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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay

, , , , , and .

NIH Chemical Genomics Center, 9800 Medical Center Dr., Building B, Bethesda, MD, 20892-3370

Received: ; Last Update: September 2, 2010.

The ERK phosphorylation signaling pathway plays a significant role in regulation of cellular functions. The anomalous activation of the ERK pathway in cancer has been shown to promote cell proliferation, differentiation, development and survival. The probe ML102 (CID-2303746) has been identified in a cell-based ERK phosphorylation assay as an inhibitor of the ERK phosphorylation signaling pathway. In addition, the probe has satisfied the project goals of a novel chemotype that is a highly potent and cell-membrane permeable selective inhibitor of EGFR kinase.

Assigned Assay Grant #: X01 MH082406-01

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher Austin

Assay Submitter & Institution: Wei Zheng, NCGC/NIH

PubChem Summary Bioassay Identifier (AID): AID-1742

Probe Structure & Characteristics

Image ml102fu1
CID/MLTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID-2303746/ML102EGFR710 nM [SID-22409543, AID-1731]c-Raf, Mek-1>100µM [SID-22409543, AID-1732]>100EGFR-T790M: 320 nM [SID-22409543, AID-1729]
EGFR-L858R: 630 nM [SID-22409543, AID-1727]

Recommendations for the scientific use of this probe

CID-2303746 has been identified in a cell-based ERK phosphorylation assay as an inhibitor for the ERK phosphorylation pathway. The subsequent enzyme assay confirmed its selective inhibitory activity on the EGFR tyrosine kinase. In addition, CID-2303746 inhibits the kinase activities of several EGFR mutants (EGFR L858R, EGFR T790M, EGFR L858R T790M) with the potency equal to or up to 4 times greater than what was observed with the wild type EGFR receptor. CID-2303746 satisfied the project goal of a novel chemotype that is a selective inhibitor of EGFR, is cell membrane permeable, and was the most potent member of this series.

Introduction

A cell-based assay of kinase activity

The ERK phosphorylation signaling pathway plays a significant role in regulation of cellular functions. The anomalous activation of the ERK pathway in cancer has been shown to promote cell proliferation, differentiation, development and survival (4, 7). ERK phosphorylation can be activated by upstream receptor tyrosine kinases (RTK), and therefore provides a convenient tool to screen members of these receptor families. Advent of a novel cell-based assay detecting ERK phosphorylation allows for the screening of membrane permeable inhibitors of the ERK signaling pathway.

In the present assay, signaling of the ERK pathway is triggered by the activation of the epidermal growth factor receptor (EGFR). Currently, there are three well-known EGFR antagonists in clinical use: Erlotinib, Gefitinib and Lapatinib. Lapatinib is an orally active drug used in the treatment of solid tumors, such as breast cancer. Erlotinib and Gefitinib are being used to treat cancer patients with metastatic non-small cell lung cancer (NSCLC), where EGFR over-expression is the target of inhibition. However, mutations in EGFR of NSCLC-carrying patients have been shown to render the enzyme non-respondent to Erlotinib and Gefitinib treatments (2). While screening for inhibitors of the ERK pathway using a novel cell-based ERK phosphorylation assay, we identified a probe that inhibited EGFR, and more significantly, also inhibited clinically observed EGFR mutants (EGFR L858R, EGFR T790M, EGFR L858R T790M) with similar or even greater potencies.

Project Description

The advent of a new cell-based assay specific for ERK phosphorylation allows for screening of cell membrane permeable inhibitors of the ERK signaling pathway in the physiological context (6). The goal of this project was to evaluate this technology and screen the MLPCN’s compound collection (the MLSMR) to identify novel cell-membrane permeable inhibitors of the ERK signaling pathway.

EGFR stimulation of the cells results in the cascade that activates Ras, Raf and MEK kinases, which in turn phosphorylates ERK protein. The phosphorylated ERK protein from the cell lysate is captured by two specific antibodies - one anti-phosphorylated site antibody and one anti non-phosphorylated site antibody. These two antibodies are then captured by the acceptor and donor AlphaScreen™ beads, respectively, to form a proximity signal upon excitation by a laser light. HTRF based kinase assays against Raf, MEK, EGFR and several clinically relevant mutants of EGFR were used as secondary assays for identifying the molecular targets of the ERK phosphorylation cascade inhibitors.

The process of discovering a novel ERK Signaling Pathway inhibitor has been summarized as a flow diagram in Figure 1.

