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EGFR and EGFR Inhibitors

It is well known that the epidermal growth factor receptor (EGFR) signaling pathway plays an important role in the the generation of various cancers. EGFR is a central regulator of cancer cell proliferation and progression in several human cancer types. The expression of EGFR is significantly up-regulated in these types of cancers. Therefore, EGFR was validated as a potent molecular target for treating cancers. The clinical efficacy of EGFR inhibitors (cetuximab, panitumumab, erlotinib, gefitinib and vandetanib) applied in the clinical practice for the treatment of metastatic cancers is restrained to a subgroup of patients with the majority of cancer patients indicating either intrinsic or acquired resistance to these drugs.

Part of biological fundament of cancers
The recent progresses in the knowledge of cancer biology and drug-resistance mechanisms have characterized, among the intracellular signaling pathways, that serve as down-stream to the EGFR, the AKT and RAS/RAF/ mitogen-activated protein kinase (MAPK) pathways as major responsible for the development of cancer cell resistance to EGFR inhibitors. However, treatment with several agents known to target directly or indirectly the AKT signalling pathway, such as LY294002, deguelin and everolimus, was not efficacious in preventing erlotinib- (ERL-) and gefitinib- (GEF-) resistant cancer cell proliferation in our in vitro non small cell lung cancer (NSCLC) model of acquired resistance to erlotinib and gefitinib.[1]
On the other side, mutations of the K-RAS gene has been reported both in NSCLC and colorectal cancer (CRC) patients as responsible for a poor prognosis and poor response to EGFR inhibitors. These mutations cause KRAS proteins to accumulate in the GTP-bound, active form resulting in constitutive, growth-factor-receptor independent activation of KRAS downstream signaling in tumor cells. The development of therapeutic approaches for patients with KRAS mutations is thus an important clinical goal. RAF serine-threonine kinases are the principal effectors of RAS in the MAPK signaling pathway and is therefore a potential target for cancer therapy. So far, the most successful clinical inhibitor of RAF activity is sorafenib (Nexavar, BAY 43-9006), an orally available multi-targeted kinase inhibitor, which inhibits the activation of C-RAF, B-RAF (both the wild-type and the activated V600E mutant), c-KIT, FLT-3, RET and vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, as well as platelet-derived growth factor receptor ? (PDGFR-?). And now sorafenib was currently approved for treating metastatic renal cell carcinoma (RCC) and advanced hepatocellular carcinoma (HCC), and under investigation in other malignancies. Sorafenib impacts the growth of tumor by directly preventing tumor cell proliferation and increasing apoptosis in a variety of tumor types as well as by preventing tumor-mediated neoangiogenesis.

EGFR inhibitors
As previously described, there is a synergistic interaction between sorafenib and erlotinib or between sorafenib and cetuximab, which is a monoclonal antibody targeting the extracellular domain of the EGF receptor, in a panel of NSCLC and colorectal cancer (CRC) cell lines, in vitro and in vivo, which is accompanied by a marked and sustained prevention of the MAPK- and AKT-dependent intracellular signals. Treatment with sorafenib could overcome the induced EGFR TKI-resistance by its ability to prevent several growth factor receptor-driven signals. Moreover, because sorafenib inhibits B-RAF, and it could be effective in cancer cell lines expressing activating K-RAS mutations. In addition, sorafenib decreased the activation of MEK and MAPK and caused an prevention of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines. Resistance to EGFR inhibitors is predominantly mediated by the RAS/RAF/MAPK signaling pathway and can be overcame by treatment with sorafenib.[2]

References:
[1] N Engl J Med 13: 1160–74
[2] Clin Cancer Res 9: 2316–26

Let's talk about the EGFR inhibitors.

