XANTHENE COMPOUNDS FOR CANCER CHEMOTHERAPY
BACKGROUND OF THE INVENTION
The ras gene was discovered as an oncogene of the Harvey (rasH) and Kirsten (rasK) rat sarcoma viruses. In humans, characteristic mutations in the cellular ras gene (c-ras) have been associated with many different types of cancers. These mutant alleles, which render ras constitutively active, have been shown to transform cells, such as the murine cell line NIH 3T3, in culture.
The ras gene product binds to guanine triphosphate (GTP) and guanine diphosphate
(GDP) and hydrolyzes GTP to GDP. It is the GTP-bound state of ras that is active. An accessory molecule, GTPase-activating protein (GAP) also binds to Ras and accelerates the hydrolysis of GTP. The Ras proto-oncogene requires a functionally intact raf-1 proto-oncogene in order to transduce growth and differentiation signals initiated by receptor and non-receptor tyrosine kinases in higher eukaryotes. Activated Ras is necessary for the activation of the c-raf-
1 proto-oncogene, but the biochemical steps through which Ras activates the Raf-1 protein
(Ser/Thr) kinase are not well characterized.
U.S. Patent Nos. 5,582,995; 5,736,337; 5,763,571; 5,767,075 and 6,103,692, the contents of which are incorporated herein by reference in their entirety, disclose a method of screening to identify compounds which inhibit direct binding of Ras to Raf, Raf activation, and proliferation.
Given the likely role of Ras and Raf in the development of excessive cellular proliferation and cancer, it is desirable to determine which compounds can disrupt the interaction of Ras with Raf.
SUMMARY OF THE INVENTION
It has now been found that a number of 3,6-dihydroxyxanthenes and oxidized variations thereof (e.g., 6-hydroxyxanthen-3-ones) are effective in preventing interaction of Ras with Raf.
The invention includes a method of treating cancer in a mammal, comprising administering to said mammal a compound represented by Structural Formula (I):
or a pharmaceutically acceptable salt thereof or an oxidized variation thereof; wherein:
Ri and R are independently -ORio;
R3 and t are independently -H, -ORio, halogen, or a substituted or unsubsituted alkyl group;
R5-R8 are independently -H, -ORio, halogen, or a substituted or unsubstituted alkyl group;
R9 is a polar functional group;
Rio is -H, a substituted or unsubsituted alkyl group, or a substituted or unsubstituted acyl group; and
L is a bond or a substituted or unsubstituted hydrocarbyl group. In another aspect, the invention is a method of inhibiting cellular proliferation (e.g., excessive or unwanted cellular proliferation) in a mammal or a method of inhibiting the interaction of Raf protein or fragments, mutants or 80%- 100% homologs thereof with Ras
protein or fragments, mutants or 80%- 100% homologs thereof in a mammal, comprising administering to the mammal one of the compounds disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows initial hit structures from the Ras-Raf screen.
Fig. 2 shows the 3,6-dihydroxy-9-carboxyxanthene core, and its oxidized and reduced forms.
Fig. 3 shows a scanning library of structurally-related compounds. Fig. 4 shows potential substitution patterns of the 3,6-dihydroxy-9-carboxyxanthene core.
Fig. 5 shows expression and purification of the K-Ras protein. Fig. 6 shows the structure of Raf and truncated Raf fusion proteins in used the assays described herein.
Fig. 7 shows the purification of biotinylated Raf-50-220 proteins. Fig. 8 shows the configuration and optimization of the Ras-Raf protein-protein interaction assay.
Fig. 9 shows the results of the high throughput screen for inhibitors of Ras-Raf protein- protein interaction.
Fig. 10 shows the phosphorylatoin of ERK following serum withdrawal and refeeding in human colon tumor cell line HCT116.