Fig. 1. Overview of screening process.

Fig. 1

Overview of screening process.

Materials and Methods

Primary Screening Assay

The cell-based ERK phosphorylation assay in SureFire assay format is obtained from PerkinElmer. The HEK-293 cell line is used for all in vivo cell based screening. The cells are stimulated with EGF to activate the ERK signaling pathway. After incubation with the agonist for 5 minutes, the cells are lysed and the phosphorylated ERK proteins are detected using the ERK SureFire kit (Fig. 2).

Fig. 2. Principle of ERK phosphorylation assay in SureFire assay format.

Fig. 2

Principle of ERK phosphorylation assay in SureFire assay format.

Two antibodies (Ab) against the ERK protein are used. One biotinylated Ab is against a non-phosphorylated site of the ERK protein, binding the streptavidin donor bead. The second Ab is against the phosphorylated site of ERK protein, and selectively binds the protein-A coated acceptor bead. These binding events bring the beads into sufficient proximity for the detection involving singlet oxygen transfer. The donor bead is excited by a laser light at 680 nm, and the energy is transferred to the acceptor bead by singlet oxygen that yields an emission of 520–620 nm. The signal will be reduced if the phosphorylation of the ERK protein is blocked. All of the reagents used for the ERK SureFire assay and their resources are listed in Table 1.

Table 1. Reagents and resources for the ERK SureFire assay.

Table 1

Reagents and resources for the ERK SureFire assay.

The HEK-293 cell line expressing endogenous EGFR was cultured at NCGC. The cells were maintained in DMEM/F12 medium containing 10 % FBS, 100 units/ml Penicillin, and 100 μg/ml Streptomycin at 37 °C, 5% CO2. Suspended HEK-293 cells were seeded into 1536-well tissue culture-treated white plates at a density of 2500 cells/well in 4 μl of Opti-MEM medium, supplemented with 1% FBS and incubated at 37 °C, 5 % CO2 for overnight. 22 nl of compound was transferred to each well and incubated at 37 °C for 10 min. 0.5 ul of agonist (EGF) at 0.2 nM final concentration added to each well and incubated at ambient for 5 min. Subsequent steps involving cell lysis and ERK detection were performed according to the AlphaScreen SureFire ERK 1/2 kit. After 6 hours incubation at ambient, plates were then read in EnVision plate reader (Perkin Elmer) using the Alpha Screen detection mode. Protocol details are provided in Table 2. 107,792 compounds were screened. The assay performed well, with a Z′ of 0.73 +/− 0.10 and signal to background of 11.6 +/− 4.1 across 531 plates. Compounds were selected for confirmation based on potency and efficacy considerations.

Table 2. ERK assay protocol for the HEK-293 cells in 1536-well plate format.

Table 2

ERK assay protocol for the HEK-293 cells in 1536-well plate format.

Cell-based Confirmatory Assay

The confirmation assay for the HEK-293 cells was performed in the same manner as the primary screen, and was used to verify the activity of the prioritized group of compounds from the primary screen in the ERK SureFire phosphorylation assay.

Purified Kinase Confirmatory Assays

Purified enzyme assays were used to de-convolute confirmed hits from the primary screen, and identify molecular targets for those compounds. Compounds were assayed against purified EGFR tyrosine kinase, Raf and MEK. In addition, EGFR inhibitors were assayed against several clinically relevant EGFR mutants (EGFR L858R, EGFR T790M, and EGFR L858R T790M). Finally, the probe molecule and analogs were profiled against related kinases to further characterize compound specificity.

The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for the EGFR and EGFR mutant enzyme assays. It uses time resolved fluorescence resonance energy transfer (TR-FRET) to detect production of a phosphorylated substrate. As illustrated in Fig. 1, a peptide substrate is labeled with a biotin that can bind to XL665 labeled streptavidin, and the anti-phosphoresidue antibody is labeled with Eu+. Upon phosphorylation of the substrate, the antibody binds to phosphorylated substrate that enables TR-FRET detection in homogenous assay format.

Fig. 3. Schematic illustration of the assay principle for EGFR kinase assay.