BIBW2992 (Afatinib): BIBW2992 (Afatinib) is an irreversible EGFR/HER2 inhibitor with an IC50 of 0.5, 0.4, 10, 14 nM for EGFRwt, EGFRL858R, EGFRL858R/T790M and HER2 in vitro, respectively. [1] In cell-free in vitro kinase assays, BIBW2992 (Afatinib) shows potent activity against wild-type and mutant forms of EGFR and HER2, similar to gefitinib in potency for L858R EGFR, but about 100-fold more active against the gefitinib resistant L858R-T790M EGFR double mutant. BIBW2992 (Afatinib) exhibits potent cellular effects on both EGFR and HER2 phosphorylation in line with the in vitro kinase results, comparing favorably to reference compounds in all cell types tested, such as human epidermoid carcinoma cell line A431 expressing wt EGFR, murine NIH-3T3 cells transfected with wt HER2, as well as breast cancer cell line BT-474 and gastric cancer cell line NCI-N87, which express endogenous HER2. In vivo studies, daily oral treatment with BIBW2992 (Afatinib) at 20 mg/kg for 25 days resulted in dramatic tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2%, and downregulation of EGFR and AKT phosphorylation, as detected by immunohistochemical staining of tissue sections. Therefore, like lapatinib and neratinib, BIBW2992 (Afatinib) is a next generation tyrosine kinase inhibitor (TKI) that irreversibly inhibits human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) kinases. BIBW2992 (Afatinib) is not only active against EGFR mutations targeted by first generation TKIs like erlotinib or gefitinib, but also against those not sensitive to these standard therapies. [2] BIBW2992 (Afatinib) is originally developed by Boehringer Ingelheim Pharmaceuticals. And a phase II clinical trial of BIBW2992 (Afatinib) for the treatment of neoplasms has been terminated.

Biochemical assay:
The wild type tyrosine kinase domain of the human EGFR as well as the EGFR L858R/T790M double mutant were fused to Glutathione-S-transferase (GST), and extracted as described in Supplementary methods. Enzyme activity was then assayed in the presence or absence of serial inhibitor, including ,BIBW2992 (Afatinib, Tomtovok, Tovok) dilutions performed in 50% Me2SO. A random polymer pEY (4:1) was used as substrate. Biotinylated pEY (bio-pEY) was added as a tracer substrate. The kinase domain of HER2 was cloned using baculovirus system and extracted similarly to that of EGFR kinase domain. Detailed procedures for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity assays are included in Supplementary information. [2]

Cell assay:
Cells (1×104) were transferred into each well of a 96-well plate and cultured over night in serum-free media for EGFR phosphorylation assay. After addition of test compounds including BIBW2992 (Afatinib, Tomtovok, Tovok) on the next day, the plates were then incubated at 37°C for 1 hour. EGF-stimulation was done at 100 ng/ml for 10 min at room temperature. Cells were washed with ice cold PBS before extraction with 120 μl per well HEPEX buffer and shaken for 1 h at room temperature. In all 2×104 cells per well was used for HER2 phosphorylation assay. Streptavidin precoated plates were coated with anti-EGFR-biotin at 1:100 dilution with blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Extracts from above steps was then transferred to the antibody-coated wells, and incubated for 1 h at room temperature. Assessment of color development is described in Supplementary information. Extinction was measured at 450 nm. [2]

Animal study:
In xenograft models, mice with established tumors (40-130mm3) were randomized and treated daily p.o. with test compounds or vehicle control on the basis of individual weights. Tumors were measured 3 times a week with calipers, and tumor volumes were calculated by the formula π/6×length×(width)2. Except for trastuzumab, experimental compounds were dissolved in 1.8% HP-beta-CD (Hydroxypropyl-β-cyclodextrin), 5% acetic acid (10%) and aqueous Natrosol (0.5%) and administered by intragastral gavage. The administration volume was 10 ml/kg body weight. Four bitransgenic mice on continuous doxycycline diets for more than 6 weeks were subjected to MRI to document the lung tumor burden. BIBW2992 (Afatinib, Tomtovok, Tovok) generated by Boehringer Ingelheim Austria GmbH) formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) was administered orally by gavage at 20 mg/kg once daily dosing schedule. Rapamycin was dissolved in 100% ethanol, freshly diluted in 5% PEG400 and 5% Tween 80 before treatment and administered by intraperitoneal injection at 2 mg/kg daily dosage. Mice were monitored by MRI every 1 or 2 weeks to determine reduction in tumor volume and killed.

References on BIBW2992 (Afatinib):
[1] Hirsh V. Future Oncol. 2011 Jul;7(7):817-25.
[2] Li D et al.Oncogene. 2008 August 7; 27(34): 4702–4711. 