DETAILED DESCRIPTION OF THE INVENTION
A "hydrocarbyl group" is an alkylene or arylene group, i.e., -(CH2)X- or -(CH2)χC6H (CH2)x-, where x is a positive integer (e.g., from 1 to about 30), preferably between 6 and about 30, more preferably between 6 and about 15. The carbon chain of the hydrocarbyl group may be optionally interrupted with one or more ether (-0-), thioether
(-S-), amine [-N(Ra)-] or ammonium [-N+(RaRD)-] linkages, or combinations thereof. Ra and
RD are independently -H, alkyl, substituted alkyl, phenyl, or substituted phenyl. Ra and R^ can be the same or different, but are preferably the same. Examples of hydrocarbyl groups include butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, dodecylene,
4-oxaoctylene, 4-azaoctylene, 4-thiaoctylene, 3,6-dioxaoctylene, 3,6-diazaoctylene, and 4,9- dioxadodecane.
An "aliphatic group" is non-aromatic, consists solely of carbon and hydrogen and may optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched, or cyclic and typically contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. Aliphatic groups include lower alkyl groups, lower alkylene and lower alkenylene groups, which include Cl-24 (preferably Cl-C 12) straight chained or branched saturated hydrocarbons.
An alkyl group is a saturated hydrocarbon in a molecule that is bonded to one other group in the molecule through a single covalent bond from one of its carbon atoms. Examples of lower alkyl groups include methyl, ethyl, w-propyl, wo-propyl, n-butyl, sec-butyl and tert-butyl. An alkoxy group is an alkyl group where an oxygen atom connects the alkyl group and one other group. An alkylene group is a saturated hydrocarbon in a molecule that is bonded to two other groups in the molecule through single covalent bonds from two of its carbon atoms. Examples of lower alkylene groups include methylene, ethylene, propylene, .-?o-propylene
(-CH(CH2)CH2-), butylene, sec-butylene (-CH(CH-)CH2CH_-), and tert-butylene
(-C(CH3)2CH_-). An alkenylene group is similar to an alkylene group, but contains one or more double bonds.
An acyl group is an alkyl, alkenyl or alkynyl group having a carbonyl group located at the terminus of the group that connects to the remainder of the molecule. Examples of acyl groups are an acetyl (CH2C(O)-) and a benzyl (C6H5C(0)-) group.
Aromatic or aryl groups include carbocyclic aromatic groups such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthacyl, and heterocyclic aromatic groups such as N-imidazolyl, 2-imidazole, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazole, 4-thiazole, 5-thiazole, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.
Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include 2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 3- indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazole, 2-benzooxazole, 2-benzimidazole, 2-
quinolinyl, 3-quinolinyl, 1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl. Phenyl is a preferred aromatic group.
"Arylene" is an aromatic ring(s) moiety in a molecule that is bonded to two other groups in the molecule through single covalent bonds from two of its ring atoms. Examples include phenylene -[-( 4)-], thienylene [-(C4H2S)-] and furanylene [-(C4H2O)-].
Examples of suitable substituents on an hydrocarbyl, aliphatic, acyl, aromatic or benzyl group may include, for example, halogen
(-Br, -CI, -I and -F), -OR, =O, =S, -CN, -N02, -NR2, -COOR, -CONR2, -SOkR (k is 0, 1 or 2) and -NH-C(=NH)-NH2. Each R is independently -H, an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group, and is preferably -H, a lower alkyl group, a benzylic group or a phenyl group. A substituted benzylic group or aromatic group can also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a benzyl, substituted benzyl, aromatic or substituted aromatic group as a substituent. A substituted aliphatic, substituted aromatic or substituted benzyl group can have more than one substituent. A preferred substituent on an aliphatic group is -OH.
Polar functional groups include halogen, alcohol, amine, nitrile, nitro, sulfonic acid, sulfinic acid, sulfenic acid, carboxylic acid, thiol, aldehyde, ester, amide, acid chloride, carbamate, carbonate, thiol acid, dithiocarbamate, dithiocarbonate, as well as pharmaceutically acceptable salts thereof.
Examples of pharmaceutically acceptable counteranions in a salt include sulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, proprionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, fumarate, maleate, benzoate, sulfonate, phenylacetate, citrate, lactate, glycolate, tartrate, carbonate, bicarbonate and the like.
