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Pharmacological targeting of the pseudokinase Her3.

Xie T, Lim SM, Westover KD, Dodge ME, Ercan D, Ficarro SB, Udayakumar D, Gurbani D, Tae HS, Riddle SM, Sim T, Marto JA, Jänne PA, Crews CM, Gray NS - Nat. Chem. Biol. (2014)

Bottom Line: Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling.Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met.These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [2] Department of Biological Chemistry &Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Her3 (also known as ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be 'undruggable' using ATP-competitive small molecules because it lacks appreciable kinase activity. Here we report what is to our knowledge the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3-dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

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Hit identification and development of the Her3 irreversible inhibitor TX1-85-1(a) Chemical structures of the representative lead compounds identified by FRET binding assay, TX1-85-1 and biotin conjugated TX1-85-1. (b) In vitro kinase FRET binding assay results for top screening hits and TX1-85-1. Purified recombinant Her3 kinase domain (665-1001) was used in conjunction with Lanthascreen® technology (Invitrogen). TX1-85-1 shows the highest potency with an IC50 of 23 nM. Each condition was tested in triplicate. Data represent mean values ± s.d. (c) Docking study of TX1-85-1 with Her3 x-ray crystallography models (PDB ID 3KEX6, 3LMG7). An acrylamide substitution is predicted to form a covalent bond with Cys721 in Her3 via Michael Addition. (d) Her3 pull down experiment with TX1-85-1-biotin. PC9 GR4 cells were pre-treated with TX1-85-1 for 8 hours before lysis or TX1-85-1 was directly added to cell lysate at the indicated concentrations. Streptavidin beads were used to recover compound labeled Her3 which was detected with an anti-Her3 antibody.
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Figure 1: Hit identification and development of the Her3 irreversible inhibitor TX1-85-1(a) Chemical structures of the representative lead compounds identified by FRET binding assay, TX1-85-1 and biotin conjugated TX1-85-1. (b) In vitro kinase FRET binding assay results for top screening hits and TX1-85-1. Purified recombinant Her3 kinase domain (665-1001) was used in conjunction with Lanthascreen® technology (Invitrogen). TX1-85-1 shows the highest potency with an IC50 of 23 nM. Each condition was tested in triplicate. Data represent mean values ± s.d. (c) Docking study of TX1-85-1 with Her3 x-ray crystallography models (PDB ID 3KEX6, 3LMG7). An acrylamide substitution is predicted to form a covalent bond with Cys721 in Her3 via Michael Addition. (d) Her3 pull down experiment with TX1-85-1-biotin. PC9 GR4 cells were pre-treated with TX1-85-1 for 8 hours before lysis or TX1-85-1 was directly added to cell lysate at the indicated concentrations. Streptavidin beads were used to recover compound labeled Her3 which was detected with an anti-Her3 antibody.

