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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2010/09/02">
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<meta name="citation_author" content="Juan Marugan">
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<meta name="citation_author" content="Seameen Dehdashti">
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<meta name="citation_author" content="Wei Zheng">
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<meta name="citation_author" content="Noel Southall">
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<meta name="citation_author" content="James Inglese">
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<meta name="citation_author" content="Christopher Austin">
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<meta name="citation_pmid" content="21433365">
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<meta name="DC.Title" content="HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay">
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<meta name="DC.Contributor" content="Juan Marugan">
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<meta name="DC.Contributor" content="Seameen Dehdashti">
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<meta name="DC.Contributor" content="Wei Zheng">
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<meta name="DC.Contributor" content="Noel Southall">
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<meta name="DC.Contributor" content="James Inglese">
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<meta name="DC.Contributor" content="Christopher Austin">
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<meta name="description" content="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.">
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<meta name="og:title" content="HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay">
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<meta name="og:description" content="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.">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK47349_"><span class="title" itemprop="name">HTS for Identification of Inhibitors against the ERK Signaling Pathway using a
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Homogenous Cell-based Assay</span></h1><p class="contribs">Marugan J, Dehdashti S, Zheng W, et al.</p><p class="fm-aai"><a href="#_NBK47349_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>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.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> X01 MH082406-01</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center,
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Christopher Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center,
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Christopher Austin</p><p><b>Assay Submitter & Institution:</b> Wei Zheng, NCGC/NIH</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1742" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1742</a></p><div id="ml102.s1"><h2 id="_ml102_s1_">Probe Structure & Characteristics</h2><div id="ml102.fu1" class="figure"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu1.jpg" alt="Image ml102fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102tu1"><a href="/books/NBK47349/table/ml102.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml102tu1" rid-ob="figobml102tu1"><img class="small-thumb" src="/books/NBK47349/table/ml102.tu1/?report=thumb" src-large="/books/NBK47349/table/ml102.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml102.tu1"><a href="/books/NBK47349/table/ml102.tu1/?report=objectonly" target="object" rid-ob="figobml102tu1">Table</a></h4></div></div></div><div id="ml102.s2"><h2 id="_ml102_s2_">Recommendations for the scientific use of this probe</h2><p>CID-2303746 has been identified in a cell-based ERK phosphorylation assay as an
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inhibitor for the ERK phosphorylation pathway. The subsequent enzyme assay confirmed
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its selective inhibitory activity on the EGFR tyrosine kinase. In addition,
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CID-2303746 inhibits the kinase activities of several EGFR mutants (EGFR L858R, EGFR
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T790M, EGFR L858R T790M) with the potency equal to or up to 4 times greater than
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what was observed with the wild type EGFR receptor. CID-2303746 satisfied the
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project goal of a novel chemotype that is a selective inhibitor of EGFR, is cell
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membrane permeable, and was the most potent member of this series.</p></div><div id="ml102.s3"><h2 id="_ml102_s3_">Introduction</h2><div id="ml102.s4"><h3>A cell-based assay of kinase activity</h3><p>The ERK phosphorylation signaling pathway plays a significant role in regulation
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of cellular functions. The anomalous activation of the ERK pathway in cancer has
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been shown to promote cell proliferation, differentiation, development and
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survival (<a class="bibr" href="#ml102.r4" rid="ml102.r4">4</a>, <a class="bibr" href="#ml102.r7" rid="ml102.r7">7</a>). ERK phosphorylation can be activated by upstream
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receptor tyrosine kinases (RTK), and therefore provides a convenient tool to
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screen members of these receptor families. Advent of a novel cell-based assay
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detecting ERK phosphorylation allows for the screening of membrane permeable
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inhibitors of the ERK signaling pathway.</p><p>In the present assay, signaling of the ERK pathway is triggered by the activation
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of the epidermal growth factor receptor (EGFR). Currently, there are three
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well-known EGFR antagonists in clinical use: Erlotinib, Gefitinib and Lapatinib.
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Lapatinib is an orally active drug used in the treatment of solid tumors, such
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as breast cancer. Erlotinib and Gefitinib are being used to treat cancer
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patients with metastatic non-small cell lung cancer (NSCLC), where EGFR
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over-expression is the target of inhibition. However, mutations in EGFR of
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NSCLC-carrying patients have been shown to render the enzyme non-respondent to
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Erlotinib and Gefitinib treatments (<a class="bibr" href="#ml102.r2" rid="ml102.r2">2</a>). While screening for inhibitors of the ERK pathway using a novel
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cell-based ERK phosphorylation assay, we identified a probe that inhibited EGFR,
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and more significantly, also inhibited clinically observed EGFR mutants (EGFR
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L858R, EGFR T790M, EGFR L858R T790M) with similar or even greater potencies.</p></div><div id="ml102.s5"><h3>Project Description</h3><p>The advent of a new cell-based assay specific for ERK phosphorylation allows for
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screening of cell membrane permeable inhibitors of the ERK signaling pathway in
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the physiological context (<a class="bibr" href="#ml102.r6" rid="ml102.r6">6</a>). The
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goal of this project was to evaluate this technology and screen the
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MLPCN’s compound collection (the MLSMR) to identify novel
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cell-membrane permeable inhibitors of the ERK signaling pathway.</p><p>EGFR stimulation of the cells results in the cascade that activates Ras, Raf and
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MEK kinases, which in turn phosphorylates ERK protein. The phosphorylated ERK
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protein from the cell lysate is captured by two specific antibodies - one
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anti-phosphorylated site antibody and one anti non-phosphorylated site antibody.