Examples of pharmaceutically acceptable cations in a salt include metal cations, such as alkali (e.g., Li, Na, K), alkaline earth (e.g., Ca, Mg), transition metal (e.g., Cu, Fe, Zn) or heavy metal (e.g., Bi) cations. Other suitable cations include the acids of the anions listed in the previous paragraph.
Effective amounts of a compound disclosed herein to be administered will be determined on an individual basis, and will be determined at least in part, by consideration of the individual's size, the severity of symptoms to be treated and the result sought. As used herein, an effective amount refers to an appropriate amount of active ingredient to obtain therapeutic effect and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
The compound can be administered alone or in a pharmaceutical composition comprising the compound, a pharmaceutically acceptable carrier, and optionally, one or more additional drugs. The compound can be administered in a manner including but not limited to, for example, topically, ophthalmically, vaginally, orally, buccally, intranasally, rectally, transdermally, parenterally (e.g., intramuscular injection, intraperitoneal injection, subcutaneous injection) or pulmonarily. The form in which the compound is administered includes but is not limited to, for example, powder, tablet, capsule, solution, or emulsion, depends in part on the route by which it is administered. The compound may be aerosolized or nebulized. Suitable carriers and diluents will be immediately apparent to persons skilled in the art. These carrier and diluent materials, either inorganic or organic in nature, include, for example, gelatin, albumin, lactose, starch, magnesium stearate preservatives (stabilizers), melting agents, emulsifying agents, salts and buffers. For topical administration, examples of pharmaceutically acceptable carriers include, for example, commercially available inert gels, or liquids supplemented with albumin, methyl cellulose or a collagen matrix. Typical of such formulations are ointments, creams and gels. The effective amount can be administered in a series of doses separated by appropriate time intervals such as minutes or hours.
Screening of the initial 2900-member diversity library obtained from the National Cancer Institute has generated 36 hits. Five of the hits have an extremely high degree of structural similarity, shown in Fig. 1. From these hits a common core has been delineated which consists of 3,6-dihdroxy-9-carboxy xanthene or its oxidized counterpart as shown in Fig. 2.
The potential for flat, polycyclics structure to intercalate into DNA with toxic effects is well known. Similar structures to those disclosed herein have been accepted for clinical trials for the treatment of cancer (Jameson, M.B. et al. Proc. Am. Soc. Clin. Oncol., 2000, 19, 182a), so this will most likely not be problematic for the parent structure.
Four of the five hits initially discovered are of the oxidized form (Fig. 2). Oxidations of systems such as these are known to those skilled in the art, so that generation of both oxidation states should be possible. There is a great deal of symmetry associated with all five of the hits. A small subset of structural variants related to those shown in Fig. 1 is outlined in Fig. 3. An NMR study can be used to determine functional groups in the scaffold that make contacts with the Ras or c-Raf polypeptide.
The xanthene core presents a number of sites for the generation of structural diversity.
One starting point is the substituent on position 9 of the xanthene core. All of the initial hits possess a carboxylic acid on this side chain while utilizing a variety of linking motifs, however, substitution of other hydrophilic groups such as alcohols, secondary amines, amides, and carbamates can be made.
The distance from the xanthene core to the pendant carboxylic acid or other polar functional group and the nature of the linking group can be varied. Suitable linkers include 1,2, and 3 carbon aliphatic linkers; aromatic linkers containing carboxyl substitution at position 2', 3', and 4'; cycloalkyl linkers containing carboxyl substitution at position 2', 3', and 4'; and a 2' benzyl linker.
On the xanthene core, positions 1, 2, 4, 5, 7, and 8 can be substituted with electron withdrawing (CI), electron donating (OMe), and neutral (Me) substituents at these positions. Specific substitution patterns include substitution of CI, Me, and OMe at position 1, 2, and 4 as well as 2,7-symmetric-disubstitution and 4,5-dihydroxyl substitutions.
Variations of the common structure are outlined in Fig. 4. If a particular compound is difficult to synthesize, in the cases where available, substitution of the desired group with another of similar stereoelectronic characteristics may be necessary.
Stereochemistry is not typically a concern, however, for the series where R of Fig. 4 is aryl or aliphatic and the xanthene core is not symmetrically substituted, enantiomers are generated at C9. Furthermore, when R is cycloalkyl, diastereomers are generated between C9 and the cyloalkyl carbon bearing X.
