Abstract
A high-throughput screen for Ras–mitogen-activated protein kinase (MAPK) signaling inhibitors identified two series (class 1 and 2) of substituted 4-anilino-3-quinolinecarbonitriles as potent (IC50s <10 nmol/L) mitogen-activated protein/extracellular signal-regulated kinase 1 (MEK1) kinase inhibitors. These compounds had cyanoquinoline cores, but differed in their respective aniline groups [1a, 1b: 4-phenoxyphenylaniline; 2a, 2b: 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline]. These compounds were competitive inhibitors of ATP binding by MEK1 kinase, and they had minimal or no effect on Raf, epidermal growth factor receptor (EGFR), Akt, cyclin-dependent kinase 4 (CDK4), or MK2 kinases at concentrations >100-fold higher than those that inhibited MEK1 kinase. Both class 1 and 2 compounds inhibited in vitro growth of human tumor cell lines. A class 2 compound (2b) was the most potent inhibitor of human tumor cell growth in vitro, and this effect was linked to distinct suppression of MAPK phosphorylation in cells. Compound 2b did not affect phosphorylation status of other kinases, such as EGFR, Akt, and stress-activated protein (SAP)/c-jun-NH kinase (Jnk); nor did it affect overall tyrosine phosphorylation level in cells. However, compound 2b did inhibit MEK1 phosphorylation in cells. Inhibition of MEK1 phosphorylation by 2b was not due to a major effect on Raf kinase activity, because enzyme assays showed minimal Raf kinase inhibition. We believe compound 2b inhibits kinase activity upstream of Raf, and thereby affects MEK1 phosphorylation in cells. Even with the dual effect of 2b on MEK and MAPK phosphorylation, this compound was well tolerated and significantly inhibited growth of the human colon tumor cell line LoVo (at 50 and 100 mg/kg BID, i.p.) in a nude mouse xenograft model.
Introduction
Raf/mitogen-activated protein/extracellular signal-regulated kinase (MEK)/mitogen-activated protein kinase (MAPK) proteins play crucial roles in cellular signaling processes. The Ras-MAPK signaling cascade (MAPK module) is found in all eukaryotic organisms and is involved in transmitting signals from the extracellular compartment into the cytosol and nucleus (1, 2). This cascade is activated by GTP-loaded Ras that recruits Raf proteins (A, B, and C) to the inner cell membrane where Raf is activated by phosphorylation. Activated Raf phosphorylates and activates the dual specificity kinases MEK1 and MEK2 (MAP kinase kinase; refs. 3, 4). Activated MEK phosphorylates and activates the MAPKs extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), which can translocate to the nucleus, and through the phosphorylation of a variety of substrates, modulate cytoplasmic events, such as cell proliferation and differentiation (4, 5).
Because oncogenic forms of Ras are associated with 30% of all cancers, Ras and the downstream kinase effectors of Ras represent attractive targets for pharmacologic intervention (6). Both in vitro and in vivo studies have shown that Raf and/or MEK are important pharmacologic targets (7-9). Raf/MEK/MAPK cascade assays, as well as individual Raf, MEK, or MAPK activity assays, have been described (10-12). We developed a sensitive, high-throughput Raf/MEK1/MAPK cascade ELISA (13), and extensively screened small molecule libraries to identify inhibitors of this signaling cascade. Using the Raf/MEK1/MAPK cascade ELISA, several potent and specific MEK1 inhibitors were found. For example, both 10E-hymenialdisine and 10Z-hymenialdisine were potent MEK1 inhibitors (IC50s of 3 and 6 nmol/L, respectively; ref. 14). In cellular assays, 10E-hymenialdisine and 10Z-hymenialdisine inhibited growth of the human colon tumor line LoVo (14). Further screening also identified 4-anilino-3-quinolinecarbonitriles (CNQ) as potent and specific inhibitors of MEK1 kinase. Two series of CNQ compounds inhibited MEK1 kinase enzymatic activity with IC50s <10 nmol/L. These were the 4-phenoxyphenylaniline (class 1) CNQs (15, 16) and the 3-chloro-4-(1-methylimidazol-2-ylsulfanyl)aniline (class 2) CNQs. In this study, we examined the specificity of these compounds for MEK1 kinase inhibition, and characterized the nature of CNQ compound inhibition of MEK1 kinase (i.e., allosteric versus competitive). We examined the potency of these compounds concerning their ability to inhibit the growth of a diverse panel of human tumor cell lines in vitro. Additionally, in cellular assays, we evaluated the CNQ compound effect on phosphorylation status of RAS-MAPK module proteins, as well as other non-Ras-MAPK module signaling proteins. In vivo antitumor activity, and a potent and apparently Ras-MAPK module specific activity profile, for a class 2 CNQ compound suggests that this type of MEK1 inhibitor could become a candidate for clinical evaluation in treatment of tumors with an activated Ras-MAPK pathway.