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These two antibodies are then captured by the acceptor and donor
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AlphaScreen™ beads, respectively, to form a proximity signal upon
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excitation by a laser light. HTRF based kinase assays against Raf, MEK, EGFR and
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several clinically relevant mutants of EGFR were used as secondary assays for
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identifying the molecular targets of the ERK phosphorylation cascade
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inhibitors.</p><p>The process of discovering a novel ERK Signaling Pathway inhibitor has been
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summarized as a flow diagram in <a class="figpopup" href="/books/NBK47349/figure/ml102.f1/?report=objectonly" target="object" rid-figpopup="figml102f1" rid-ob="figobml102f1">Figure
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1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml102f1" co-legend-rid="figlgndml102f1"><a href="/books/NBK47349/figure/ml102.f1/?report=objectonly" target="object" title="Fig. 1" class="img_link icnblk_img figpopup" rid-figpopup="figml102f1" rid-ob="figobml102f1"><img class="small-thumb" src="/books/NBK47349/bin/ml102f1.gif" src-large="/books/NBK47349/bin/ml102f1.jpg" alt="Fig. 1. Overview of screening process." /></a><div class="icnblk_cntnt" id="figlgndml102f1"><h4 id="ml102.f1"><a href="/books/NBK47349/figure/ml102.f1/?report=objectonly" target="object" rid-ob="figobml102f1">Fig. 1</a></h4><p class="float-caption no_bottom_margin">Overview of screening process. </p></div></div></div></div><div id="ml102.s6"><h2 id="_ml102_s6_">Materials and Methods</h2><div id="ml102.s7"><h3>Primary Screening Assay</h3><p>The cell-based ERK phosphorylation assay in SureFire assay format is obtained
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from PerkinElmer. The HEK-293 cell line is used for all <i>in vivo</i>
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cell based screening. The cells are stimulated with EGF to activate the ERK
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signaling pathway. After incubation with the agonist for 5 minutes, the cells
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are lysed and the phosphorylated ERK proteins are detected using the ERK
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SureFire kit (<a class="figpopup" href="/books/NBK47349/figure/ml102.f2/?report=objectonly" target="object" rid-figpopup="figml102f2" rid-ob="figobml102f2">Fig. 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml102f2" co-legend-rid="figlgndml102f2"><a href="/books/NBK47349/figure/ml102.f2/?report=objectonly" target="object" title="Fig. 2" class="img_link icnblk_img figpopup" rid-figpopup="figml102f2" rid-ob="figobml102f2"><img class="small-thumb" src="/books/NBK47349/bin/ml102f2.gif" src-large="/books/NBK47349/bin/ml102f2.jpg" alt="Fig. 2. Principle of ERK phosphorylation assay in SureFire assay format." /></a><div class="icnblk_cntnt" id="figlgndml102f2"><h4 id="ml102.f2"><a href="/books/NBK47349/figure/ml102.f2/?report=objectonly" target="object" rid-ob="figobml102f2">Fig. 2</a></h4><p class="float-caption no_bottom_margin">Principle of ERK phosphorylation assay in SureFire assay
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format. </p></div></div><p>Two antibodies (Ab) against the ERK protein are used. One biotinylated Ab is
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against a non-phosphorylated site of the ERK protein, binding the streptavidin
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donor bead. The second Ab is against the phosphorylated site of ERK protein, and
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selectively binds the protein-A coated acceptor bead. These binding events bring
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the beads into sufficient proximity for the detection involving singlet oxygen
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transfer. The donor bead is excited by a laser light at 680 nm, and the energy
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is transferred to the acceptor bead by singlet oxygen that yields an emission of
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520–620 nm. The signal will be reduced if the phosphorylation of the
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ERK protein is blocked. All of the reagents used for the ERK SureFire assay and
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their resources are listed in <a class="figpopup" href="/books/NBK47349/table/ml102.t1/?report=objectonly" target="object" rid-figpopup="figml102t1" rid-ob="figobml102t1">Table
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1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t1"><a href="/books/NBK47349/table/ml102.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml102t1" rid-ob="figobml102t1"><img class="small-thumb" src="/books/NBK47349/table/ml102.t1/?report=thumb" src-large="/books/NBK47349/table/ml102.t1/?report=previmg" alt="Table 1. Reagents and resources for the ERK SureFire assay." /></a><div class="icnblk_cntnt"><h4 id="ml102.t1"><a href="/books/NBK47349/table/ml102.t1/?report=objectonly" target="object" rid-ob="figobml102t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the ERK SureFire assay. </p></div></div><p>The HEK-293 cell line expressing endogenous EGFR was cultured at NCGC. The cells
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were maintained in DMEM/F12 medium containing 10 % FBS, 100 units/ml
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Penicillin, and 100 μg/ml Streptomycin at 37 °C,
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5% CO2. Suspended HEK-293 cells were seeded into 1536-well tissue
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culture-treated white plates at a density of 2500 cells/well in 4 μl
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of Opti-MEM medium, supplemented with 1% FBS and incubated at 37
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°C, 5 % CO<sub>2</sub> for overnight. 22 nl of compound
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was transferred to each well and incubated at 37 °C for 10 min. 0.5
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ul of agonist (EGF) at 0.2 nM final concentration added to each well and
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incubated at ambient for 5 min. Subsequent steps involving cell lysis and ERK
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detection were performed according to the AlphaScreen SureFire ERK 1/2 kit.