EXEMPLIFICATION
AlphaScreen detection technology is based on a signal generated by the forced proximity of two types of 100 nM beads, so-called Acceptor and Donor beads. This proximity is mediated by interactions between molecules under study that are bound to each bead. When the two beads are brought within 100 nM of each other in a stable intermolecular complex, a unique light signature is produced. Employing this methodology, Applicants developed an assay for the interaction between Ras and Raf-1, and to monitor the inhibition of this interaction by small molecules. The AlphaScreen assay is further described below. In order to configure a high throughput assay sensitive to small molecules that interfere with the intermolecular interaction between Ras oncogene products and c-Raf, each protein must first be expressed and purified in stable fashion to greater than 95% purity as evidenced by polyacrylamide gel electrophoresis.
Example 1
Expression of K-Ras in insect cells and affinity purification.
The full length, GTPase-deficient valine-12 substituted K-Ras containing 6 His residues at the N-terminus (21 kd) was expressed in insect SF9 cells using a baculovirus vector and purified by affinity chromatography on a Ni-NTA-cellulose column. A typical yield in this expression system is approximately 20 μg of purified K-Ras from a 15 cm diameter plate containing 107 SF9 cells. Typically, ten 10 cm plates of cells are infected at a time with a yield of -200 μg of >95% pure K-Ras protein. A polyacrylamide gel detailing the purification of V12-K-Ras from baculovirus infected cells is shown in Fig. 5. Sf9 cells were infected (MOI of 5) with a recombinant baculovirus encoding
His6-V12-K-Ras. Cells were lysed 48 hr post-infection and Ras protein was purified by affinity chromatography on a Ni-NTA-agarose column and eluted with increasing concentrations of imidazole. Each eluted fraction was 2 ml with 20 μl loaded in each lane. The material in the 350 mM and 500 mM fractions was pooled for use in the Ras-Raf protein-protein interaction assay.
Amino acids 39-220 constitute the Ras binding domain of Raf-1. This domain can be subdivided into two regions. The amino terminal segment, amino acids 50-150, contains a high affinity site (Kd <10 nM) that binds Ras in a GTP-dependent manner. The second region, amino acids 150-220, encodes a hydrophobic zinc finger domain, which binds Ras in a GTP- independent manner at low affinity (Kd>20 μM). This low affinity binding requires the isoprenyl group at the carboxy terminus of Ras proteins (Fig. 6).
Applicants engineered both the high affinity Ras binding site (Raf AAs 50-150) and the complete Ras binding site (AAs50-220) containing both Ras interaction regions and expressed these truncated Raf proteins as a fusion constructs with a domain that permits stoichiometric biotinylation in vivo in E. coli (Fig. 6). The Raf fusion proteins, expressed in E. coli, are convenient to express and several hundred micrograms of protein of either fragment can be purified from a liter of bacteria.
Example 2
Purification of Biotinylated Raf-50-220 Proteins.
E. coli cells were transformed with a plasmid vector expressing the fusion protein in Fig. 6. Raf fusion protein was purified by affinity chromatography on monomeric avidin- sepharose and eluted with free biotin.
Example 3
Demonstration of a GTP-dependent binding in the AlphaScreen format of c-Raf 50-150 to K-Ras.
Assay conditions were established initially with a series of titrations of Ras and Raf protein concentrations, and anti-Ras antibody dilution (Fig. 8, Panels A, B, C). The effectiveness of binding was evaluated with three parameters: i) the absolute level of AlphaScreen signal that was produced had to be above 10,000 counts per second (cps) for statistical confidence; ii) the relative Alpha signal of Ras-GTP bound to Raf had to be at least two-fold greater than Ras-GDP binding to Raf; and iii) the signal had to be depleted substantially by competition with an excess of non-biotinylated Raf protein.