BMS-599626 is a highly selective pan-HERKinase inhibitor with IC50 of 20 and 30 nM for the inhibition of HER1and HER2, respectively. HER1 expression has been shown in as much as 80% to 90% of colon and non–small- cell lung cancer. HER2 gene amplification and overexpression of the protein have been shown to occur in f30% of all breast cancer. [1] BMS-599626 was highly selective when tested against a broad panel of diverse protein kinases. BMS-599626 abrogated HER1and HER2 signaling. BMS-599626 inhibited the proliferation of tumor cell lines that are dependent on these receptors, with IC50 in the range of 0.24 to 1 μM. [2]

References on BMS-599626 (AC480):

[1] Slamon DJ et al. Science. 1987 Jan 9;235(4785):177-82
[2] Wong TW et al. Clin Cancer Res. 2006 Oct 15;12(20 Pt 1):6186-93

Erlotinib Hydrochloride: Preclinical studies have shown that very low concentrations are required to inhibit activity against isolated tyrosine kinase(IC50, 2 nmol/L), to reduce HER1/EGFR autophosphorylation in intact human tumor cells in vitro (IC50, 20 nmol/L), and to inhibit the EGF-dependent proliferation of cells (10). It acts by inducing the expression of the cell-cycle inhibitor p27, and suppressing the expression of the cell-cycle promoter cyclin D1, thereby blocking cell-cycle progression at the G1 phase [1] . Assessment of the effect of various oral doses of erlotinib on tumor growth in the HN5 head and neck tumor xenograft model indicated a marked improvement in antitumor effect between doses of 1.6 and 12.5 mg/kg; since the 12.5 mg/kg dose resulted in no substantive tumor growth, relative improvements in antitumor effect at higher doses are not readily apparent. [2]

Gefitinib (ZD-1839, Iressa) is a novel potent EGFR tyrosine kinase and Akt phosphorylations inhibitor with IC50 of 37, 26 and 57 nM for Tyr1173, Tyr1173 and Tyr992 in, respectively, the low and high EGFR expressing cell lines. Immunoblot analysis of whole cell lysates revealed that in general gefitinib effectively inhibited all tyrosine phosphorylation sites on EGFR in both the high and low-EGFR-expressing cell lines. However, the phosphorylation sites Tyr1173 and Tyr992 were less sensitive requiring higher concentrations of gefitinib for inhibition. As was the case for ERK, gefitinib fails to effectively inhibit AKT phosphorylation in the high-EGFR-
expressing cell line indicating that EGFR is not the major activator of AKT in this cell line. The low IC50 (7 nM), however show that the weak induction of AKT phosphorylation by EGFR in this cell line is efficiently blocked by gefitinib. Gefitinib inhibits AKT phosphorylations, with IC50 values of 220 and 263 nM, in the low-EGFR- and –EGFRvIII-expressing cell lines, respectively. [1] MCF10A cells are nontransformed breast epithelial cells that require EGF to proliferate. The monolayer growth of these EGF-driven untransformed cells is inhibited by ZD1839 with an IC50 of 20 nM, similar to its IC50 in vitro for EGFR and consistent with effective inhibition of EGFR in vivo. [2] Cell line characteristics and sensitivity to ZD1839 at 1uM are 59% inhibition for MDA-MB-231,74% inhibition for A431, 81% inhibition for SKBr3,60% inhibition for SKOV3, 33% inhibition for BT474,52% inhibition for MCF-7, 28% inhibition for T47D, respectively. [3]

References on Gefitinib(Iressa):
[1] Pedersen MW et al. Br J Cancer. 2005 Oct 17; 93(8):915-23.
[2] Mark M. Moasser et al. Cancer Res. 2001 October 1;61:7184-7188
[3] Neil G. Anderson et al. International Journal of Cancer.2001 Sep 23;94(6):774-782

Lapatinib (Tykerb) was found to have 50% inhibitory concentration (IC50) values against purified EGFR and HER2 of 10.2 and 9.8 nM, respectively. And IC50 >10000nM against c-Raf-1,MEK,ERK,CDK1CDK2,p38 and VEGFR-2. The IC50s for inhibition of cell growth by 72h treatment with GW2016 are 0.16μM for A431,0.12 μM for HN5,0.10 μM for BT474,0.09 μM for N87.
Lapatinib (Tykerb) undergoes first-pass metabolism catalyzed by CYP3A4/5 and does not appear to be a substrate for P-glycoprotein. [2]

References on Lapatinib (Tykerb):
[1] David W. Rusnak et al. Mol Cancer Ther December. 2001 Dec;1(2):85-94
[2] Michael H Nelson,Christian R Dolder. The Annals of Pharmacotherapy.40(2):261-269