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After 6 hours incubation at ambient, plates were then read in EnVision plate
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reader (Perkin Elmer) using the Alpha Screen detection mode. Protocol details
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are provided in <a class="figpopup" href="/books/NBK47349/table/ml102.t2/?report=objectonly" target="object" rid-figpopup="figml102t2" rid-ob="figobml102t2">Table 2</a>. 107,792
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compounds were screened. The assay performed well, with a Z′ of 0.73
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+/− 0.10 and signal to background of 11.6
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+/− 4.1 across 531 plates. Compounds were selected for
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confirmation based on potency and efficacy considerations.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t2"><a href="/books/NBK47349/table/ml102.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml102t2" rid-ob="figobml102t2"><img class="small-thumb" src="/books/NBK47349/table/ml102.t2/?report=thumb" src-large="/books/NBK47349/table/ml102.t2/?report=previmg" alt="Table 2. ERK assay protocol for the HEK-293 cells in 1536-well plate format." /></a><div class="icnblk_cntnt"><h4 id="ml102.t2"><a href="/books/NBK47349/table/ml102.t2/?report=objectonly" target="object" rid-ob="figobml102t2">Table 2</a></h4><p class="float-caption no_bottom_margin">ERK assay protocol for the HEK-293 cells in 1536-well plate
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format. </p></div></div></div><div id="ml102.s8"><h3>Cell-based Confirmatory Assay</h3><p>The confirmation assay for the HEK-293 cells was performed in the same manner as
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the primary screen, and was used to verify the activity of the prioritized group
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of compounds from the primary screen in the ERK SureFire phosphorylation
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assay.</p></div><div id="ml102.s9"><h3>Purified Kinase Confirmatory Assays</h3><p>Purified enzyme assays were used to de-convolute confirmed hits from the primary
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screen, and identify molecular targets for those compounds. Compounds were
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assayed against purified EGFR tyrosine kinase, Raf and MEK. In addition, EGFR
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inhibitors were assayed against several clinically relevant EGFR mutants (EGFR
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L858R, EGFR T790M, and EGFR L858R T790M). Finally, the probe molecule and
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analogs were profiled against related kinases to further characterize compound
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specificity.</p><p>The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for the
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EGFR and EGFR mutant enzyme assays. It uses time resolved fluorescence resonance
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energy transfer (TR-FRET) to detect production of a phosphorylated substrate. As
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illustrated in <a class="figpopup" href="/books/NBK47349/figure/ml102.f1/?report=objectonly" target="object" rid-figpopup="figml102f1" rid-ob="figobml102f1">Fig. 1</a>, a peptide
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substrate is labeled with a biotin that can bind to XL665 labeled streptavidin,
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and the anti-phosphoresidue antibody is labeled with Eu<sup>+</sup>.
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Upon phosphorylation of the substrate, the antibody binds to phosphorylated
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substrate that enables TR-FRET detection in homogenous assay format.</p><div id="ml102.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47349/bin/ml102f3.jpg" alt="Fig. 3. Schematic illustration of the assay principle for EGFR kinase assay." /></div><h3><span class="label">Fig. 3</span><span class="title">Schematic illustration of the assay principle for EGFR kinase
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assay</span></h3></div><p>All the reagents used for the EGFR and EGFR mutant assays including their
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resources are listed in <a class="figpopup" href="/books/NBK47349/table/ml102.t3/?report=objectonly" target="object" rid-figpopup="figml102t3" rid-ob="figobml102t3">Table 3</a>. An
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HTRF Kinase TK assay kit (Cisbio, Bedford, MA) was used to develop the EGFR and
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EGFR mutant assays (<a class="bibr" href="#ml102.r3" rid="ml102.r3">3</a>). EGFR and
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EGFR mutants were obtained from Invitrogen. The assay buffer was composed of 50
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mM HEPES (pH 7.0), 5 mM MgCl<sub>2</sub>, 5mM DTT, 0.1 %
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NaN<sub>3</sub>, 0.1 % BSA and 0.1 mM orthovanadate. The HTRF
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assays were preformed according to the HTRF Kinase TK kit. Optimization for each
