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Experimental Therapeutics |
Departments of Cancer Biology [D. W. R., R. J. M., K. J. A., B. R. K., R. M. C., D. M. M., T. M. G.], Molecular Biochemistry [E. R. W., B. R. R., W. B. K.], Protein Sciences [P. S. B.], Molecular Sciences [S. H. K.], and Medicinal Chemistry [K. L.], GlaxoSmithKline, Research Triangle Park, North Carolina 27709; Departments of Respiratory Systems [K. A., R. H., R. J. S.], Discovery Chemistry [S. G. C., S. B. G., K. J. S., M. C. C.], Oncology [C. S., M. P., J. S.], Molecular Recognition [A. J.], and Cellular Immunology [P. T.], GlaxoSmithKline, Stevenage, Herts SG1 2NY, United Kingdom; Arrow Therapeutics, Ltd., Carlshalton, Surrey SM5 4DS, United Kingdom [S. G. C., J. S.]; Oxford Glycosciences Abindgdon Science Park, Abingdon, Oxon OX14 3YS, United Kingdom [C. S., M. P.]; Oxford BioMedica, Oxford Science Park, Oxford OX4 4GA, United Kingdom [R. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, correlates with recurrence or poor prognosis in bladder and pancreatic cancer patients (5
, 6)
. ErbB-2 also plays an important role in the development and progression of many types of cancer. Overexpression of ErbB-2 occurs in 
30% of breast cancers and correlates with metastasis (7, 8, 9, 10)
, early relapse, and shorter survival in these patients (11)
. ErbB-2 expression also correlates with poor prognosis in cancers of the colon, ovary, and bladder (12, 13, 14)
. Amplification of the erbB-2 oncogene or overexpression of ErbB-2 protein is documented in a variety of other neoplastic tissues including cancers of the esophagus, stomach, lung, uterus, and prostate (reviewed in Ref. 15
). Coexpression of elevated levels of EGFR and ErbB-2 in ovarian cancer patients appears to result in a poorer prognosis than overexpression of either enzyme alone, implying cooperation of the two proteins in the development of serious neoplasia (16)
. The biochemical pathways involved in type I receptor signaling have been studied extensively. ErbB family proteins have functional tyrosine kinase catalytic domains, with the exception of ErbB-3, which acts as a noncatalytic partner to other ErbB family members. Catalytic activity is initiated after ligand binds to the receptor. Although ErbB-2 has no known ligand, it participates in type I receptor signaling by heterodimerization with ligand-bound members of the ErbB receptor family. Dimerization causes a conformational change that activates the kinase domain, leading to autophosphorylation and initiation of divergent signal transduction cascades (17) . The type I receptors signal through the well-characterized Ras/Raf/MEK/ERK pathway, stimulating cell division (18) . Evidence also suggests that the type I receptors modulate cell survival through activation of the phosphoinositol 3-kinase pathway (19) . Both of these effects implicate the catalytic activity of EGFR and ErbB-2 in the development and maintenance of the neoplastic phenotype, suggesting that inhibition of this activity could provide a therapeutic opportunity in patients with tumors expressing either or both of these receptors.
Several therapies targeting type I receptors have been studied in the clinic. Antibodies or small molecule inhibitors targeted to ErbB-2 or EGFR are either approved for therapy or are showing encouraging safety and efficacy profiles in clinical trials. Herceptin, monoclonal antibody-based therapy targeted to ErbB-2, is presently approved in breast cancer patients and increases median survival time in metastatic breast cancer patients overexpressing ErbB-2 (20) . IMC-C225, a monoclonal antibody targeted to EGFR, is in Phase III clinical trials for head and neck and lung cancer (21 , 22) . Small molecule inhibitors of EGFR, OSI-774 (CP 358,774), and Iressa, have progressed to clinical trials in EGFR-expressing cancers. Folliculitis is the most common adverse event associated with anti-EGFR therapy and appears to be reversible (23, 24, 25, 26) . The emerging efficacy and relative safety of these therapies suggest that inhibitors of EGFR and ErbB-2 may play an important role in the future treatment of cancer.
