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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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TX1-85-1 is insufficient to inactivate the Her3/PI3K/AKT pathway explaining lack of growth inhibition for Her3 dependent cells(a) EC50s of TX1-85-1 for several Her3 dependent cell lines. Anti-proliferative activity was achieved by TX1-85-1 at micromolar concentrations. Each condition was tested in triplicate. Data represent mean values ± s.d. (b) Her3/PI3K/AKT signaling analysis on the PC9 GR4 cell line. Western blotting for proteins in the Her3 signaling axis showed lack of specific inhibition in the low micromolar range.
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Figure 2: TX1-85-1 is insufficient to inactivate the Her3/PI3K/AKT pathway explaining lack of growth inhibition for Her3 dependent cells(a) EC50s of TX1-85-1 for several Her3 dependent cell lines. Anti-proliferative activity was achieved by TX1-85-1 at micromolar concentrations. Each condition was tested in triplicate. Data represent mean values ± s.d. (b) Her3/PI3K/AKT signaling analysis on the PC9 GR4 cell line. Western blotting for proteins in the Her3 signaling axis showed lack of specific inhibition in the low micromolar range.

Mentions: We next evaluated the ability of TX1-85-1 to inhibit Her3-dependent signaling and growth. We utilized two established lung cancer cell lines, PC9 GR4 (EGFR E746_A750/T790M), HCC827 GR6 (EGFR E746_A750/MET amplification)18,37 and an ovarian cancer cell line, Ovcar8 that we reconfirmed to be ‘addicted’ to Her3 using siRNA mediated depletion of Her3 (Supplementary Fig. 5). TX1-85-1 possessed an anti-proliferation EC50 of greater than or equal to approximately 10 μM for all three cell lines (Fig. 2a). At a concentration of TX1-85-1 sufficient to fully label Her3 in cells (5 μM) there was no growth inhibition of PC9 GR4 cells and no inhibition of the phosphorylation of Akt, an important downstream effector of Her3 (Fig. 2b). These results suggest that despite successful target engagement of Her3 in cells by TX1-85-1, the compound is not capable of inhibiting Her3-dependent function under the conditions investigated.


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)

TX1-85-1 is insufficient to inactivate the Her3/PI3K/AKT pathway explaining lack of growth inhibition for Her3 dependent cells(a) EC50s of TX1-85-1 for several Her3 dependent cell lines. Anti-proliferative activity was achieved by TX1-85-1 at micromolar concentrations. Each condition was tested in triplicate. Data represent mean values ± s.d. (b) Her3/PI3K/AKT signaling analysis on the PC9 GR4 cell line. Western blotting for proteins in the Her3 signaling axis showed lack of specific inhibition in the low micromolar range.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: TX1-85-1 is insufficient to inactivate the Her3/PI3K/AKT pathway explaining lack of growth inhibition for Her3 dependent cells(a) EC50s of TX1-85-1 for several Her3 dependent cell lines. Anti-proliferative activity was achieved by TX1-85-1 at micromolar concentrations. Each condition was tested in triplicate. Data represent mean values ± s.d. (b) Her3/PI3K/AKT signaling analysis on the PC9 GR4 cell line. Western blotting for proteins in the Her3 signaling axis showed lack of specific inhibition in the low micromolar range.
Mentions: We next evaluated the ability of TX1-85-1 to inhibit Her3-dependent signaling and growth. We utilized two established lung cancer cell lines, PC9 GR4 (EGFR E746_A750/T790M), HCC827 GR6 (EGFR E746_A750/MET amplification)18,37 and an ovarian cancer cell line, Ovcar8 that we reconfirmed to be ‘addicted’ to Her3 using siRNA mediated depletion of Her3 (Supplementary Fig. 5). TX1-85-1 possessed an anti-proliferation EC50 of greater than or equal to approximately 10 μM for all three cell lines (Fig. 2a). At a concentration of TX1-85-1 sufficient to fully label Her3 in cells (5 μM) there was no growth inhibition of PC9 GR4 cells and no inhibition of the phosphorylation of Akt, an important downstream effector of Her3 (Fig. 2b). These results suggest that despite successful target engagement of Her3 in cells by TX1-85-1, the compound is not capable of inhibiting Her3-dependent function under the conditions investigated.

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