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enzyme was performed in 384 well format (data not shown). All reagents were
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dispensed into 1536 well plates according to <a class="figpopup" href="/books/NBK47349/table/ml102.t4/?report=objectonly" target="object" rid-figpopup="figml102t4" rid-ob="figobml102t4">table 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t3"><a href="/books/NBK47349/table/ml102.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml102t3" rid-ob="figobml102t3"><img class="small-thumb" src="/books/NBK47349/table/ml102.t3/?report=thumb" src-large="/books/NBK47349/table/ml102.t3/?report=previmg" alt="Table 3. Reagents and resources for the various enzymatic assays of purified kinases." /></a><div class="icnblk_cntnt"><h4 id="ml102.t3"><a href="/books/NBK47349/table/ml102.t3/?report=objectonly" target="object" rid-ob="figobml102t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the various enzymatic assays of purified
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kinases. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t4"><a href="/books/NBK47349/table/ml102.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml102t4" rid-ob="figobml102t4"><img class="small-thumb" src="/books/NBK47349/table/ml102.t4/?report=thumb" src-large="/books/NBK47349/table/ml102.t4/?report=previmg" alt="Table 4. HTRF assay protocol for the EGFR enzymes in 1536-well plate format." /></a><div class="icnblk_cntnt"><h4 id="ml102.t4"><a href="/books/NBK47349/table/ml102.t4/?report=objectonly" target="object" rid-ob="figobml102t4">Table 4</a></h4><p class="float-caption no_bottom_margin">HTRF assay protocol for the EGFR enzymes in 1536-well plate
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format. </p></div></div><p>From the 63 compounds tested in confirmation, 23 were active against EGFR,
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including several known inhibitors of EGFR. From this set of active compounds
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two analogs happened to be more active against the EGFR mutants than EGFR w.t.,
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which contrasts with existing inhibitors of EGFR. We tested three mutant
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variations of EGFR w.t.; EGFR L858R, EGFR T790M, and EGFR L858 T790M.</p></div><div id="ml102.s10"><h3>Kinase Profiling</h3><p>The probe molecule and analogs were sent to Ambit and Reaction Biology for
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external confirmation of their inhibition, and to assay the compounds against
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related targets. The results compare favorably with the in-house determination
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of activity. The results are presented in table 6. Assay details can be found at <a href="http://www.ambitbio.com/technology" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.ambitbio.com/technology</a> and <a href="http://www.reactionbiology.com/pages/kinase.htm" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.reactionbiology.com/pages/kinase.htm</a>.</p></div><div id="ml102.s11"><h3>PubChem Data Deposition</h3><p><a class="figpopup" href="/books/NBK47349/table/ml102.t5/?report=objectonly" target="object" rid-figpopup="figml102t5" rid-ob="figobml102t5">Table 5</a> lists the assays run and
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data deposited into PubChem during the course of this project.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t5"><a href="/books/NBK47349/table/ml102.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml102t5" rid-ob="figobml102t5"><img class="small-thumb" src="/books/NBK47349/table/ml102.t5/?report=thumb" src-large="/books/NBK47349/table/ml102.t5/?report=previmg" alt="Table 5. Data deposited into PubChem." /></a><div class="icnblk_cntnt"><h4 id="ml102.t5"><a href="/books/NBK47349/table/ml102.t5/?report=objectonly" target="object" rid-ob="figobml102t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Data deposited into PubChem. </p></div></div></div></div><div id="ml102.s12"><h2 id="_ml102_s12_">Results</h2><div id="ml102.s13"><h3>Cell-based ERK signaling pathway assay recapitulates activity of known
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inhibitors</h3><p>The cell-based assay was developed to identify novel cell-permeable inhibitors of
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|
kinases in the ERK pathway, and avoid chemical scaffolds that possess
|
|
non-specific kinase activity or kinase activity only in a purified <i>in
|
|
vitro</i> 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
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|
inhibitors gefitinib, erlotinib, and lapatinib.</p></div><div id="ml102.s14"><h3>CID-2303746 is a novel inhibitor of EGFR</h3><p>Several novel compounds also demonstrated activity in the cell-based assay. Upon
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interrogation in purified kinase assays, one compound, CID 2303746, was shown to
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inhibit EGFR at nanomolar concentrations.</p></div><div id="ml102.s15"><h3>CID-2303746 inhibits clinically-relevant mutants of EGFR</h3><p>By itself, EGFR kinase inhibition is not a novel activity for a chemical series.