The epidemiological evidence implicating EGFR and ErbB-2 in patients with cancers from a variety of tumor types suggest that a dual inhibitor, targeting both ErbB-2 and EGFR kinase activity, could benefit a large and diverse patient population. EGFR and ErbB-2 have homologous kinase catalytic domains that share many similar biochemical and kinetic properties6 (Brignola et al., 2001). Most small molecule inhibitors of these enzymes target the conserved ATP binding site. We therefore postulated that molecules that inhibit both enzymes would be feasible to design. In an effort to take advantage of this therapeutic intervention opportunity, we have synthesized a large number of potent small molecule inhibitors that target both the ErbB-2 and EGFR catalytic domains. Many of these compounds inhibit the growth of human tumor cell lines in monolayer and show efficacy in s.c. human tumor xenograft growth assays. In this report, we will describe the biochemical and biological activity of a representative set of seven quinazoline or pyridopyrimidine dual inhibitors showing efficacy in EGFR or ErbB-2 positive cell-based and tumor xenograft models. In addition, we will offer evidence to support inhibition of either ErbB-2 or EGFR catalytic activity as the mechanism of growth inhibition.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kinase Assays.
ErbB-2 and EGFR intracellular domains were purified from a baculovirus expression system, and enzyme selectivity screens were performed as described.6
Substrate concentrations were maintained below Km so that IC50s approximate the Ki for the inhibitor. Enzyme concentration was kept below 10 nM. Reactions were performed in 96-well polystyrene round-bottomed plates in a final volume of 45 µl. Reaction mixtures contained 50 mM 4-morpholine propanesulfonic acid (pH 7.5), 2 mM MnCl2, 10 µM ATP, 1.0 µCi [
-33P]ATP per reaction, 50 µM peptide substrate, and 1 mM DTT. The peptide substrate, Biotin-(amino hexonoic acid)-EEEEYFELVAKKK-CONH2, was synthesized by Quality Controlled Biochemicals, Inc. (Hopkinton, MA) and optimized for EGFR phosphorylation using a synthetic library approach (33)
. The concentrations of dissolved peptide stocks were determined by amino acid analysis. The reaction was initiated by adding the indicated purified type-1 receptor intracellular domain. Reactions were terminated after 10 min at 23°C by adding 45 µl of 0.5% phosphoric acid in water. Seventy-five µl of the terminated reaction mix was transferred to MAPH phosphocellulose filter plates (Millipore, Marlborough, MA). The plates were filtered and washed three times with 200 µl of 0.5% phosphoric acid. Fifty µl of scintillation mixture (Optiphase; Wallac, Turku, Finland) was added to each well, and the assay was quantified by counting in a Packard Topcount (Packard Instrument Co., Meriden, CT).
Cells and Cell Culture.
Normal HFFs were isolated by digesting human neonatal foreskins with a solution of 2.5% trypsin and 1 mM EDTA. The LICR-LON-HN5 head and neck carcinoma cell line (HN5) was a gift from Helmout Modjtahedi at the Institute of Cancer Research (Surrey, United Kingdom). The breast carcinoma cell line, BT474, and the gastric carcinoma cell line, NCI-N87 (N87), were obtained from the American Type Culture Collection (Rockville, MD). HB4a c5.2 cells were generated as described (34)
. HB4a r4.2 cells were generated by transfecting HB4a parental cells with plasmid pEJ containing a 6.6-Kb genomic Ha-ras sequence. HFF, BT474, HN5, and N87 were maintained by subculturing in 75-cm2 tissue culture flasks in Low Glucose DMEM containing 10% fetal bovine serum (Hyclone) until ready for use. HB4a r4.2 and HB4a c5.2 cells were maintained in RPMI 1640 supplemented with 10% FBS, 5 µg/ml hydrocortisone, 5 µg/ml insulin, and 50 µg/ml hygromycin B.
In Vitro Growth Inhibition Assays.