Materials and Methods
Materials
The 4-phenoxyphenylaniline series (class 1 compounds) were prepared by previously described methods (15, 16). A similar method was used to prepare the 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline series (class 2 compounds; ref. 17).
Raf/MEK1/MAPK ELISA
Production of proteins and ELISA protocol was as described previously (13). Activated Raf was used to phosphorylate and activate glutathione S-transferase (GST)-MEK1 that in turn phosphorylated GST-MAPK. The phosphorylation of GST-MAPK by GST-MEK1 was detectable in ELISA format by a phospho-specific MAPK antibody (Sigma-Aldrich Corp., St. Louis, MO). The Sigma phospho-specific MAPK antibody recognized phosphorylation of the TEY sequence at amino acids 202 to 204 on MAPK. The ELISA protocol has been described (13). Various kinase inhibitors or random small molecules were prepared in 100% DMSO and were diluted from stock solutions into assay reactions so that DMSO concentration never exceeded 1% of total reaction volume. Reactions were incubated (0 to 300 min) at 25°C, and reactions were stopped by addition of EDTA (final concentration = 70 mmol/L). Data were collected from a Wallac Victor model Plate Reader and analyzed in Excel for single point percentage inhibition and IC50 determinations. The ELISA was DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay) in format.
Assays for Raf and MEK1 Enzyme Selectivity
Raf. Activated Raf phosphorylation of MEK1 (30 nmol/L) was carried out in assay buffer with 100 μmol/L ATP. Kinase inhibitors, when present, were at 1 to 10,000 nmol/L, with final DMSO (compound solvent) concentration of 1% in all reactions. Raf kinase reactions were run for 0 to 300 minutes at 25°C, and reactions were stopped by addition of EDTA (final concentration = 70 mmol/L). MEK1 phosphorylation was determined by Western analysis using a-phospho-MEK1 antibody (Cell Signaling Technology, Beverly, MA). MEK1 phosphorylation was quantified by densitometry of X-ray film from MEK1 Western blots (13).
MEK1. MEK1 phosphorylation of MAPK was directly measured by use of activated MEK1 (Upstate Biotech, Lake Placid, NY) in an ELISA protocol that omitted addition of activated Raf. All other aspects of the MEK1/MAPK ELISA were identical to the Raf/MEK1/MAPK ELISA. When kinase inhibitor compounds were tested in the MEK1/MAPK ELISA, they were at 1 to 1,000 nmol/L, with final DMSO (compound solvent) concentration of 1% in all reactions.
Cellular Assays
Human tumor cell lines from American Type Culture Collection used were: LoVo (colon), CaCo-2 (colon), HCT116 (colon), LNCap (prostate), T24 (bladder), HT119 (bladder), MiaPaca (pancreas), and BXPC3 (pancreas). These cell lines were exposed to varying concentrations of CNQ compounds. Cells were grown in RPMI 1640 or DMEM with 10% fetal bovine serum supplemented with l-glutamine and penicillin/streptomycin. Cyanoquinoline compounds were prepared as a 10 mg/ml stock solution in 100% DMSO, and then diluted with complete growth media to desired concentrations and applied to cells; final DMSO concentration never exceeded 0.25%. Growth inhibition was measured by staining cells with sulforhodamine B (14).