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|
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
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|
<a class="figpopup" href="/books/NBK47349/figure/ml102.f4/?report=objectonly" target="object" rid-figpopup="figml102f4" rid-ob="figobml102f4">figure 4</a> and <a class="figpopup" href="/books/NBK47349/table/ml102.t6/?report=objectonly" target="object" rid-figpopup="figml102t6" rid-ob="figobml102t6">table 6</a>. GW-583340 is shown in lieu of lapatinib in
|
|
<a class="figpopup" href="/books/NBK47349/figure/ml102.f4/?report=objectonly" target="object" rid-figpopup="figml102f4" rid-ob="figobml102f4">figure 4</a>, 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
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chemotype for kinase inhibition, has a promising profile as an EGFR kinase
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inhibitor.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml102f4" co-legend-rid="figlgndml102f4"><a href="/books/NBK47349/figure/ml102.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml102f4" rid-ob="figobml102f4"><img class="small-thumb" src="/books/NBK47349/bin/ml102f4.gif" src-large="/books/NBK47349/bin/ml102f4.jpg" alt="Figure 4. Activity of probe and other EGFR inhibitors against clinical EGFR mutants." /></a><div class="icnblk_cntnt" id="figlgndml102f4"><h4 id="ml102.f4"><a href="/books/NBK47349/figure/ml102.f4/?report=objectonly" target="object" rid-ob="figobml102f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Activity of probe and other EGFR inhibitors against clinical EGFR
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mutants. GW-583340 is a close analog of lapatinib. Black = L858R, Cyan
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= T790M, Blue = T790M/L858R, Grey =
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wild type enzyme. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102t6"><a href="/books/NBK47349/table/ml102.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml102t6" rid-ob="figobml102t6"><img class="small-thumb" src="/books/NBK47349/table/ml102.t6/?report=thumb" src-large="/books/NBK47349/table/ml102.t6/?report=previmg" alt="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." /></a><div class="icnblk_cntnt"><h4 id="ml102.t6"><a href="/books/NBK47349/table/ml102.t6/?report=objectonly" target="object" rid-ob="figobml102t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Summary of CID-2303746’s activity against EGFR,
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clinically-observed mutants of EGFR, and homologous enzymes ERBB2,
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ERBB3, and ERBB4 based on data generated in-house, and at Ambit and
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Reaction Biology. </p></div></div><p>Regarding the SAR of the series, from <a class="figpopup" href="/books/NBK47349/table/ml102.t6/?report=objectonly" target="object" rid-figpopup="figml102t6" rid-ob="figobml102t6">table
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6</a> it can be seen that introduction of a methyl functional group at
|
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the meta position of the aromatic ring of the benzodiazepine bicycle does not
|
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have an impact on the activity. In other hand, introduction of a methyl
|
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functional group at the para position of the acyclic aromatic ring reduces the
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activity both against wild type and mutant receptors. Unusually, MLS-000391787
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shows better activity for mutant receptors than wild type, and it is selective
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for within the ERBB family towards the EGFR.</p></div></div><div id="ml102.s16"><h2 id="_ml102_s16_">Probe description</h2><div id="ml102.s17"><h3>a. Chemical name</h3><p>(3Z,4Z)-4-methyl-3-(2-phenylhydrazono)-1H-benzo[b][1,4]diazepin-2(3H)-one
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<b>[<a href="/pcsubstance/?term=ML102[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML102</a></b>]</p></div><div id="ml102.s18"><h3>b. Probe chemical structure</h3><div id="ml102.fu2" class="figure"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu6.jpg" alt="Image ml102fu6" /></div></div></div><div id="ml102.s19"><h3>c. Structural Verification Information of probe SID</h3><p>Structural verification and initial purity quantification was performed by
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<sup>1</sup>H NMR analysis using a Varian spectrometer dissolving the sample
|
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in deuterated DMSO.</p><p><sup>1</sup>H δ (ppm): 11.18 (s, 1H), 10.43 (s, 1H), 7.70 (s, 1H),
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|
7.31 (m, 3H), 7.17 (m, 5H), 6.90 (t, 1H, J= 8.0 Hz), 3.48 (br s,
|
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2H).</p><p>In addition, further analysis was carried out by LC/MS using an Agilent system in
|
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the following conditions:</p><ul class="simple-list"><li class="half_rhythm"><div>Column: 3 x 75 mm Luna C18, 3 micron</div></li><li class="half_rhythm"><div>Run time: 4.5 minutes</div></li><li class="half_rhythm"><div>Gradient: 4% to 100% over 2.8 minutes</div></li><li class="half_rhythm"><div>Mobile phase: acetonitrile (0.025% TFA), water
|
|
(0.05% TFA)</div></li><li class="half_rhythm"><div>Flow rate: 0.8 to 1.0 mL/min</div></li><li class="half_rhythm"><div>Temperature: 50 C</div></li><li class="half_rhythm"><div>UV Wavelength: 220 nm, 254 nm</div></li></ul><p>The retention time of MLS-000391787 (CID-2303746, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>) in those
|
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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.</p></div><div id="ml102.s20"><h3>d. PubChem CID (corresponding to the SID)</h3><p>CID-2303746</p></div><div id="ml102.s21"><h3>e. Vendor availability</h3><p>MLS-000391787 (CID-2303746, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>) can be purchased from the following
|
|
vendor:</p><p>Company Info: Enamine</p><p>Email: <a href="mailto:dev@null" data-email="ten.enimane@enimane" class="oemail">ten.enimane@enimane</a></p><p>Catalog Name: Enamine Screening Library</p><p>Order Number: T0500-2111</p></div><div id="ml102.s22"><h3>f. MLS submission</h3><p>CID-2303746 is registered with the MLSMR as MLS-000391787.</p><p>Analogs CID-2303119 and CID-2322338 are registered with the MLSMR as
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|
MLS-000391788 and MLS-000391794, respectively.</p></div><div id="ml102.s23"><h3>g. Mode of Action</h3><p>MLS-000391787 is able to disrupt the ERK phosphorylation through the inhibition
|
|
of the EGFR kinase.</p></div><div id="ml102.s24"><h3>h. Synthetic Pathway</h3><p>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). <a class="figpopup" href="/books/NBK47349/figure/ml102.f5/?report=objectonly" target="object" rid-figpopup="figml102f5" rid-ob="figobml102f5">Figure 5</a> shows the
|
|
general synthetic methodology for synthesizing these compounds:</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml102f5" co-legend-rid="figlgndml102f5"><a href="/books/NBK47349/figure/ml102.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml102f5" rid-ob="figobml102f5"><img class="small-thumb" src="/books/NBK47349/bin/ml102f5.gif" src-large="/books/NBK47349/bin/ml102f5.jpg" alt="Figure 5. Synthesis of 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one." /></a><div class="icnblk_cntnt" id="figlgndml102f5"><h4 id="ml102.f5"><a href="/books/NBK47349/figure/ml102.f5/?report=objectonly" target="object" rid-ob="figobml102f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Synthesis of
|
|
2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one. </p></div></div></div><div id="ml102.s25"><h3>i. Probe Properties</h3><p>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.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml102tu2"><a href="/books/NBK47349/table/ml102.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml102tu2" rid-ob="figobml102tu2"><img class="small-thumb" src="/books/NBK47349/table/ml102.tu2/?report=thumb" src-large="/books/NBK47349/table/ml102.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml102.tu2"><a href="/books/NBK47349/table/ml102.tu2/?report=objectonly" target="object" rid-ob="figobml102tu2">Table</a></h4></div></div></div></div><div id="ml102.bib"><h2 id="_ml102_bib_">Bibliography</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml102.r1">El-Kerdawy MM, Ismaiel AM, Yousif MY, El-Sherbeny MA. 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-ones,
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as potential psychosedative and anxiolytic agents. <span><span class="ref-journal">Pakistan Journal of Scientific and Industrial Research. </span>1989;<span class="ref-vol">32</span>(8):510–512.</span></div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml102.r2">Engelman JA, Jänne PA. Mechanisms of acquired resistance to epidermal growth factor
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receptor tyrosine kinase inhibitors in non-small cell lung