For assessment of cell-based potency, cells were plated in 96-well Falcon plates (Becton Dickinson) in the growth medium described above. Plating densities that resulted in logarithmic growth of vehicle-treated cells for the duration of the assay were used: HFF, 15,000 cells/cm2; BT474 30,000 cells/cm2; HN5, 10,000 cells/cm2; N87, 30,000 cells/cm2, HB4a c5.2, 10,000 cells/cm2; HB4a r4.2, 10,000 cells/cm2. After 24 h, cells were exposed to compounds at the concentrations indicated in Fig. 1
. HFF, BT474, HN5, and N87 cells were treated in DMEM containing 5% FBS, 50 mg/ml gentamicin, and 0.3% v/v DMSO. HB4a c5.2 and HB4a r4.2 cells were treated in 50% DMEM, 50% RPMI 1640 supplemented with 5% fetal bovine serum, 2.5 µg/ml hydrocortisone, 2.5 µg/ml insulin, 25 µg/ml hygromycin B, 50 µg/ml gentamicin, and 0.3% v/v DMSO. After 3 days, relative cell number was estimated using methylene blue staining. The medium was removed, and 100 µl of 0.5% w/v methylene blue dissolved in 50% ethanol and 50% water were added to each well. Plates were washed by immersion in deionized water and allowed to air dry. One hundred µl of 1% w/v n-lauroylsarcosine dissolved in PBS were added to each well, and plates were incubated for 30 min at room temperature. The optical density at 620 nm was read in a Molecular Devices UVMax microplate reader. Data were analyzed using curve-fitting macros written for Microsoft Excel. Concentrations that inhibit 50% of cell growth (IC50) were interpolated using the method of Levenberg and Marquardt (35)
and the equation, 
, where K is equal to the concentration that inhibits 50% of cell growth (IC50).
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In Vivo Receptor Phosphorylation Assays.
Mice were sacrificed by CO2 asphyxiation. Tumors were removed, flash-frozen in liquid nitrogen, pulverized while frozen, and protein was extracted in boiling sample buffer (70 mM Tris, pH 6.8; 3% SDS). Two hundred µg of total extract were loaded on duplicate 6% gels for SDS-PAGE. After transfer to nitrocellulose, resolved extracts were immunoblotted for receptor phosphorylation or receptor expression using the following antibodies: anti-pTyr polyclonal antibody (generated in-house), anti-ErbB-2 Ab-3 (Lab Vision), or anti-EGFR Ab-12 (Lab Vision). ECF (Amersham) was used for detection and quantitation of receptor phosphorylation using the STORM phosphorimager and ImageQuant software (Molecular Devices). ECL (Amersham) was used for detection of receptor expression.
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RESULTS |
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contains structures and IC50s for the inhibition of enzyme activity for seven representative dual inhibitors of ErbB-2 and EGFR kinase activity. These compounds represent a structurally diverse set of quinazolines and pyridopyrimidines, which inhibited kinase activity by 50% or more at concentrations <70 nM and are nearly equipotent on ErbB-2 and EGFR. The compounds were >50-fold selective for ErbB-2 and EGFR versus other proliferative kinases that we have investigated including cRaf-1, CDK1, CDK2, c-Src, ERK2, MEK, p38, Tie2, VEGFR2, and c-Fms (data not shown). The EGFR inhibitor, OSI-774, is included in Table 1
for comparison. OSI-774 was reported to be EGFR selective (36)
and was 40-fold more potent on EGFR than ErbB-2 (17 and 680 nM, respectively) in our in vitro enzyme assays.
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and Table 2
). Growth of each of the tumor cell lines is inhibited by 50% at concentrations >1.0 µM. The most potent compounds GW2974 and GW9263 have IC50s <0.4 µM. Selectivity for tumor versus normal tissue was determined by comparing efficacy on tumor cells to efficacy on HFF cells. The value for cellular selectivity was calculated by dividing the IC50 for the HFF cells by the average of all tumor cell IC50s. The compounds were 9- to >75-fold more potent on the transformed cell lines than on the HFF cells.
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and Table 3
). HB4a cells transfected with the genomic sequence of mutant Ha-ras, HB4a r4.2, were 7- to >40-fold less sensitive to ErbB-2 and EGFR inhibition than HB4a c5.2.