Mentions: In order to develop potent and selective covalent Her3 ligands, we first identified ATP-competitive ligands using high-throughput screening and then employed structure-guided drug design to introduce reactive moieties that could form a covalent bond with Cys721. Kinome-wide sequence alignments suggest that Cys721 is uniquely present in Her3 and therefore provides an excellent potential means to achieve selectivity27. A covalent inhibitor provides a number of potential advantages including higher potency, improved selectivity due to Cys721 being highly unusual across the kinome, and the ability to address pharmacological specificity by performing ‘rescue’ experiments with a Cys721 to serine mutation. We developed an ATP-competitive ligand binding assay using the fluorescence resonance energy transfer (FRET) based LanthaScreen™ Eu methodology28 and screened a 1,500 member library consisting of both known and novel ATP-competitive kinase inhibitors. The most potent Her3 binders to emerge from the screen were five previously reported Src-family inhibitors: KIN001-111(A-770041)(1)29, KIN001-051 (2)30,31, (Fig. 1a) dasatinib32, bosutinib33 and KIN001-30 (3), (Supplementary Results, Supplementary Fig. 1a) which possessed IC50s below one hundred nanomolar (Fig. 1b). In order to develop covalent binders of Her3 that exploit the unique Cys721 residue, we docked these compounds to the previously reported 2.80 Å Her3 crystal structures (3KEX6 and 3LMG7, Fig. 1c and Supplementary Fig. 1b). This modeling suggested that introducing a meta-acrylamide from the phenyl substituent of KIN001-111 might afford the correct trajectory for a conjugate addition with Cys721. After the iterative synthesis and assaying of approximately 100 pyrazolopyrimidine acrylamides, we developed the ‘lead’ compound TX1-85-1 (4) which possessed an IC50 of 23 nM in the binding assay (Fig. 1a, b). TX1-85-1 can be viewed as a molecular amalgam of the hinge binding moiety of KIN001-051 and the solubility enhancing tail of KIN001-111, combined with an acrylamide arm to reach Cys721 (Fig. 1c). As expected for a covalent inhibitor, the apparent IC50 decreases upon longer incubation with the protein and reaches a plateau at about 3 hours (Supplementary Fig. 2). Mass spectrometry was used to confirm the covalent addition of TX1-85-1 to recombinant Her3 kinase domain protein and subsequent proteolysis of Her3 with trypsin and MS2 analysis revealed unique modification of Cys721 (Supplementary Fig. 3). We next investigated whether TX1-85-1 could form a covalent bond with Her3 in cells using a cellular competition binding assay. To enable these experiments we synthesized a biotinylated derivative (TX1-85-1-biotin) (5) that still preserves the ability to covalently bind to Her3 (Fig. 1a, Supplementary Fig. 4, IC50 = 50.7 nM). Cells were incubated with TX1-85-1 and then lysates were labeled with TX1-85-1-biotin in order to quantify the amount of Her3 that had escaped labeling by the initial incubation with TX1-85-1. We determined that incubation of PC9 GR4 cells (EGFR E746_A750/T790M) with 5 μM of TX1-85-1 for 8 hours resulted in complete protection of Her3 from subsequent labeling with TX1-85-1-biotin (Fig. 1d). This result suggests that TX1-85-1 (MW = 580Da) can pass through the cell membrane and is capable of complete ‘target engagement’ with Her3 intracellularly. To examine the specificity with which TX1-85-1 modifies Her3 we performed a live cell chemical proteomics experiment using the KiNativ® approach34–36 which demonstrated potent binding to Her3 as well as to Lyn, Her2 and several other Src family kinases (Supplementary Table. 1). Binding to Lyn, Her2 and several other Src family kinases was expected as these kinases were equipotently bound by the non-acrylamide screening hit compound KIN001-11129, suggesting that interaction with these targets is non-covalent. Collectively these results suggest that TX1-85-1 is capable of covalently modifying Cys721 of Her3 in vitro and in cells.


Pharmacological targeting of the pseudokinase Her3.

Xie T, Lim SM, Westover KD, Dodge ME, Ercan D, Ficarro SB, Udayakumar D, Gurbani D, Tae HS, Riddle SM, Sim T, Marto JA, Jänne PA, Crews CM, Gray NS - Nat. Chem. Biol. (2014)

Hit identification and development of the Her3 irreversible inhibitor TX1-85-1(a) Chemical structures of the representative lead compounds identified by FRET binding assay, TX1-85-1 and biotin conjugated TX1-85-1. (b) In vitro kinase FRET binding assay results for top screening hits and TX1-85-1. Purified recombinant Her3 kinase domain (665-1001) was used in conjunction with Lanthascreen® technology (Invitrogen). TX1-85-1 shows the highest potency with an IC50 of 23 nM. Each condition was tested in triplicate. Data represent mean values ± s.d. (c) Docking study of TX1-85-1 with Her3 x-ray crystallography models (PDB ID 3KEX6, 3LMG7). An acrylamide substitution is predicted to form a covalent bond with Cys721 in Her3 via Michael Addition. (d) Her3 pull down experiment with TX1-85-1-biotin. PC9 GR4 cells were pre-treated with TX1-85-1 for 8 hours before lysis or TX1-85-1 was directly added to cell lysate at the indicated concentrations. Streptavidin beads were used to recover compound labeled Her3 which was detected with an anti-Her3 antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232461&req=5