PD153035 hydrochloride is a hydrochloride acid salt form of PD153035 which is a ATP-competitive EGFR inhibitor with an IC50 and Ki of 25 and 6 pM.PD153035 effectively blocks the enhancement of mitogenesis, induction of early gene expression, and oncogenic transformation that occur in response to EGF receptor stimulation. With human fibroblasts and epidermoid carcinoma cells, PD153035 at nanomolar concentrations rapidly inhibits EGFR autophosphorylation. With breast and ovarian cancer cells, PD153035 not only blocks cell growth via inhibition of EGFR, but also upregulates the expression of the tumor suppressor retinoic acid receptor-beta 2 (RAR-beta2). [1][2]

References on PD153035:
[1] Bridges AJ et al. J Med Chem. 1996 Jan 5; 39(1):267-76.
[2] Bos M et al. Clin Cancer Res. 1997 Nov; 3(11):2099-106.

Vandetanib (Zactima) is a VEGFR and EGFR antagonist and a tyrosine kinase inhibitor with IC50 of 60, 90, 40 nM for HUVEC proliferation, PC-9 cells and tyrosine kinase activity, respectively. In vitro, two cell lines (OZ and HuCCT1), both of which harboured KRAS mutation, were refractory to vandetanib, one cell line (TGBC24TKB) was somewhat resistant, and another cell line (TKKK) was sensitive. The most sensitive cell line (TKKK) had EGFR amplification. Vandetanib (25 mg /kg/ day) significantly prolonged the time to metastasis in an intravenous model of TKKK metastasis. [1][2]

References on Vandetanib (Zactima):
[1] Yoshikawa D et al. Br J Cancer. 2009 Apr 21;100(8):1257-66.
[2] http://en.wikipedia.org/wiki/Vandetanib

XL647: The activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo[1,2] .EXEL-7647 potently inhibits the EGFR and ErbB2 with IC50 0.3 and 16 nM respectively[2] . EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647 substantially inhibited the growth and vascularization of MDAMB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors.[2]

References on XL647:
[1] Trowe T, et al. Clin Cancer Res. 2008 Apr 15;14(8):2465-75.
[2] Gendreau SB, et al. Clin Cancer Res. 2007 Jun 15;13(12):3713-23

AEE788 is a novel multitargeted human epidermal receptor (HER) 1/2 and vascular endothelial growth factor receptor (VEGFR) 1/2 receptor family tyrosine kinases inhibitor with IC50 of 2, 6, 77, 59 nM for EGFR, ErbB2, KDR, and Flt-1. In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited with IC50s of 11 and 220 nM, respectively. It efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, It also inhibited VEGF-induced angiogenesis in a murine implant model. It has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. [1][2][3]

References on AEE788:
[1] Traxler P et al. Cancer Res. 2004 Jul 15;64(14):4931-41.
[2] Juengel E et al. BMC Cancer. 2009 May 27;9:161.
[3] Meco D et al. Transl Oncol. 2010 Oct 1;3(5):326-35.

AG 490 is a potent epidermal growth factor receptor kinase autophosphorylation inhibitor with an IC50 of 100 nM and 56.8μM for EGFR and JAK, respectively. It inhibits cytokine-independent cell growth in vitro and tumor cell invasion in vivo. It selectively blocks leukemic cell growth in vitro and in vivo by inducing programmed cell death, with no harmful effect on normal hematopoiesis. It inhibits the constitutive activation of STAT-3 DNA binding and IL-2-induced growth of MF tumor cells. It displays apoptotic and antiproliferative properties with IC50 of 1.7, 2.8 and 6.1 µM in 2E8, Baf/3 and Jurkat cells, respectively. [1][2]

References on AG-490:
[1] J Immunol. 1999 Apr 1;162(7):3897-904.
[2] Meydan N et al. Nature. 1996 Feb 15;379(6566):645-8.

CUDC-101 is a potent multitargeted HDAC, EGFR and HER2 inhibitor with IC50 of 4.4, 2.4, and 15.7 nM, respectively. CUDC-101 has novel structure incorporating HDAC inhibitory functionality into the pharmacophore of the EGFR and HER2 inhibitors. In most tumor cell lines tested, CUDC-101 exhibits efficient antiproliferative activity with greater potency than vorinostat (SAHA), erlotinib, lapatinib, and combinations of vorinostat/erlotinib and vorinostat/lapatinib. In vivo, CUDC-101 promotes tumor regression or inhibition in various cancer xenograft models including nonsmall cell lung cancer (NSCLC), liver, breast, head and neck, colon, and pancreatic cancers. [1][2]

References on CUDC-101:
[1] Cai X et al. J Med Chem. 2010 Mar 11; 53(5):2000-9.
[2] Lai CJ et al. Cancer Res. 2010 May 1; 70(9):3647-56.