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. BT474 and HN5 cells were grown as models for ErbB-2- and EGFR-driven tumor growth, respectively. Established tumors (200 to 250 mg in size) were treated orally, b.i.d. for the duration of the assay. GW2974 caused significant inhibition of tumor growth in both models in a dose-dependent manner. The 30-mg/kg dose of GW2974 inhibited the average tumor growth in the BT474 xenograft by >95% after 21 days of treatment. Complete inhibition of tumor growth was observed in the HN5 model by treatment with 30 mg/kg of GW2974, orally b.i.d. Regression, defined by >50% reduction in initial tumor volume, was seen in 6 of 48 implants from multiple experiments on the HN5 tumors at 30 mg/kg. Treatment of s.c. human tumor implants with 10 mg/kg, orally b.i.d., GW0277, resulted in significant inhibition of tumor growth, where 95% inhibition was observed in the HN5 (P = 0.0001) model, and 50% was observed in the BT474 model (P = 0.011).
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. Maintenance of weight in CD-1 nude mice treated with GW0277 indicates that this compound is also well tolerated by these animals at therapeutically relevant doses. However, C.B-17 SCID mice treated with GW0277 were sensitive to the compound. Mean animal weight for animals treated with 10 mg/kg GW0277 for the entire experiment was 90% of untreated control animals. Four of 20 C.B-17 SCID mice treated with GW0277 were culled from experiments because of >25% weight loss. Necropsy was not performed on these animals.
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. Phosphorylation of both receptors was inhibited by 90% with treatment, whereas the expression level of both the ErbB-2 and EGFR protein remained unchanged.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The seven molecules described in this report are potent inhibitors of both the EGFR and ErbB-2 catalytic domains. The quinazoline and pyridopyridimine series have proven to be excellent scaffolds to build potent ErbB family tyrosine kinase inhibitors. A plethora of patents and publications have emerged over the last decade describing structure-activity relationships derived principally from the 4-anilino substitutions and 6,7-position functionalization (44
, 45)
. Although the work in the quinazoline and pyrolopyrimidine series focused principally on EGFR or vascular EGFR tyrosine kinases, we wished to expand the compound profiles to include dual inhibitory properties, specifically for EGFR and ErbB-2. The key to dual inhibition appears to lie with the 4-anilino substituents, for example, 3-chloroaniline (AG1478), 3-ethynylanilino (OSI-774), and 4-flouro-3-chloroanilino (Iressa) inhibit only EGFR tyrosine kinase activity. Several examples are shown in Table 1
. N1-benzylindazolyl, 4-benzyloxyphenyl, and phenylsulfonylphenyl substituents all confer dual inhibition. Substitutions on the terminal benzyl or phenyl ring seem to reduce the potency with the exception of small groups such as fluorine. There was no significant difference in enzyme potency between the quinazoline and pyridopyrimidine series. A wide range of substituents was introduced at the 6-position to improve compound properties, while having very little effect on enzyme activity. Compounds listed in Table 1
demonstrate the diversity of tolerated groups for maintaining dual inhibition: disubstituted amines, aminomethyl furans, oxadiazoles, and amino alkyl ethers.
We assessed the ability of these compounds to inhibit the proliferation of the EGFR-overexpressing HN5 cell line, the ErbB-2-overexpressing cell line, BT474, and a cell line that overexpresses both receptors, N87. In a cell-based growth inhibition assay, the compounds are effective at inhibiting the growth of all three phenotypes. The ability of these compounds to inhibit the growth of HN5 cells in culture is similar to that of the EGFR tyrosine kinase inhibitor, OSI-774, and as their efficacy against ErbB-2 would predict, these molecules are more potent on ErbB-2-expressing cell lines than OSI-774. By comparison, Herceptin has shown activity in cell-based assays only on cells overexpressing significant amounts of ErbB-2. Cell lines expressing only EGFR are not responsive to growth inhibition by Herceptin (46) . Similarly, in these studies, the two cell lines overexpressing ErbB-2, BT474, and N87 are 25-fold less responsive to the EGFR inhibitor, OSI-774, than the EGFR overexpressing cell line, HN5. These data suggest that a dual inhibitor of EGFR and ErbB-2 will provide therapeutic benefit to a larger patient population than a therapy that targets either receptor alone.