Using GTP-Ras binding to c-Raf (50-150) as the benchmark signal and GDP-Ras binding to c-Raf as the noise, the calculated Z' values, a statistical measure of the robustness of an assay which can be used across all assay platforms, are typically between 0.7 and 0.8. By way of reference, if the Z' value for an assay is above 0.5, the assay is considered to be highly robust and reliable. In addition, if a non-biotinylated c-Raf protein is used as a competitive inhibitor, the Alpha signal shows a dose dependent decrease (Fig. 8, Panel D). The calculated Z' value for this assay is 0.77, using the maximum inhibitory concentration of the c-Raf competitor as the basal level of expression.
All samples were assayed in triplicate. Ras-Raf binding reactions were in 5 μl for 60 min at room temperature followed by the addition of 5 μl of AlphaScreen beads and anti-Ras antibody mixture. Binding assays were run and read using Perkin Elmer 384 well Proxiplates. Letters A-D correspond to the panels of Fig. 8. A. Titration of K-Ras. All assays were done in the presence of 80 nM biotinylated c-Raf(50-150), 1:1000 antibody dilution. B. Titration of biotinylated c-Raf(50-l 50), 1 : 1000 antibody dilution. Assays were run in the presence of 80 nM V12 K-Ras. C. Titration of anti-K-Ras antibody. Assays were run with Ras at 160 nM and Raf at 80 nM. D. Inhibition of the Ras-Raf AlphaScreen Signal in the presence of competing nonbiotinylated peptide. Assays were performed under optimal conditions, 160 nM Ras, 80 nM b-Raf, 1 : 1000 antibody dilution. Raf proteins were mixed prior to the addition of Ras.
Example 4
High throughput screening of 2900 compounds for inhibitors in a Ras-Raf protein-protein interaction.
A . Val idation of the Ras-Raf protein-protein HTS
Applicants next evaluated the feasibility of the Ras-Raf binding assay for use in high throughput screening (HTS) for inhibitors of the Ras-Raf interaction. Based on the results of the optimization study outlined above and in Fig. 8, standardized conditions for the Ras-Raf protein-protein interaction assay were established:
• K-Ras - 80 nM
• c-Raf(50-150) - 80 nM • Anti-K-Ras antibody dilution (commercially available anti-K-Ras polyclonal antibody, Santa Cruz Biologicals) - 1:1000
• AlphaScreen beads were used at the manufacturer's (Perkin Elmer, Inc.) recommended concentration of 20 μg/ml.
Approximately 2900 compounds acquired from the Developmental Therapeutics
Program at the National Cancer Institute were screened at 20 μM for inhibition of the Ras-Raf interaction. The results of a typical 384 well plate that was part of a 10 plate screen is shown in Fig. 9. This plate includes 8 inhibitor controls using the GST-c-Raf competitor polypeptide at 1 μM, three wells on each side of the plate. The data in Fig. 9 is plotted as the reciprocal of the raw counts. This allows the easy visual identification of Hits as peaks that stand out in a 3- dimensional plot. If the data were to be plotted directly, the infrequent hit would be buried and nearly unnoticeable. A Hit is defined in this assay as any compound that can inhibit the Ras-Raf protein-protein interaction at a level at least 50% of that incurred by the control inhibitor peptide at 1 μM. Based on this criterion, there were three Hits identified on this plate, two with high efficacy and one with partial efficacy. At least two Hits were verified upon retest. For comparison of the robustness of the high throughput screen to the original assay run by hand in triplicate, the Z value (equivalent to Z', but used when calculating noise in an actual screen with compounds present) was calculated to be 0.75.
GTP-Ras was mixed with biotinylated c-Raf in the presence of 20 μM concentration of each of the NCI compounds in the screen. The order of addition, using a 384-well TomTec Quadra3 liquid handler, was:
1. Add 1 μl of a 100 μM stock of test compound.
2. Add 2 μl ofa 2.5X stock of K-Ras.
3. Add 2 μl of a 2.5X stock of biotinylated-c-Raf.
4. Incubate at room temperature for 60 minutes.
5. Add 5 μl of 2X AlphaScreen beads containing a 1 :500 dilution of the anti-Ras antibody.
6. Incubate 60 minutes.
7. Measure AlphaScreen signal on Perkin Elmer Fusion Alpha Instrument.
B. Results of screening of the 2900 sample set from the National Cancer Institute.
Applicants assayed 2926 compounds from the Special Plated Sets provided by the Developmental Therapeutics Program of the National Cancer Institute against both V12 K-Ras binding to biotin-c-Raf(50-150) as well as Ras binding to the complete Ras binding domain of Raf-1 : biotin-c-Raf(50-220).