Fig. 3Schematic illustration of the assay principle for EGFR kinase assay

All the reagents used for the EGFR and EGFR mutant assays including their resources are listed in Table 3. An HTRF Kinase TK assay kit (Cisbio, Bedford, MA) was used to develop the EGFR and EGFR mutant assays (3). EGFR and EGFR mutants were obtained from Invitrogen. The assay buffer was composed of 50 mM HEPES (pH 7.0), 5 mM MgCl2, 5mM DTT, 0.1 % NaN3, 0.1 % BSA and 0.1 mM orthovanadate. The HTRF assays were preformed according to the HTRF Kinase TK kit. Optimization for each enzyme was performed in 384 well format (data not shown). All reagents were dispensed into 1536 well plates according to table 4.

Table 3. Reagents and resources for the various enzymatic assays of purified kinases.

Table 3

Reagents and resources for the various enzymatic assays of purified kinases.

Table 4. HTRF assay protocol for the EGFR enzymes in 1536-well plate format.

Table 4

HTRF assay protocol for the EGFR enzymes in 1536-well plate format.

From the 63 compounds tested in confirmation, 23 were active against EGFR, including several known inhibitors of EGFR. From this set of active compounds two analogs happened to be more active against the EGFR mutants than EGFR w.t., which contrasts with existing inhibitors of EGFR. We tested three mutant variations of EGFR w.t.; EGFR L858R, EGFR T790M, and EGFR L858 T790M.

Kinase Profiling

The probe molecule and analogs were sent to Ambit and Reaction Biology for external confirmation of their inhibition, and to assay the compounds against related targets. The results compare favorably with the in-house determination of activity. The results are presented in table 6. Assay details can be found at http://www.ambitbio.com/technology and http://www.reactionbiology.com/pages/kinase.htm.

PubChem Data Deposition

Table 5 lists the assays run and data deposited into PubChem during the course of this project.

Table 5. Data deposited into PubChem.

Table 5

Data deposited into PubChem.

Results

Cell-based ERK signaling pathway assay recapitulates activity of known inhibitors

The cell-based assay was developed to identify novel cell-permeable inhibitors of kinases in the ERK pathway, and avoid chemical scaffolds that possess non-specific kinase activity or kinase activity only in a purified in vitro system. The current assay was able to demonstrate cellular activity of known inhibitors such as staurosporine, U0126 (an inhibitor of the c-Raf:MEK interaction), and when stimulated with EGF, the clinical EGFR inhibitors gefitinib, erlotinib, and lapatinib.

CID-2303746 is a novel inhibitor of EGFR

Several novel compounds also demonstrated activity in the cell-based assay. Upon interrogation in purified kinase assays, one compound, CID 2303746, was shown to inhibit EGFR at nanomolar concentrations.

CID-2303746 inhibits clinically-relevant mutants of EGFR

By itself, EGFR kinase inhibition is not a novel activity for a chemical series. There are three such compounds now approved for marketing: gefitinib, erlotinib, and lapatinib. A well-known limitation of such compounds (Li 2008) is that certain mutations in EGFR prevent the existing compounds from inhibiting the enzyme. Strikingly, CID-2303746 does not suffer from this limitation as shown in figure 4 and table 6. GW-583340 is shown in lieu of lapatinib in figure 4, but it is known that lapatinib also lacks activity against these mutants (Li 2008). Staurosporine also retains potency against these mutants, but its lack of selectivity against other kinases as well as its pharmacodynamic properties has made it a challenging candidate for development. CID-2303746, which represents a novel chemotype for kinase inhibition, has a promising profile as an EGFR kinase inhibitor.

Figure 4. Activity of probe and other EGFR inhibitors against clinical EGFR mutants.

Figure 4

Activity of probe and other EGFR inhibitors against clinical EGFR mutants. GW-583340 is a close analog of lapatinib. Black = L858R, Cyan = T790M, Blue = T790M/L858R, Grey = wild type enzyme.

Table 6. Summary of CID-2303746’s activity against EGFR, clinically-observed mutants of EGFR, and homologous enzymes ERBB2, ERBB3, and ERBB4 based on data generated in-house, and at Ambit and Reaction Biology.

Table 6

Summary of CID-2303746’s activity against EGFR, clinically-observed mutants of EGFR, and homologous enzymes ERBB2, ERBB3, and ERBB4 based on data generated in-house, and at Ambit and Reaction Biology.

Regarding the SAR of the series, from table 6 it can be seen that introduction of a methyl functional group at the meta position of the aromatic ring of the benzodiazepine bicycle does not have an impact on the activity. In other hand, introduction of a methyl functional group at the para position of the acyclic aromatic ring reduces the activity both against wild type and mutant receptors. Unusually, MLS-000391787 shows better activity for mutant receptors than wild type, and it is selective for within the ERBB family towards the EGFR.