Neratinib (HKI-272) is an orally available, irreversible tyrosine kinase inhibitor with IC50 of 59 nM and 92 nM for HER2 and EGFR, respectively.[1] Neratinib binds to the HER-2 receptor irreversibly, thereby reducing autophosphorylation in cells, apparently by targeting a cysteine residue in the ATP-binding pocket of the receptor. Neratinib also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells.[2]

Pelitinib (EKB-569) is a 3-cyanoquinoline pan-ErbB tyrosine kinase inhibitor with potential antineoplastic activity. Pelitinib irreversibly binds covalently to epidermal growth factor receptors (EGFR) ErbB-1, -2 and -4, thereby inhibiting receptor phosphorylation and signal transduction and resulting in apoptosis and suppression of proliferation in tumor cells that overexpress these receptors. Pelitinib inhibits EGF-induced phosphorylation of EGF-R and the growth of tumors that overexpress EGF-R in animal models.

References on Pelitinib (EKB-569):
[1] http://clincancerres.aacrjournals.org/content/14/1/215.full.pdf+html
[2] Wissner A et al. J Med Chem. 2003 Jan 2;46(1):49-63

References on WZ3146:

[1] Zhou W et al. Nature. 2009 Dec 24;462(7276):1070-4

WZ4002 is an irreversiblely inhibitor against EGFR T790M (mutation of the gatekeeper T790 residue) which is detected in 50% of clinically resistant patients to gefitinib or erlotinib. WZ4002 has a basic chemical framework (covalent pyrimidine) which is different from that of other EGFR inhibitors. [1]This agent is 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wildtype EGFR, than quinazoline-based EGFR inhibitors (HKI-272 and CL-387,785) in vitro. [1] This agent has a 300-fold lower half-maximum inhibitory concentration (IC50<20nM) against the PC9GR(delE746_A750/T790M, gefitinib-resistant) cells compared with clinical-stage inhibitors such as HKI-272. [1] In a 2-week efficacy study, WZ4002 treatment resulted in significant tumour regressions compared with vehicle alone in both T790M-containing murine models. [1]

References on WZ4002:
[1] Zhou W et al. Nature. 2009 Dec 24;462(7276):1070-4

WZ8040 is an irreversiblely inhibitor against EGFR T790M  (IC50<10nM, mutation of the gatekeeper T790 residue) which is detected in 50% of clinically resistant patients to gefitinib or erlotinib. WZ8040 has a basic chemical framework (covalent pyrimidine) which is different from that of other EGFR inhibitors. [1]
This agent is 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wildtype EGFR, than quinazoline-based EGFR inhibitors (HKI-272 and CL-387,785) in vitro. [1]
WZ8040 has a 300-fold lower half-maximum inhibitory concentration (IC50<10nM) against the PC9GR(delE746_A750/T790M, gefitinib-resistant) cells compared with clinical-stage inhibitors such as HKI-272. [1]

References on WZ8040:
[1] Zhou W et al. Nature. 2009 Dec 24;462(7276):1070-4

AV-412 is a second-generation, orally bioavailable dual EGFR/HER2 tyrosine kinase inhibitor. AV-412 possesses potential antineoplastic activity. AV-412 binds to and inhibits the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor 2 (HER2), which may result in the inhibition of tumor growth and angiogenesis, and tumor regression in EGFR/HER2-expressing tumors. AV-412 may be active against EGFR/HER2-expressing tumor cells that are resistant to first-generation kinase inhibitors. EGFR and HER2 are receptor tyrosine kinases that play major roles in tumor cell proliferation and tumor vascularization. [1] In a study, at both 1 and 3 days following treatment, AV412-treated tumors revealed significantly more apoptotic regions compared with tumors from mice treated with vehicle alone. Furthermore, the number of carcinoma cells expressing HER2 was decreased following treatment with AV-412. [2]AV-412 is originally developed by AVEO Pharmaceuticals, Inc. The phase I clinical trials for AV-412 has been terminated in the treatment of tumor.