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cancer. <span><span class="ref-journal">Clin Cancer Res. </span>2008;<span class="ref-vol">14</span>(10):2895–2899.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18483355" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18483355</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml102.r3">Harbert C, Marshall J, Soh S, Steger K. Development of a HTRF Kinase Assay for Determination of Syk
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Activity. <span><span class="ref-journal">Current Chemical Genomics. </span>2008;<span class="ref-vol">1</span>:20–26.</span> [<a href="/pmc/articles/PMC2774622/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2774622</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20161824" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20161824</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml102.r4">Lee JT Jr, McCubrey JA. The Raf/MEK/ERK signal transduction cascade as a target for
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chemotherapeutic intervention in leukemia. <span><span class="ref-journal">Leukemia. </span>2002;<span class="ref-vol">16</span>:486–507.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11960326" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11960326</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml102.r5">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
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preclinical lung cancer models. <span><span class="ref-journal">Oncogene. </span>2008;<span class="ref-vol">27</span>(34):4702–4711.</span> [<a href="/pmc/articles/PMC2748240/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2748240</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18408761" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18408761</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml102.r6">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
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protein-coupled receptors. <span><span class="ref-journal">J Biomol Screen. </span>2005;<span class="ref-vol">10</span>:730–737.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16129779" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16129779</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml102.r7">Thompson N, Lyons J. Recent progress in targeting the Raf/MEK/ERK pathway with
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inhibitors in cancer drug discovery. <span><span class="ref-journal">Curr Opin Pharmacol. </span>2005;<span class="ref-vol">5</span>:350–356.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15955734" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15955734</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK47349_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Juan Marugan</span>,<sup>1</sup> <span itemprop="author">Seameen Dehdashti</span>,<sup>1</sup> <span itemprop="author">Wei Zheng</span>,<sup>1</sup> <span itemprop="author">Noel Southall</span>,<sup>1</sup> <span itemprop="author">James Inglese</span>,<sup>1</sup> and <span itemprop="author">Christopher Austin</span><sup>1</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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NIH Chemical Genomics Center, 9800 Medical Center Dr.,
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Building B, Bethesda, MD, 20892-3370</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">May 18, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Marugan J, Dehdashti S, Zheng W, et al. HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Homogenous Cell-based Assay. 2009 May 18 [Updated 2010 Sep 2]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml103/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml101/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml102fu1"><div id="ml102.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102fu1.jpg" alt="Image ml102fu1" /></div></div></article><article data-type="table-wrap" id="figobml102tu1"><div id="ml102.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml102.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml102.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml102.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (nM) [SID,
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AID]</th><th id="hd_h_ml102.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml102.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM)
|
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[SID, AID]</th><th id="hd_h_ml102.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml102.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
|
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[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml102.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-2303746/<a href="/pcsubstance/?term=ML102[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML102</a></td><td headers="hd_h_ml102.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR</td><td headers="hd_h_ml102.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">710 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>,
|
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1731" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1731</a>]</td><td headers="hd_h_ml102.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">c-Raf, Mek-1</td><td headers="hd_h_ml102.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1732" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1732</a>]</td><td headers="hd_h_ml102.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100</td><td headers="hd_h_ml102.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR-T790M: 320 nM
|
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[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1729" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1729</a>]<br />EGFR-L858R:
|
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630 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-22409543</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1727</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml102f1"><div id="ml102.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102f1.jpg" alt="Fig. 1. Overview of screening process." /></div><h3><span class="label">Fig. 1</span><span class="title">Overview of screening process</span></h3></div></article><article data-type="fig" id="figobml102f2"><div id="ml102.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102f2.jpg" alt="Fig. 2. Principle of ERK phosphorylation assay in SureFire assay format." /></div><h3><span class="label">Fig. 2</span><span class="title">Principle of ERK phosphorylation assay in SureFire assay
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format</span></h3></div></article><article data-type="table-wrap" id="figobml102t1"><div id="ml102.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Reagents and resources for the ERK SureFire assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Reagents</th><th id="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Resources</th><th id="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Catalog Number</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AlphaScreen SureFire Phospho-ERK 1/2
|
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Kit</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PerkinElmer</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">TGRES50K</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AlphaScreen General IgG (Protein A)
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Detection Kit</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PerkinElmer</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">6760617M</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGF</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Millipore</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">01–107</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DMEM/F12</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Invitrogen</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">11320</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fetal Bovine Serum</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Invitrogen</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10091</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Penicillin-Streptomycin</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Invitrogen</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">15140</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Opti-MEM</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Invitrogen</td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">11058</td></tr><tr><td headers="hd_h_ml102.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HEK-293</td><td headers="hd_h_ml102.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"></td><td headers="hd_h_ml102.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"></td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml102t2"><div id="ml102.t2" class="table"><h3><span class="label">Table 2</span><span class="title">ERK assay protocol for the HEK-293 cells in 1536-well plate
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format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Step</th><th id="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Parameter</th><th id="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Value</th><th id="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Cells</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">4 μl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">200 cells/well</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Culture</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">16–20 hr</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">37°C, 5%