Probe description

a. Chemical name

(3Z,4Z)-4-methyl-3-(2-phenylhydrazono)-1H-benzo[b][1,4]diazepin-2(3H)-one [ML102]

b. Probe chemical structure

Image ml102fu6

c. Structural Verification Information of probe SID

Structural verification and initial purity quantification was performed by 1H NMR analysis using a Varian spectrometer dissolving the sample in deuterated DMSO.

1H δ (ppm): 11.18 (s, 1H), 10.43 (s, 1H), 7.70 (s, 1H), 7.31 (m, 3H), 7.17 (m, 5H), 6.90 (t, 1H, J= 8.0 Hz), 3.48 (br s, 2H).

In addition, further analysis was carried out by LC/MS using an Agilent system in the following conditions:

  • Column: 3 x 75 mm Luna C18, 3 micron
  • Run time: 4.5 minutes
  • Gradient: 4% to 100% over 2.8 minutes
  • Mobile phase: acetonitrile (0.025% TFA), water (0.05% TFA)
  • Flow rate: 0.8 to 1.0 mL/min
  • Temperature: 50 C
  • UV Wavelength: 220 nm, 254 nm

The retention time of MLS-000391787 (CID-2303746, SID-22409543) in those conditions was of 2.93 minutes. The purity of the Mass spectra was recorded in the positive ionization mode using an electrospray (API-ES) ionizing source with nitrogen as drying gas. Both NMR and LC/MS analysis showed purity greater than 99% for those batches of MLS-000391787 use in the biological evaluation.

d. PubChem CID (corresponding to the SID)

CID-2303746

e. Vendor availability

MLS-000391787 (CID-2303746, SID-22409543) can be purchased from the following vendor:

Company Info: Enamine

Email: ten.enimane@enimane

Catalog Name: Enamine Screening Library

Order Number: T0500-2111

f. MLS submission

CID-2303746 is registered with the MLSMR as MLS-000391787.

Analogs CID-2303119 and CID-2322338 are registered with the MLSMR as MLS-000391788 and MLS-000391794, respectively.

g. Mode of Action

MLS-000391787 is able to disrupt the ERK phosphorylation through the inhibition of the EGFR kinase.

h. Synthetic Pathway

There are several papers addressing the synthesis of 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one analogues (El-Kerdawy 1989). Figure 5 shows the general synthetic methodology for synthesizing these compounds:

Figure 5. Synthesis of 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one.

Figure 5

Synthesis of 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one.

i. Probe Properties

MLS-000391787 and the rest of the compounds of this series are ease to dissolve in organic solvents such us MeOH, Cl2CH2, acetone or DMSO. As pure compound, MLS000391787 is a yellow powder at room temperature, chemically stable and with no apparent reactivity with air.

Molecular Weight278.30856 [g/mol]
XLogP2.4
tPSA:65.85
H-Bond Donor2
H-Bond Acceptor4

Bibliography

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El-Kerdawy MM, Ismaiel AM, Yousif MY, El-Sherbeny MA. 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-ones, as potential psychosedative and anxiolytic agents. Pakistan Journal of Scientific and Industrial Research. 1989;32(8):510–512.
2.
Engelman JA, Jänne PA. Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clin Cancer Res. 2008;14(10):2895–2899. [PubMed: 18483355]
3.
Harbert C, Marshall J, Soh S, Steger K. Development of a HTRF Kinase Assay for Determination of Syk Activity. Current Chemical Genomics. 2008;1:20–26. [PMC free article: PMC2774622] [PubMed: 20161824]
4.
Lee JT Jr, McCubrey JA. The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic intervention in leukemia. Leukemia. 2002;16:486–507. [PubMed: 11960326]
5.
Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR, Padera RF, Shapiro GI, Baum A, Himmelsbach F, Rettig WJ, Meyerson M, Solca F, Greulich H, BIBW, Wong KK. 2992 an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008;27(34):4702–4711. [PMC free article: PMC2748240] [PubMed: 18408761]
6.
Osmond RI, Sheehan A, Borowicz R, Barnett E, Harvey G, Turner C, Brown A, Crouch MF, Dyer AR. GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors. J Biomol Screen. 2005;10:730–737. [PubMed: 16129779]
7.
Thompson N, Lyons J. Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery. Curr Opin Pharmacol. 2005;5:350–356. [PubMed: 15955734]
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