Figure 1: Hit identification and development of the Her3 irreversible inhibitor TX1-85-1(a) Chemical structures of the representative lead compounds identified by FRET binding assay, TX1-85-1 and biotin conjugated TX1-85-1. (b) In vitro kinase FRET binding assay results for top screening hits and TX1-85-1. Purified recombinant Her3 kinase domain (665-1001) was used in conjunction with Lanthascreen® technology (Invitrogen). TX1-85-1 shows the highest potency with an IC50 of 23 nM. Each condition was tested in triplicate. Data represent mean values ± s.d. (c) Docking study of TX1-85-1 with Her3 x-ray crystallography models (PDB ID 3KEX6, 3LMG7). An acrylamide substitution is predicted to form a covalent bond with Cys721 in Her3 via Michael Addition. (d) Her3 pull down experiment with TX1-85-1-biotin. PC9 GR4 cells were pre-treated with TX1-85-1 for 8 hours before lysis or TX1-85-1 was directly added to cell lysate at the indicated concentrations. Streptavidin beads were used to recover compound labeled Her3 which was detected with an anti-Her3 antibody.
Mentions: In order to develop potent and selective covalent Her3 ligands, we first identified ATP-competitive ligands using high-throughput screening and then employed structure-guided drug design to introduce reactive moieties that could form a covalent bond with Cys721. Kinome-wide sequence alignments suggest that Cys721 is uniquely present in Her3 and therefore provides an excellent potential means to achieve selectivity27. A covalent inhibitor provides a number of potential advantages including higher potency, improved selectivity due to Cys721 being highly unusual across the kinome, and the ability to address pharmacological specificity by performing ‘rescue’ experiments with a Cys721 to serine mutation. We developed an ATP-competitive ligand binding assay using the fluorescence resonance energy transfer (FRET) based LanthaScreen™ Eu methodology28 and screened a 1,500 member library consisting of both known and novel ATP-competitive kinase inhibitors. The most potent Her3 binders to emerge from the screen were five previously reported Src-family inhibitors: KIN001-111(A-770041)(1)29, KIN001-051 (2)30,31, (Fig. 1a) dasatinib32, bosutinib33 and KIN001-30 (3), (Supplementary Results, Supplementary Fig. 1a) which possessed IC50s below one hundred nanomolar (Fig. 1b). In order to develop covalent binders of Her3 that exploit the unique Cys721 residue, we docked these compounds to the previously reported 2.80 Å Her3 crystal structures (3KEX6 and 3LMG7, Fig. 1c and Supplementary Fig. 1b). This modeling suggested that introducing a meta-acrylamide from the phenyl substituent of KIN001-111 might afford the correct trajectory for a conjugate addition with Cys721. After the iterative synthesis and assaying of approximately 100 pyrazolopyrimidine acrylamides, we developed the ‘lead’ compound TX1-85-1 (4) which possessed an IC50 of 23 nM in the binding assay (Fig. 1a, b). TX1-85-1 can be viewed as a molecular amalgam of the hinge binding moiety of KIN001-051 and the solubility enhancing tail of KIN001-111, combined with an acrylamide arm to reach Cys721 (Fig. 1c). As expected for a covalent inhibitor, the apparent IC50 decreases upon longer incubation with the protein and reaches a plateau at about 3 hours (Supplementary Fig. 2). Mass spectrometry was used to confirm the covalent addition of TX1-85-1 to recombinant Her3 kinase domain protein and subsequent proteolysis of Her3 with trypsin and MS2 analysis revealed unique modification of Cys721 (Supplementary Fig. 3). We next investigated whether TX1-85-1 could form a covalent bond with Her3 in cells using a cellular competition binding assay. To enable these experiments we synthesized a biotinylated derivative (TX1-85-1-biotin) (5) that still preserves the ability to covalently bind to Her3 (Fig. 1a, Supplementary Fig. 4, IC50 = 50.7 nM). Cells were incubated with TX1-85-1 and then lysates were labeled with TX1-85-1-biotin in order to quantify the amount of Her3 that had escaped labeling by the initial incubation with TX1-85-1. We determined that incubation of PC9 GR4 cells (EGFR E746_A750/T790M) with 5 μM of TX1-85-1 for 8 hours resulted in complete protection of Her3 from subsequent labeling with TX1-85-1-biotin (Fig. 1d). This result suggests that TX1-85-1 (MW = 580Da) can pass through the cell membrane and is capable of complete ‘target engagement’ with Her3 intracellularly. To examine the specificity with which TX1-85-1 modifies Her3 we performed a live cell chemical proteomics experiment using the KiNativ® approach34–36 which demonstrated potent binding to Her3 as well as to Lyn, Her2 and several other Src family kinases (Supplementary Table. 1). Binding to Lyn, Her2 and several other Src family kinases was expected as these kinases were equipotently bound by the non-acrylamide screening hit compound KIN001-11129, suggesting that interaction with these targets is non-covalent. Collectively these results suggest that TX1-85-1 is capable of covalently modifying Cys721 of Her3 in vitro and in cells.

Bottom Line: Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling.Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met.These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [2] Department of Biological Chemistry &Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Her3 (also known as ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be 'undruggable' using ATP-competitive small molecules because it lacks appreciable kinase activity. Here we report what is to our knowledge the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3-dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

Show MeSH
Related in: MedlinePlus