Cellular Targets of Gefitinib

Brehmer, Dirk; Greff, Zoltán; Godl, Klaus; Blencke, Stephanie; Kurtenbach, Alexander; Weber, Martina; Müller, Stefan; Klebl, Bert; Cotten, Matt; Kéri, Gÿorgý; Wissing, Josef; Daub, Henrik

doi:10.1158/0008-5472.379.65.2

Cited by 207 publications

(33 citation statements)

References 16 publications

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“…This cysteine residue has been previously reported to be located next to the highly conserved DFG motif, which in an active kinase state coordinates magnesium ion in the ATP binding site . Interestingly, a structurally similar analog of dacomitinib, FDA-approved EGFR inhibitor gefitinib, has also been demonstrated to target GAK. ,, Additionally, GAK C190 has been recently characterized as a target of a SM1-71, a promiscuous covalent kinase inhibitor …”

Section: Resultsmentioning

confidence: 97%

“…57 Interestingly, a structurally similar analog of dacomitinib, FDA-approved EGFR inhibitor gefitinib, has also been demonstrated to target GAK. 32,58,59 Additionally, GAK C190 has been recently characterized as a target of a SM1-71, a promiscuous covalent kinase inhibitor. 60 Another cysteine residue that was observed to undergo modulation upon treatment with dacomitinib was C218, localized in close proximity to the active site of glycogen synthase kinase-3 beta (GSK3B).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

2019

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Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely used chemically cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using the iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, we evaluated the key sample preparation steps. We also performed a mass tolerant database search strategy using MSFragger software. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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Section: Resultsmentioning

confidence: 97%

Section: ■ Results and Discussionmentioning

confidence: 99%

Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

2019

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“…Kinase inhibitors, while being valuable drugs and powerful molecular probes, have been recognized to display widely varying degrees of target selectivity, ,− which may lead to toxicity or conversely enable repurposing of clinical compounds. , Understanding a kinase inhibitor’s cellular target profile also has important implications for the correct evaluation of its biological effects, as noncanonical targets can either decrease or enhance cellular efficacy and assist in dissecting the wiring maps of the targeted signaling networks.…”

Section: Discussionmentioning

confidence: 99%

Dual Targeting of WEE1 and PLK1 by AZD1775 Elicits Single Agent Cellular Anticancer Activity

et al. 2017

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Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775’s anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 by WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.

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“…The T790M mutation was originally identified from patients treated with gefitinib and later found in other patients receiving erlotinib treatment . Clinical studies demonstrated that the gatekeeper mutation (T790M) resulted in a dramatic reduction in therapeutic efficacy of EGFR-selective inhibitors . Recently novel inhibitors are designed to target inhibitor resistant mutants including afatinib and neratinib, both of which have advanced to phase III clinical trials.…”

Section: Introductionmentioning

confidence: 99%

“…12 Clinical studies demonstrated that the gatekeeper mutation (T790M) resulted in a dramatic reduction in therapeutic efficacy of EGFR-selective inhibitors. 13 Recently novel inhibitors are designed to target inhibitor resistant mutants including afatinib 14 and neratinib, 15 both of which have advanced to phase III clinical trials.…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Analysis of Binding of Kinase Inhibitors to WT EGFR and the T790M Mutant

Park

McDonald²,

Petter³

et al. 2016

J. Chem. Theory Comput.

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Epidermal growth factor receptor (EGFR) inhibitors interrupt EGFR-dependent cellular signaling pathways that lead to accelerated tumor growth and proliferation. Mutation of a threonine in the inhibitor binding pocket, known as the "gatekeeper", to methionine (T790M) confers acquired resistance to several EGFR-selective inhibitors. We studied interactions between EGFR inhibitors and the gatekeeper residues of the target protein. Thermodynamic integration (TI) with Amber14 indicates that the binding energies of gefitinib and AEE788 to the active state of the T790M mutant EGFR is 3 kcal/mol higher than to the wild type (WT), whereas ATP binding energy to the mutant is similar to the WT. Using metadynamics MD simulations with NAMD v2.9, the conformational equilibrium between the inactive resting state and the catalytically competent activate state was determined for the WT EGFR. When combined with the results obtained by Sutto and Gervasio, our simulations showed that the T790M point mutation lowers the free energy of the active state by 5 kcal/mol relative to the inactive state of the enzyme. Relative to the WT, the T790M mutant binds gefitinib more strongly. The T790M mutation is nevertheless resistant due to its increased binding of ATP. By contrast, the binding of AEE788 to the active state causes a conformational change in the αC-helix adjacent to the inhibitor binding pocket, that results in a 2 kcal/mol energy penalty. The energy penalty explains why the binding of AEE788 to the T790M mutant is unfavorable relative to binding to WT EGFR. These results establish the role of the gatekeeper mutation on inhibitor selectivity. Additional molecular dynamics (MD) simulations, TI, and metadynamics MD simulations reveal the origins of the changes in binding energy of WT and mutants.

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Cellular Targets of Gefitinib

Cited by 207 publications

References 16 publications

Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

Dual Targeting of WEE1 and PLK1 by AZD1775 Elicits Single Agent Cellular Anticancer Activity

Molecular Dynamics Analysis of Binding of Kinase Inhibitors to WT EGFR and the T790M Mutant

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