in our internal c-Met program.

The observations made during our screening endeavor and examination of known c-Met inhibitors convinced us to examine our internal kinase library for additional inhibitors of the unphosphorylated form. The internal library is a compound collection that resulted from medicinal chemistry efforts on several unrelated kinase targets. All chemical series were shown to be ATP competitive against their respective kinase target.

Using a 30 µM cutoff for potency when defining a hit, the hit rate for phosphorylated c-Met was 1.0 % (73 total hits) while the hit rate for unphosphorylated c-Met was 0.89 % (62 total hits). The hit rate for unphosphorylated c-Met was similar to the phosphorylated c-Met. While screening a random library led us discover potential lead molecules that inhibit unphosphorylated c-Met, screening our internal kinase inhibitors (Table 3) resulted in identifying seven compounds that were 5-fold or more potent against the phosphorylated form (ratio significantly <1.0). For example, while Hit 1 and 2 inhibit the phosphorylated form with IC₅₀s of 11 and 4.2 µM respectively, they do not inhibit the unphosphorylated form. There are also compounds which do not inhibit the phosphorylated form but are relatively potent against unphosphorylated c-Met (Hit 3 and 4). In fact six compounds from the 62 hits were identified to be 5-fold or more selective for unphosphorylated form. The screening of our internal kinase collection has shown that it is possible to obtain compounds that are more potent on the phosphorylated c-Met. Most of these compounds had a common core of benzoimidazo (or thiozolo) isoquiolinon, a known kinse scaffold. These compounds presumably bind a conformation (or induce a conformation) that is preferred by the phosphorylated form and/or disfavored by the unphosphorylated form.