Cell exposure to compounds for Western blot analysis of phosphoproteins was for either short (4 hours) or long (96 hours) duration. Exposure of cells to compounds was in complete media with serum. Antibodies used were from Sigma or Cell Signaling Technology.
In vivo Tumor Xenografts
Athymic nu/nu female mice were implanted s.c. with LoVo (colon) human tumor cells. When tumors reached an average mass between 80 and 200 mg (day 0), animals were randomized into treatment groups each containing either 5 or 10 animals. Mice were treated i.p. with the following: compound 1b or 2b prepared in a vehicle containing 2% Tween 80/D5W or vehicle alone (2% Tween 80/D5W). Compound was given to mice at 50 and 100 mg/kg doses twice daily (BID). Tumor mass [(length × width2)/2] was determined approximately once a week for up to 28 days. Animal body weight and behavior was monitored for the duration of in vivo experiments.
Results
Inhibition of the Raf/MEK1/MAPK Cascade ELISA by 1a, 1b, 2a, and 2b
The Raf/MEK1/MAPK cascade ELISA found CNQ class 1 (1a, 1b) and class 2 (2a, 2b) compounds (Fig. 1) to be potent inhibitors of assay signal. The ELISA end point was the measurement of MAPK phosphorylation on Threonine (T) 202 and Tyrosine (Y) 204 by a phospho-specific monoclonal antibody. The sensitivity of the Raf/MEK1/MAPK ELISA was confirmed by testing specific and generic kinase inhibitors, such as staurosporine, tyrphostin, olomoucine, genistein, lavendustin A, and apigenin (Table 1). The Raf/MEK1/MAPK ELISA sensitivity to staurosporine (IC50 = 3 nmol/L), and lack of response to tyrphostin, olomoucine, genistein, lavendustin A, and apigenin confirmed assay performance. Table 1 lists ELISA data for 1a, 1b, 2a, and 2b. All of these compounds had IC50 values below 10 nmol/L. In comparison, the commercially available MEK1 inhibitors PD98059 and U0126 had IC50s of 2800 and 800 nmol/L in the Raf/Mek1/MAPK ELISA.
High-throughput screening efforts identified potent Ras-MAPK pathway inhibitors. MEK1 specific CNQ leads found by high-throughput screening include class 1 compounds 1a and 1b (4-phenoxyphenylaniline compounds) (A) and class 2 compounds 2a and 2b [3-chloro-4-(1-methylimidazol-2-ylsulfanyl)aniline compounds] (B).
Raf/MEK1/MAPK ELISA signal inhibition by various kinase inhibitors and CNQ compounds
Specificity of Class 1 and Class 2 Compounds
The specific target of compound inhibition in the Raf/MEK1/MAPK ELISA was identified by kinase assays that directly measured Raf phosphorylation of MEK1, or MEK1 phosphorylation of MAPK, using either MEK1 or MAPK phospho-specific antibodies. Additionally, this group of cyanoquinoline compounds was tested in epidermal growth factor receptor (EGFR), MK2, Akt, and cyclin-dependent kinase 4 (CDK4) kinase assays. Table 2 lists data from this battery of kinase assays. It was found that 1a, 1b, 2a, and 2b all inhibited MEK1 at IC50 values of <10 nmol/L, in good agreement with cascade ELISA data (Table 1). Detectable, but less potent, inhibition of both Raf kinase and EGFR kinase (IC50s = 700 to 850 nmol/L) was observed for these compounds. Only one compound, 1a, had MEK1 and EGFR IC50 values that were within 100-fold of each other (Table 2).