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CO2</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Compound</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">22 nl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Library Compounds in 0.128 to 10 mM
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titration series or control</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10 min</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">37°C, 5%
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CO2</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.5 μl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Agonist 0.2 nM in final</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">5 min</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ambient</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.5 μl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Lysis buffer</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">15 min</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ambient</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.5 μl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Activation buffer</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10 min</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Room temperature</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1 μl</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reaction buffer with beads (50
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ng/well)</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">6 hour</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ambient/Dark</td></tr><tr><td headers="hd_h_ml102.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td><td headers="hd_h_ml102.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Detector</td><td headers="hd_h_ml102.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ex 680, Em 520–620</td><td headers="hd_h_ml102.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">EnVision plate reader</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml102f3"><div id="ml102.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102f3.jpg" alt="Fig. 3. Schematic illustration of the assay principle for EGFR kinase assay." /></div><h3><span class="label">Fig. 3</span><span class="title">Schematic illustration of the assay principle for EGFR kinase
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assay</span></h3></div></article><article data-type="table-wrap" id="figobml102t3"><div id="ml102.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Reagents and resources for the various enzymatic assays of purified
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kinases</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagents</th><th id="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Resources</th><th id="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Catalog Number</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">KinEASE TK Kit</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cisbio</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">62TK0PEC</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR w.t.</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PV3872</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR L858R</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PV4128</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR T790M</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PV4803</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EGFR L858R, T790M</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PV4879</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">RAF1 (c-RAF)</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Carna Bio</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">09–125</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MEK1 (MAP2K1)</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Millipore</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SGT220</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">A1852</td></tr><tr><td headers="hd_h_ml102.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DTT</td><td headers="hd_h_ml102.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml102.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">D9779</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml102t4"><div id="ml102.t4" class="table"><h3><span class="label">Table 4</span><span class="title">HTRF assay protocol for the EGFR enzymes in 1536-well plate
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format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t4_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Step</th><th id="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Parameter</th><th id="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Value</th><th id="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Enzyme</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">2 nl</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">EGFR at 3 nM final</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Compound</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">22 nl</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Library Compounds in 0.128 to 10 mM
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titration series or control</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1.5 μl</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10 µM ATP and 0.25 µM substrate
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final</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">30 min</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ambient</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Reagent</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">3 μl</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">antibody and XL-665</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Incubation</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">30 min</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ambient</td></tr><tr><td headers="hd_h_ml102.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml102.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Detector</td><td headers="hd_h_ml102.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Ex 320, Em 615,665</td><td headers="hd_h_ml102.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">EnVision plate reader</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml102t5"><div id="ml102.t5" class="table"><h3><span class="label">Table 5</span><span class="title">Data deposited into PubChem</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t5_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">AID</th><th id="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay-Type</th><th id="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compounds Tested</th><th id="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Bioassay Name</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/995" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">995</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Primary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">70,907</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">qHTS Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay
|
|
[Confirmatory]</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1454" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1454</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Primary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">131,339</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">qHTS Assay for Inhibitors of the ERK
|
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Signaling Pathway using a Homogeneous Screening Assay;
|
|
Stimulation with EGF [Confirmatory]</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1724" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1724</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Confirm DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">113</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmation Concentration-Response
|
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Assay for Inhibitors of the ERK Signaling Pathway using a
|
|
Homogeneous Screening Assay</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1725" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1725</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Confirm DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">60</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmation Concentration-Response
|
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Assay for Inhibitors of the ERK Signaling Pathway using a
|
|
Homogeneous Screening Assay; Stimulation with EGF</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1730" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1730</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Counterscreen</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">87</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Counterscreen Assay for Inhibitors of
|
|
the ERK Signaling Pathway using a Homogeneous Screening
|
|