References on AV-412:
[1] http://www.cancer.gov/drugdictionary?cdrid=513167
[2] Wu M, et al. Proc Natl Acad Sci U S A. 2009 April 28; 106(17): 7022–7027

AZD8931 is a novel potent reversible small molecule epidermal growth factor receptor, ERBB2 (HER2) and ERBB3 inhibitor with an IC50 of 4, 3, 4 nM, respectively. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non-small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation. It has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. [1][2][3]

References on AZD8931:
[1] Hickinson DM et al. Clin Cancer Res. 2010 Feb 15;16(4):1159-69.
[2] http://www.cancer.gov/drugdictionary/?CdrID=593099
[3] http://meeting.ascopubs.org/cgi/content/abstract/27/15S/11072

Chrysophanic acid (Chrysophanol) is a EGFR/mTOR pathway inhibitor. Chrysophanic acid (Chrysophanol) is a natural anthraquinone, has anticancer activity in EGFR-overexpressing SNU-C5 human colon cancer cells. Chrysophanic acid (Chrysophanol) preferentially blocked proliferation in SNU-C5 cells but not in other cell lines (HT7, HT29, KM12C, SW480, HCT116 and SNU-C4) with low levels of EGFR expression. Chrysophanic acid (Chrysophanol) treatment in SNU-C5 cells inhibited EGF-induced phosphorylation of EGFR and suppressed activation of downstream signaling molecules, such as AKT, extracellular signal-regulated kinase (ERK) and the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K). Chrysophanic acid (80 and 120 µM) significantly blocked cell proliferation when combined with the mTOR inhibitor, rapamycin. [1]

References on Chrysophanic acid (Chrysophanol):
[1] Lee MS et al. Phytother Res. 2010 Nov 19. doi: 10.1002/ptr.3323.

Mubritinib(TAK 165) is a potent EGFR and p34cdc2 inhibitor with IC50 of 6 nM and 0.2 µM, respectively. Mubritinib(TAK 165) also inhibits p33cdk2 and p33cdk5. Mubritinib(TAK 165) displays > 4000-fold selectivity over EGFR, FGFR, PDGFR, JAK1 and Src. Mubritinib(TAK 165) exhibits potent antiproliferative effects in ErbB2-overexpressing cancer cell lines (IC50 = 5 nM in BT474 breast cancer cells) and significantly inhibits bladder, breast and prostate cancer xenograft growth in vivo. [1][2]

References on Mubritinib(TAK 165):
[1] http://en.wikipedia.org/wiki/Mubritinib
[2] Havlícek L et al. J Med Chem. 1997 Feb 14;40(4):408-12.

OSI-420 (Desmethyl Erlotinib,CP-473420) is an active metabolite of erlotinib which is an orally active EGFR tyrosin kinase inhibitor with IC50 of 2 and 20 nM for the inhibition of human EGFR and EGFR autophosphorylation in tumor cells. Erlotinib disappearance from plasma after a short IV infusion was biexponential with a mean terminal half-life of 5.2 h and a mean clearance of 128 ml/min per m2. OSI-420 exposure (AUC) in plasma was 30% (range 12–59%) of erlotinib, and OSI-420 clearance was more than 5-fold higher than erlotinib. Erlotinib and OSI-420 were detectable in CSF. The CSF penetration (AUCCSF:AUCplasma) of erlotinib and OSI-420 was <5% relative to total plasma concentration, but CSF drug exposure was ∼30% of plasma free drug exposure, which was calculated from published plasma protein binding values. The intravenous administration of erlotinib was well tolerated. [1][2][3]

References on OSI-420 (Desmethyl Erlotinib,CP-473420):
[1] Zhao M et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Aug 15;793(2):413-20.
[2] Ling J et al. Anticancer Drugs. 2008 Feb;19(2):209-16.
[3] Broniscer A et al. Clin Cancer Res. 2007 Mar 1;13(5):1511-5.