The selectivity of potential chemotherapeutic agents for transformed versus normal cells in culture is thought to be indicative of their tolerability in animal models. HFF cells, which are from normal tissue and express low levels of EGFR and ErbB-2, were included as a control cell line for nonspecific toxicity. The compounds range from 16- to >75-fold more potent on the tumor cells than the normal fibroblasts, implying that these compounds should be well tolerated by host animals during in vivo studies and, ultimately, in human patients. In addition, tumor cell lines expressing lower amounts of EGFR and ErbB-2, including HT29, MCF7, and T47D, are less responsive to the compounds (data not shown). Finally, these molecules are selective for EGFR and ErbB-2 compared with several other kinases known to be important in the growth of cells in culture including cRAF-1, CDK1, CDK2, c-Src, ERK2, MEK, p38, Tie2, VEGFR2, and c-Fms. These data suggest that these compounds are inhibiting cell growth because of their specificity for EGFR and ErbB-2.
To confirm this we tested the compounds in a transfected cell system in which proliferation is driven by either ErbB-2 or mutant Ha-Ras, one of the downstream effectors of ErbB-2. The type I receptors have been shown to transduce a mitogenic signal after autophosphorylation by activating Ras, which then signals through Raf, MEK1, and ERK2, resulting in translation of immediate early-response genes and ultimately, cell division (47) . A cell line that is overexpressing enough of an activated form of the Ras oncogene, therefore, should be resistant to the antiproliferative effects of a selective EGFR or ErbB-2 inhibitor. In fact, the erbB-2-transfected HB4a c5.2 cell line is up to 40-fold more responsive to the inhibitory effects of these compounds than the Ha-ras-transfected HB4a r4.2 cell line. The ramification of this for patients with tumors containing a mutant Ras oncogene is unknown. Ciardiello et al., (48) have reported that an Ha-ras-transfected clone of the MCF10A cell line is as responsive to treatment with Iressa as human tumor-derived cell lines overexpressing EGFR. It is important to note that the type I receptors signal not only through the Ras pathway but also through cell survival pathways downstream of PI3K. Perturbation of EGFR or ErbB-2 signaling cascades may inhibit these survival pathways and could result in selective cell death regardless of the ras status of a tumor. Therefore, although HB4a r4.2 is a suitable control for the assessment of compound specificity in cell-based assays, it may or may not be a reliable predictor of the effects of mutated ras oncogenes in the treatment of EGFR- or ErbB-2-positive tumors in the clinical setting. These results suggest that the effect of mutant ras on the efficacy of type I receptor inhibitors merits investigation.
We present data for representative compounds that were studied in tumor xenograft models. Twice daily oral treatment with the most potent of these enzyme inhibitors, GW2974, resulted in significant inhibition of average tumor growth at 10 mg/kg and complete inhibition of average tumor growth at 30 mg/kg. Furthermore, tumor regression was observed at the higher dose. The compound was well tolerated. Weight was maintained throughout treatment, and no mice treated with 30 mg/kg GW2974 were culled from the experiment. The only evidence of toxicity in the xenograft models treated with GW2974 was observed at high doses (
50 mg/kg; data not shown). Another compound, GW0277, yielded a similar antitumor effect to that of GW2974 at 10 mg/kg. Dose escalation was not performed with this compound because 3 of 20 C.B-17 SCID mice treated with GW0277 were culled from experiments because of >25% weight loss. It should be emphasized that even with the toxicity observed, a therapeutic window that resulted in inhibition of tumor growth could be achieved.
Although GW2974 was selective in enzyme, cell-based, and tumor xenograft assays, it was important to verify that the compound was specifically inhibiting EGFR or ErbB-2 activity in tumor samples. The primary early event in type I receptor signaling after ligand binding and dimerization is autophosphorylation among receptor family members (1 , 17) . For this reason, the inhibition of receptor autophosphorylation is an indicator of compound potency and selectivity. Western blot analysis was performed to detect inhibition of receptor autophosphorylation by GW2974 in tumor samples extracted from mouse xenografts. Short-term exposure of HN5 and BT474 xenografts to GW2974 resulted in dramatic inhibition of autophosphorylation of EGFR and ErbB-2, respectively. There was no effect on the amount of EGFR or ErbB-2 protein expressed in these samples. Together, these data suggest that the biological responses to GW2974 treatment were attributable specifically to inhibition of EGFR or ErbB-2 catalytic activity.