Assay</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1731" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1731</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">28</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: EGFR
|
|
Kinase Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1732" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1732</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">72</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: MEK
|
|
Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1728</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">72</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: c-Raf
|
|
Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1727</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: EGFR
|
|
L858R Kinase Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1729" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1729</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: EGFR
|
|
T790M Kinase Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1726" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1726</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Secondary DR</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay for Inhibitors of the ERK
|
|
Signaling Pathway using a Homogeneous Screening Assay: EGFR
|
|
L858R;T790M Kinase Inhibition</td></tr><tr><td headers="hd_h_ml102.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1742" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1742</a></td><td headers="hd_h_ml102.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Summary</td><td headers="hd_h_ml102.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">N/A</td><td headers="hd_h_ml102.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Identification of Inhibitors against
|
|
the ERK Signaling Pathway using a Homogenous Cell-based
|
|
Assay</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml102f4"><div id="ml102.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102f4.jpg" alt="Figure 4. Activity of probe and other EGFR inhibitors against clinical EGFR mutants." /></div><h3><span class="label">Figure 4</span><span class="title">Activity of probe and other EGFR inhibitors against clinical EGFR
|
|
mutants</span></h3><div class="caption"><p>GW-583340 is a close analog of lapatinib. Black = L858R, Cyan
|
|
= T790M, Blue = T790M/L858R, Grey =
|
|
wild type enzyme.</p></div></div></article><article data-type="table-wrap" id="figobml102t6"><div id="ml102.t6" class="table"><h3><span class="label">Table 6</span><span class="title">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</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.t6_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Profiling Source</th><th id="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Enzyme</th><th id="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu2.jpg" alt="Image ml102fu2.jpg" /></div><br />CID 2303746</th><th id="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu3.jpg" alt="Image ml102fu3.jpg" /></div><br />CID 2303119</th><th id="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu4.jpg" alt="Image ml102fu4.jpg" /></div><br />CID 2322338</th><th id="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47349/bin/ml102fu5.jpg" alt="Image ml102fu5.jpg" /></div><br />Staurosporine</th></tr></thead><tbody><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>In-House</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<i>EGFR w.t.</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">710</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4460</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">710</td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">447</td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Reaction Bio.</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">221</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1443</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">102.8</td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">210</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1300</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>In-House</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<i>EGFR L858R</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">631</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3500</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">631</td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">398</td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Reaction Bio.</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">120</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>In-House</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<i>EGFR T790M</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">316</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>50000</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">355</td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5.6</td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Reaction Bio.</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>In-House</i>
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</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">
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<i>EGFR L858R T790M</i>
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</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3548</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>50000</td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3162</td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">45</td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Reaction Bio.</i>
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</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Ambit</i>
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</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">180</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Ambit</i>
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</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(E746-A750del)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
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|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(G719C)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">89</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Ambit</i>
|
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</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(G719S)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">92</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(L747-E749del, A750P)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">130</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Ambit</i>
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|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(L747-S752del, P753S)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">90</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(L747-T751del,Sins)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">120</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(L861Q)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">250</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<i>Ambit</i>
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|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EGFR(S752-1759del)</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">110</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
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|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ERBB2</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">40000</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
|
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</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ERBB3</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">17000</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml102.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<i>Ambit</i>
|
|
</td><td headers="hd_h_ml102.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ERBB4</td><td headers="hd_h_ml102.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1900</td><td headers="hd_h_ml102.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml102.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml102fu2"><div id="ml102.fu2" class="figure"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102fu6.jpg" alt="Image ml102fu6" /></div></div></article><article data-type="fig" id="figobml102f5"><div id="ml102.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47349/bin/ml102f5.jpg" alt="Figure 5. Synthesis of 2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one." /></div><h3><span class="label">Figure 5</span><span class="title">Synthesis of
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2,3-dihydro-3-arylhydrazono-4-methyl-1H-1,5-benzodiazepin-2-one</span></h3></div></article><article data-type="table-wrap" id="figobml102tu2"><div id="ml102.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47349/table/ml102.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml102.tu2_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">278.30856
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[g/mol]</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">tPSA:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">65.85</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4</td></tr></tbody></table></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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