Specificity of class 1 (1a, 1b) and class 2 (2a, 2b) CNQs in kinase assays
Kinetic Studies: Site of Action of Class 1 (1b) and Class 2 (2b) Compounds
To determine what type of MEK1 inhibitors class 1 and class 2 compounds were, we carried out double reciprocal analysis varying protein substrate, ATP, and inhibitor concentrations in kinase enzyme experiments. Data were analyzed by using the Enzyme Kinetics module of Sigmaplot 8.0. Figure 2 shows a representative graph indicating that compound 2b behaved as a competitive inhibitor of ATP binding by MEK1 when ATP concentration was varied and MAPK held constant. A Ki value of 3.9 nmol/L was derived by plotting the slopes of the double reciprocal analysis against the concentration of 2b (not shown). When MEK1 activity was assayed at various MAPK concentrations (ATP constant) and increasing amounts of 2b, this class 2 compound was not competitive for MAPK binding by MEK1 (not shown). Similar analysis of 1a, 1b, and 2a showed that they also were competitive inhibitors of ATP binding by MEK1, and they were not competitive for MAPK substrate binding (data not shown).
Compound 2b (class 2) was a competitive inhibitor of ATP binding by MEK1. Data were analyzed by using the Enzyme Kinetics module of Sigmaplot 8.0. Double reciprocal plots from experiments in which MEK1 and MAPK concentrations were held constant, whereas ATP concentration was varied, yielded lines that intersected the Y axis in unison, indicating competitive inhibition.
Cell Growth Inhibition by Class 1 and Class 2 Compounds
Cell growth inhibition by these MEK1 inhibitors was tested in a group of human tumor cell lines that were both wild-type (wt) mutant at the ras locus. Previously, we found that the human colon tumor line LoVo was sensitive to low nanomolar concentrations of FTI-276, a protein farnesyl transferase inhibitor that impairs Ras function (14, 18). In contrast CaCo-2 (wt at the ras locus) were >20-fold less sensitive to FTI-276 (14). We, therefore, assessed whether the MEK1 inhibitors described here had the same effect on LoVo and CaCo-2, and we also evaluated compound effect on the several additional tumor cell lines (wt and mutant at the ras locus) listed in Table 3 .
Humlan tumor cell line growth inhibition by class 1 (1a, 1b) and class 2 (2a, 2b) CNQs
None of the MEK1 inhibitors described here were potent inhibitors of CaCo-2 growth (IC50s > 1,000 nmol/L). In contrast, the class 1 and class 2 compounds did inhibit growth of the other tumor cell lines listed in Table 3. Growth inhibition varied between compound classes, with compound 2b showing the most potent and broadest range of tumor cell growth inhibition. Tumor cell lines affected by 2b were from a diverse group of tissues that varied in ras status. LoVo (colon) was the most sensitive cell line (IC50 = 5 nmol/L), and HCT116, another human colon tumor line, was the least sensitive (IC50 = 450 nmol/L) to the growth inhibitory effects of 2b. Compound 2b also potently inhibited growth of LNCap (prostate) and BXPC3 (pancreas) cell lines (IC50s ≤ 50 nmol/L).
Additional class 1 and class 2 compound analogues3 tested in cellular assays confirmed that class 2 compounds were indeed more potent inhibitors of tumor cell line cell growth than class 1 compounds. The class 2 compound exception was 2a, which did show good activity versus LoVo (IC50 = 15 nmol/L), but due to poor solubility in aqueous solutions (i.e., cell growth media) had limited in vitro activity versus most tumor cell lines (Table 3).
Class 1 and Class 2 Compound Specificity in Cells
Compounds 1b and 2b were examined for their ability to suppress MAPK phosphorylation in LoVo cells. Cell exposure to these compounds was for either short (4 hours) or long (96 hours) duration. With either protocol, both of these compounds effectively suppressed MAPK phosphorylation (data shown in Figs. 3 and 4 are from 96 hours exposure to compounds). Figure 3 shows that the class 1 CNQ, 1b, suppressed MAPK phosphorylation (p-MAPK) in LoVo cells, without diminishing total MAPK protein. The IC50 for this effect on p-MAPK was 300 nmol/L (Table 4), in good agreement with data showing 1b inhibitory effect on LoVo cell growth listed in Table 3. Compound 1b had no effect on MEK1 phosphorylation (p-MEK1) levels, nor did it affect overall MEK1 protein levels (Fig. 3). These data indicate no gross toxicity by 1b.