We have presented seven compounds that inhibit both the EGFR and ErbB-2 kinase domain. The compounds were potent on cells overexpressing either receptor. The most potent and selective compound, GW2974, was well tolerated by host animals in xenograft assays. The chemical diversity displayed in these compounds suggests that opportunities exist for modulating compound properties such as potency and selectivity. For these reasons, we believe that small-molecule, dual EGFR/ErbB-2 kinase inhibitors have potential use as cancer therapy for a broad range of tumors.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at GlaxoSmithKline, Building RC2, Room 809, 5 Moore Drive, Research Triangle Park, NC 27709. 
2 Present address: Arrow Therapeutics, Ltd., Carlshalten, Surrey SM5 4DS, United Kingdom.
3 Present address: Oxford Glycosciences, Abingdon Science Park, Abingdon, Oxon OX14 3YS, United Kingdom.
4 Present address: Oxford Biomedica, Oxford Science Park, Oxford OX4 4GA, United Kingdom.
5 The abbreviations used are: EGFR, epidermal growth factor receptor; HFF, human foreskin fibroblast; b.i.d., twice per day; SCID, severe combined immunodeficient.
6 P. S. Brignola, K. Lackey, S. Kadwell, C. Hoffman, E. Horne, H. L. Carter, W. B. Knight, and E. R. Wood. Comparison of biochemical and kinetic properties of the type-1 receptor tyrosine kinase intracellular domains: demonstration of differential sensitivity to kinase inhibitors, submitted for publication, 2001.
Received 4/ 6/01. Accepted 7/26/01.
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T. M. Grana, C. I. Sartor, and A. D. Cox Epidermal Growth Factor Receptor Autocrine Signaling in RIE-1 Cells Transformed by the Ras Oncogene Enhances Radiation Resistance Cancer Res., November 15, 2003; 63(22): 7807 - 7814. [Abstract] [Full Text] [PDF]
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R. Nahta and F. J. Esteva HER-2-Targeted Therapy: Lessons Learned and Future Directions Clin. Cancer Res., November 1, 2003; 9(14): 5078 - 5084. [Abstract] [Full Text] [PDF]
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E. G. Barbacci, L. R. Pustilnik, A. M. K. Rossi, E. Emerson, P. E. Miller, B. P. Boscoe, E. D. Cox, K. K. Iwata, J. P. Jani, K. Provoncha, et al. The Biological and Biochemical Effects of CP-654577, a Selective erbB2 Kinase Inhibitor, on Human Breast Cancer Cells Cancer Res., August 1, 2003; 63(15): 4450 - 4459. [Abstract] [Full Text] [PDF]
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R. Nahta, G. N. Hortobagyi, and F. J. Esteva Growth Factor Receptors in Breast Cancer: Potential for Therapeutic Intervention Oncologist, February 1, 2003; 8(1): 5 - 17. [Abstract] [Full Text] [PDF]
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V. G. Keshamouni, R. R. Mattingly, and K. B. Reddy Mechanism of 17-beta -Estradiol-induced Erk1/2 Activation in Breast Cancer Cells. A ROLE FOR HER2 AND PKC-delta J. Biol. Chem., June 14, 2002; 277(25): 22558 - 22565. [Abstract] [Full Text] [PDF]
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A. B. Motoyama, N. E. Hynes, and H. A. Lane The Efficacy of ErbB Receptor-targeted Anticancer Therapeutics Is Influenced by the Availability of Epidermal Growth Factor-related Peptides Cancer Res., June 1, 2002; 62(11): 3151 - 3158. [Abstract] [Full Text] [PDF]
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P. S. Brignola, K. Lackey, S. H. Kadwell, C. Hoffman, E. Horne, H. L. Carter, J. D. Stuart, K. Blackburn, M. B. Moyer, K. J. Alligood, et al. Comparison of the Biochemical and Kinetic Properties of the Type 1 Receptor Tyrosine Kinase Intracellular Domains. DEMONSTRATION OF DIFFERENTIAL SENSITIVITY TO KINASE INHIBITORS J. Biol. Chem., January 4, 2002; 277(2): 1576 - 1585. [Abstract] [Full Text] [PDF]
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