Compounds were screened for inhibition of an AlphaScreen signal at 20 μM. Compounds testing positive were rescreened in triplicate, with >90% of positives from the initial screen testing positive upon rescreen. Two assays were then performed on confirmed positives: i) the IC50 values were determined for all confirmed positives; and ii) confirmed positives were screened for nonspecific inhibition of the Alpha signal using positive control Alpha beads in which the biotin residue is conjugated directly onto the Acceptor Beads to facilitate high affinity complex formation between the Streptavidin bound Donor beads and the biotinylated Acceptor beads. Any inhibition of this strong positive control signal is evidence for nonspecific quenching by the compounds. So as not to disqualify a compound simply because it showed some degree of Alpha signal quenching, the IC50 values for quenchers in this nonspecific screen were determined. If the IC50 value of a compound for the Ras-Raf assay was at least 10-fold lower that the IC50 for nonspecific quenching, the compound would remain on the active list.
Of 41 number of repeat actives, it was determined that 5 were nonspecific quenchers of the Alpha signal. The remaining 36 compounds, which showed specific inhibition in the Ras- Raf assay are listed in Table 1 below according to their potencies.
A number of active compounds were identified through this screen that specifically inhibited the Ras-Raf protein-protein interaction. These compounds fall into different categories based on their extent of inhibition, their IC50 values, their tendency to inhibit the AlphaScreen signal non-specifically, and their ability to inhibit the binding of Ras to both the c- Raf 50-150 and 50-220 segments. A summary of the results for the high throughput screen against the 50-150 Ras fragment are given below. It has been noted that many of the compounds identified as actives in this screen share a structural scaffold. As a result, an early SAR pattern was developed based on the varied potencies of the compounds as well as the varying selectivity for the two different Raf segments. These structures are inhibitors of Ras signaling. Even in this small set of 36 actives, there were differences among the compounds in how they inhibited the Raf segment containing the single high affinity site versus the Raf segment that contained the low affinity zinc finger domain.
Table I
Example 5
Preliminary configuration of a cell-based assay sensitive to MAP kinase pathway activation.
Compounds that have been identified as inhibitors of the Ras-Raf interaction in the cell- free high throughput screen are further characterized in one or more cell-based assays. Applicants run two types of cellular assays, one that monitors the IC50 for cell proliferation and the second indicates the level of activation of the MAP kinase pathway by measuring the level of phosphorylation of MAP kinase (ERK 1 &2) and/or MAP kinase kinase (MEK), the direct Raf substrate. Applicants are testing a number of cell lines for their ability undergo activation of the MAP kinase pathway under conditions of serum starvation (16 h) followed by 15 minutes of refeeding in the presence or absence of the MAP kinase kinase inhibitor compound UO126 (Fig. 10). The results of this experiment indicate that in a human colon cancer cell line expressing a mutant K-Ras protein, a 15 minute pretreatment with the MAP kinase kinase inhibitor U0126 before refeeding with serum completely blocks ERK phosphorylation.
K-Ras oncogene bearing human colon tumor cell line HCT116 was grown in 6-well plates in the absence of serum for 16 hours followed by 15 minutes of refeeding with 10% FBS with or without a 15 minute pretreatment with the MAP kinase kinase inhibitor U0126 at 20 μM. Cell extracts were prepared and parallel aliquots were loaded on two 8% polyacrylamide gels. Both gels were electroblotted to PVDF membranes. One blot (Panel A) was probed with an antibody specific to phosphorylated ERK proteins and the second blot (Panel B) was probed with pan-specific anti-ERK antibody (to assure that comparable amounts of protein for each sample were loaded) followed by alkaline phosphatase conjugated Protein A. Blots were then developed with chromagen XYZ for visualization of the bands. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.