Compound 1b (class 1) inhibited MAPK phosphorylation in LoVo (human colon tumor) cells. A, LoVo cells were exposed to 1b for 96 hours at concentrations of (1) 0 nmol/L, (2) 10 nmol/L, (3) 30 nmol/L, (4) 100 nmol/L, (5) 300 nmol/L, and (6) 1,000 nmol/L. The IC50 for compound 1b inhibition of phosphorylated MAPK (p - MAPK) was 300 nmol/L (by densitometry). B, compound 1b had no effect on MEK1 or phosphorylated-MEK1 (p - MEK1) levels at any compound concentration tested [(1) 0 nmol/L, (2) 10 nmol/L, (3) 30 nmol/L, (4) 100 nmol/L, (5) 300 nmol/L, and (6) 1000 nmol/L].
Compound 2b (class 2) inhibited MAPK (A) and MEK1 (B) phosphorylation in LoVo (human colon tumor) cells. A, LoVo cells were exposed to 2b for 96 hours at concentrations of (1) 0 nmol/L, (2) 0.01 nmol/L, (3) 0.1 nmol/L, (4) 1.0 nmol/L, and (5) 10.0 nmol/L. Compound 2b had an IC50 value of 0.01 nmol/L (densitometry) for inhibition of phosphorylated-MAPK (p - MAPK). B, compound 2b also reduced phosphorylated-MEK1 (p - MEK1) when LoVo cells were exposed to (1) 0 nmol/L, (2) 0.01 nmol/L, (3) 0.1 nmol/L, (4) 1.0 nmol/L, and (5) 10.0 nmol/L compound concentrations. The IC50 = 8 nmol/L (by densitometry) corresponds well with concentrations that caused LoVo cell growth inhibition.
Summary IC50 (nmol/L) phospho-blot data from LoVo (Human Colon Tumor) cells exposed to CNQs
In contrast, in LoVo cells exposed to the compound 2b, not only was p-MAPK inhibited at a considerably lower concentration (IC50 = 0.01 nmol/L; Table 4), but 2b also unexpectedly suppressed p-MEK1 levels (Fig. 4). Compound 2b effect on p-MEK1 (IC50 = 8 nmol/L, Table 4) correlated with the IC50 observed for LoVo cell growth inhibition (Table 3). There was no effect on MAPK or MEK1 total protein levels by 2b (Fig. 4), indicating that compound effect on p-MAPK and p-MEK1 levels was not due to compound toxicity.
Because 2b suppressed p-MEK1 and p-MAPK in LoVo cells, yet had only minimal activity versus Raf kinase in enzyme assays, we tested specificity of both class 2 and class 1 compounds by examining the phosphorylation status of signaling proteins outside the Ras-MAPK pathway in LoVo cells. Table 4 summarizes phospho-blot data for LoVo cells exposed (96 hours) to these compounds. Compound 2b had no effect on phosphorylation status of stress-activated protein (SAP), Akt, or EGFR kinases; nor did they affect overall cell phospho-tyrosine levels at concentrations from 1 to 1,000 nmol/L. The class 1 CNQs also had no effect on phosphorylation status of stress-activated protein (SAP), Akt, or EGFR kinases; nor did they affect overall cell phospho-tyrosine levels at concentrations from 1 to 1,000 nmol/L. Poor solubility of 2a in aqueous solutions (cell culture media) prevented accurate evaluation of this compound's in vitro cellular effects. Finally, compound 2b effect on both MEK1 and MAPK phosphorylation was not confined to LoVo cells because this same effect was observed when BXPC3 (pancreas) and SW620 (colon) tumor cells were exposed to 2b (data not shown).
In vivo
Figure 5 shows that compound 2b inhibited LoVo tumor growth when given to animals by i.p. route at both 50 and 100 mg/kg BID. Compound administration was stopped at day 21, at which time there was 78% tumor growth inhibition at the 100 mg/kg dose level, and 54% tumor growth inhibition at the 50 mg/kg dose level. Tumors were measured 1 week after compound dosing stopped and tumor growth inhibition of 81% and 56% was still observed for animals treated at 100 and 50 mg/kg dose levels, respectively. All animals treated with compound 2b showed no gross toxicities, no weight loss, nor any abnormal behavior. All tumor inhibition results were found to be statistically significant compared with control group (vehicle only) as determined by Student's two-tailed t test analysis of data.