PF299804 is a potent, orally available, irreversible tyrosine kinase HER 1 (EGFR), HER2 and HER4 inhibitor with IC50 of 6, 45.7 and 73.7 nM for EGFR, ERBB2 and ERBB4, respectively. [1] PF299804 is a quinazalone-based irreversible pan-ERBB inhibitor structurally related to CI-1033. PF299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. PF299804 effectively inhibited the in vitro kinase activity of wild-type EGFR with similar efficacy as gefitinib, erlotinib, andCI-1033. In contrast to gefitinib and erlotinib, PF299804 also effectively inhibited wild-type ERBB2. LCK and SRC were the only other kinases inhibited by PF299804 although with >10 fold higher IC50 than against EGFR. PF299804 inhibited cellular EGFR and ERBB 2 with IC50 of 5.8 and 41 nM in NIH3T3/EGFR cell and NIH3T3/ERBB2 cell, respectively. PF299804 is active in E-sensitive and -resistant preclinical models. PF299804 had clinical activity in phase I/II trials in EGFR TK inhibitor (TKI)-refractory NSCLC.[2] PF299804 is originally developed by Seoul National University Hospital and Pfizer. The phase II clinical trials for PF299804 was performing in the treatment of advanced gastric cancer.

References on PF299804:
[1] Jänne PA et al. Clin Cancer Res. 2011 Mar 1;17(5):1131-9.
[2] Engelman JA et al. Cancer Res. 2007 Dec 15;67(24):11924-32.

PP121 is a multitargeted dual receptor tyrosine kinases inhibitor with IC50 of 0.052, 1.4, 0.15, 1.1, 0.06, 0.01and 0.002 μM for p110α, p110β, p110δ, p110γ, DNA-PK, mTOR and PDGFR, respectively. PP121 also inhibits Abl, Hck, Src,Src(T338I), VEGFR2, EGFR EphB4 with IC50 of 0.018, 0.008, 0.014, 0.22, 0.012, 0.26, 0.19 μM, respectively. PP121 exhibits no significant effect on receptor serine/threonine kinases (RSTKs). PP121 blocks the proliferation of tumor cells by direct inhibition of PI 3-K, mTOR, Src and the VEGF receptor. PP121 achieves its dual potency by targeting a residue (Glu310 in Src) that has been structurally conserved between kinase families. PP121 potently and dose-dependently blocked the phosphorylation of Akt, p70S6K and S6 in these cells. [1]

References on PP121:
[1] Apsel B et al. Nat Chem Biol. 2008 Nov;4(11):691-9.

AG-1478 (Tyrphostin AG-1478) is a highly potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with an IC50 of 3 nM.  Much higher concentrations are required for AG-1478’s prevention of the kinase activity of the closely-related HER2(neu/erbB2) receptor (IC50> 100 μM), the platelet-derived growth factor receptor (PDGFR) with IC50>100 μM), and p210Bcr-Abl (IC50 > 5 0 μM). That AG-1478 (Tyrphostin AG-1478) has been used in a broad range of studies contributes to its potency and selectivity. AG-1478 (Tyrphostin AG-1478) reversibly prevents rat brain Kv1.5 potassium channels (IC50 = 9.8 µM) independent of PTK activity. Additionally, AG-1478 (Tyrphostin AG-1478) also suppresses the growth of leiomyoma and myometrium cell cultures with IC50 values of 5.6 and 5.7 µM, respectively. AG-1478 (Tyrphostin AG-1478) blocke the activation of MAP kinase and strongly represses induction of fos gene expression and DNA synthesis. AG-1478 (Tyrphostin AG-1478) promotes optic nerve regeneration. [1] AG-1478 (Tyrphostin AG-1478) enhances expression of pSAPK/JNK, while selective JNK inhibitor SP 600125 elevates expression of GSK-3β (S9P). The selective EGFR inhibitor AG-1478 (Tyrphostin AG-1478) are observed to be able to block EGFR-mediated signaling pathway and prevent versican G3 induced effects on mammary cancer cell proliferation. In particular, versican G3 enhanced cell survival is prevented by selective EGFR inhibitor AG-1478 (Tyrphostin AG-1478) through mechanisms blocking G3 activated expression of pERK and GSK-3 β (S9P). AG-1478 (Tyrphostin AG-1478) blocks G3 enhanced effects on cell apoptosis. [2] AG-1478 (Tyrphostin AG-1478) is active both in vitro in cell lines such as colon cancer, non-small cell lung cancer and glioblastoma cell models and in vivo in mice xenograft models. In tumor xenograft models AG-1478 (Tyrphostin AG-1478) prevents the growth of A431 tumors and human glioblastomas which over-express a mutant EGFR. [3]

References on AG-1478 (Tyrphostin AG-1478):
[1] Robinson R, et al. ACS Nano. 2011, 5(6), 4392-4400.
[2]Du WW, et al. PLoS One. 2011, 6(11), e26396.
[3] Ellis AG, et al. Biochem Pharmacol. 2006, 71(10), 1422-34.