In vivo efficacy of compound 2b. Female nu/nu mice were injected with 1.5 × 106 LoVo colon tumor cells. Mice bearing LoVo tumors of ∼100 mg were treated BID by i.p. route with compound 2b in 2% Tween 80/D5W at 50 mg/kg (▴) and 100 mg/kg (•) for 21 days. Control group (♦) received vehicle (2% Tween 80/D5W) only. Statistical significance was determined by Student's two-tailed t test for all doses compared with vehicle control. *, P ≤ 0.02.
Compound 1b was also tested in vivo against LoVo tumors at both 50 and 100 mg/kg BID by i.p. route. This compound did not show any in vivo efficacy (data not shown). Compound 2b had poor solubility at the 100 mg/kg dose level, because dosing solutions had turbid appearance. We believe that the antitumor activity of compound 2b was directly related to its more potent profile in in vitro cellular assays.
Discussion
In this report, we have shown that two types of CNQs, the 4-phenoxyphenylaniline (class 1) and 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline (class 2) compounds were potent inhibitors of the MEK1 kinase in vitro (Tables 1 and 2). The class 1 and class 2 compounds inhibited MEK1 kinase at IC50 concentrations below 10 nmol/L (Table 1). The potency of these compounds compares favorably with the Pfizer MEK1 inhibitor, CI-1040, which has a reported IC50 of 17 nmol/L (8). CI-1040 was advanced to clinical trials (19). Unlike the MEK1 inhibitors CI-1040, PD98059, or U0126 that all apparently act by an allosteric mechanism (20), the CNQs were competitive inhibitors of ATP binding by MEK1 (Fig. 2). In direct enzyme assays, class 1 and 2 compounds inhibited MEK1 phosphorylation of MAPK, but had a comparatively modest effect on Raf kinase phosphorylation of MEK1 (Table 2).
The CNQs had different potency profiles when tested for growth inhibition of human tumor cell lines. The class 2 compound, 2b, had the best potency against the broadest range of human tumor cell lines (IC50s from 5 to 450 nmol/L; Table 3). The most sensitive lines were LoVo (colon), LNCap (prostate), BXPC3 (pancreas), and HT1197 (bladder) tumor cells (IC50s <60 nmol/L). In comparison, the farnesyl transferase inhibitor FTI-276 (18) only showed a potency advantage over 2b against the human colon tumor cell line HCT116 (Table 3). The MEK1 inhibitors U0126 and PD98059 both had IC50 values >1,000 nmol/L when tested against the cell lines listed in Table 3 (data not shown).
Compound 2b caused cell growth inhibition in cells with both mutant and wt ras, indicating that MEK1 inhibition is an important target in tumors with Ras-MAPK pathway activation whether or not such activation originates from mutant ras. It is likely that tumors that have Ras-MAPK pathway activation resulting from mutant or inappropriately activated forms of receptor tyrosine kinases (21), or mutant forms of Raf [e.g., B-Raf (22)] will be susceptible to MEK1 and/or Raf inhibitors. Pre-clinical data on the in vitro and in vivo profiles of the Bayer Raf inhibitor (BAY43-9006), and the Pfizer MEK1 inhibitor (CI-1040) on human tumor cells (both ras mutant and wt) support this hypothesis (8, 9, 23).
An unexpected result from this study was the effect of compound 2b on MEK1 phosphorylation in LoVo cells. Compound 2b inhibited MEK1 phosphorylation in LoVo cells at an IC50 of 8 nmol/L (Table 4), and this corresponded with the IC50 for cell growth inhibition by this compound on LoVo (IC50 = 5 nmol/L, Table 3). This occurred despite enzyme assay data showing minimal compound effect on Raf kinase. Additionally, inhibition of MAPK phosphorylation occurred at a >100-fold lower level than that observed for 2b effect on MEK1 phosphorylation (Table 4). Compound 2b effects on a target (or targets) other than Raf are likely the basis of these effects.
The effects of 2b on target(s) upstream of Raf must cause a reduced pool of activated MEK1 available to phosphorylate MAPK. This effect, in conjunction with the direct effect of 2b on MEK1 kinase activity, may account for the potent inhibition (IC50 = 0.01 nmol/L) of MAPK phosphorylation by 2b in LoVo cells. This observation corresponds with the predicted sensitivity of the Raf/MEK1/MAPK signaling cascade to inhibitors (i.e., Raf > MEK1 > MAPK; refs. 24, 25). This sensitivity profile results from the distributive (non-processive) mechanism of both Raf and MEK1 in which the rate of MEK1 activation depends on the concentration of Raf squared; and similarly the rate of MAPK activation is dependent on the concentration of MEK1 squared (13, 24, 25).
We have not yet determined the mechanism by which 2b suppresses MEK1 phosphorylation and also causes very potent suppression of p-MAPK in cells. Certainly a component of the effect on p-MAPK in cells must be the direct inhibition of MEK1 by 2b (Tables 1 and 2). But we expect 2b, like many kinase inhibitors, has additional activities beyond those for which it was originally targeted. Gleevec inhibits the activity of at least three kinases in vitro: Abl, c-Kit, and PDGFR (26). As an even more relevant example, a recent report showed that the MEK1 inhibitors U0126 and PD98059 inhibited KSR (kinase suppressor of Ras)associated phosphorylation of Raf in HL-60 human promyeloblastic leukemia cells (27). We are, therefore, testing 2b for inhibitory effects on KSR or non-receptor kinases implicated in Raf activation (e.g., Pak1 or C-Tak1; refs. 28-34). Additionally, we are extending analysis of 2b to examine any effects on receptor tyrosine kinases besides EGFR. These receptors are present on many tumor cell lines (35), and possible attenuation of their activity by 2b could help account for the potent inhibitory effects on MEK1 and MAPK phosphorylation.
Suppression of p-MEK1 and p-MAPK levels by 2b did not translate into general compound toxicity at the cellular or in vivo (below) levels. In vitro, two sets of experimental data support this conclusion. One, CaCo-2 cell growth was not affected by 2b at concentrations >1,000 nmol/L (Table 3). And secondly, phosphorylation status of non-Ras-MAPK module signaling proteins, including overall tyrosine phosphorylation levels, were not affected by 2b at concentrations >1,000 nmol/L (Table 4). Compound 2b inhibition of MEK1 phosphorylation was not unique to LoVo cells, because the same effect was observed in vitro in BXPC3 and SW620 (colon) tumor cells exposed to 2b (not shown).
Despite the unresolved mechanism of action in cells, significant in vivo antitumor activity of compound 2b was observed at doses of both 50 and 100 mg/kg BID (i.p.) in the LoVo xenograft model. In vivo activity of 2b correlated with both its potent inhibition of MAPK phosphorylation, and its growth inhibitory effects on the LoVo human colon tumor cell line in vitro (Tables 3 and 4). Compound 2b was well tolerated by mice at both dose levels, suggesting that the relatively selective nature of this compound for the Ras-MAPK pathway could yield a clinical candidate with an acceptable safety profile. Compound 1b had no in vivo activity, which correlated with its less potent in vitro profile (Tables 3 and 4). Compound 2b did have poor solubility at 100 mg/kg dose level concentrations. We are addressing this problem both by altering the formulation for in vivo delivery of this compound, and by structural changes to the 2b molecule.
Acknowledgments
We gratefully acknowledge the assistance of Dr. Ann Aulabaugh in the analysis of enzyme kinetic data. We also gratefully acknowledge the support and encouragement of Drs. Philip Frost and Tarek Mansour in carrying out the work presented here.
Footnotes
↵3 Mallon R, Wojciechowics D, Collins K, Berger D, Dutia M, and Powell D. In vitro and in vivo evaluation of 4-anilino-3-quinolinecarbonitrile inhibitors of MEK1 kinase, manuscript in preparation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Accepted April 12, 2004.
- Received September 8, 2003.
- Revision received April 2, 2004.
- American Association for Cancer Research
References
Volume 3, Issue 6
