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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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Adamantane conjugated compounds induce Her3 degradation(a) Chemical structure of representative adamantane tagged compound TX2-121-1, negative control without adamantane and negative control lacking a nucleophilic warhead. (b) Her3/PI3K/AKT signaling analysis. Western blots were performed on lysates from serum starved PC9 GR4 cells treated with adamantane tagged compounds followed by 30 min treatment of 100 ng/ml NRG. Her3 was induced by NRG (lane 2) but degraded by 2 μM TX2-121-1 (lane 7). This was accompanied by decreases in p-Akt and p-Erk. (c) Anti-proliferative activity of adamantane conjugated compounds. TX2-121-1 which includes the reactive acrylamide group is about 7 fold more potent against PC9 GR4 cell line than negative controls which contain an unreactive propionamide group. The EC50 was not achieved for a compound lacking the adamantane group. Each condition was tested in triplicate. Data represent mean values ± s.d. In summary these results suggest that both adamantane and the electrophilic warhead contribute to potency of the compound.
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Figure 3: Adamantane conjugated compounds induce Her3 degradation(a) Chemical structure of representative adamantane tagged compound TX2-121-1, negative control without adamantane and negative control lacking a nucleophilic warhead. (b) Her3/PI3K/AKT signaling analysis. Western blots were performed on lysates from serum starved PC9 GR4 cells treated with adamantane tagged compounds followed by 30 min treatment of 100 ng/ml NRG. Her3 was induced by NRG (lane 2) but degraded by 2 μM TX2-121-1 (lane 7). This was accompanied by decreases in p-Akt and p-Erk. (c) Anti-proliferative activity of adamantane conjugated compounds. TX2-121-1 which includes the reactive acrylamide group is about 7 fold more potent against PC9 GR4 cell line than negative controls which contain an unreactive propionamide group. The EC50 was not achieved for a compound lacking the adamantane group. Each condition was tested in triplicate. Data represent mean values ± s.d. In summary these results suggest that both adamantane and the electrophilic warhead contribute to potency of the compound.

Mentions: We next examined whether adamantane-derivatized Her3-binders could induce degradation of Her3 and thereby inhibit downstream signaling. As part of this analysis we also evaluated the importance of the electrophilic warhead by preparing TX2-135-2 (7), where the reactive acrylamide moiety is replaced with the non-reactive propyl amide (Fig. 3a). Treatment of starved PC9 GR4 cells with TX2-121-1 at concentrations of 0.5 and 2 μM for 12 hours resulted in partial degradation of Her3 and inhibition of phosphorylation of downstream Her3 effectors: Erk and Akt, after stimulation with Neuregulin (NRG) (Fig. 3b). The non-covalent analog TX2-135-2 and compounds lacking the adamantane group (e.g. TX2-120-1 (8) and TX1-85-1) were less effective at inducing Her3 degradation or blocking signaling. Growth assays also demonstrated that the acrylamide group substituted compounds possess cell growth EC50 about 7-fold lower than the propionamide containing compounds which are incapable of covalent bond formation (Fig. 3c). Analysis of the structure-activity relationships for this compound series demonstrates that degradation is dependent on the type and the length of linker.


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)

Adamantane conjugated compounds induce Her3 degradation(a) Chemical structure of representative adamantane tagged compound TX2-121-1, negative control without adamantane and negative control lacking a nucleophilic warhead. (b) Her3/PI3K/AKT signaling analysis. Western blots were performed on lysates from serum starved PC9 GR4 cells treated with adamantane tagged compounds followed by 30 min treatment of 100 ng/ml NRG. Her3 was induced by NRG (lane 2) but degraded by 2 μM TX2-121-1 (lane 7). This was accompanied by decreases in p-Akt and p-Erk. (c) Anti-proliferative activity of adamantane conjugated compounds. TX2-121-1 which includes the reactive acrylamide group is about 7 fold more potent against PC9 GR4 cell line than negative controls which contain an unreactive propionamide group. The EC50 was not achieved for a compound lacking the adamantane group. Each condition was tested in triplicate. Data represent mean values ± s.d. In summary these results suggest that both adamantane and the electrophilic warhead contribute to potency of the compound.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232461&req=5

Figure 3: Adamantane conjugated compounds induce Her3 degradation(a) Chemical structure of representative adamantane tagged compound TX2-121-1, negative control without adamantane and negative control lacking a nucleophilic warhead. (b) Her3/PI3K/AKT signaling analysis. Western blots were performed on lysates from serum starved PC9 GR4 cells treated with adamantane tagged compounds followed by 30 min treatment of 100 ng/ml NRG. Her3 was induced by NRG (lane 2) but degraded by 2 μM TX2-121-1 (lane 7). This was accompanied by decreases in p-Akt and p-Erk. (c) Anti-proliferative activity of adamantane conjugated compounds. TX2-121-1 which includes the reactive acrylamide group is about 7 fold more potent against PC9 GR4 cell line than negative controls which contain an unreactive propionamide group. The EC50 was not achieved for a compound lacking the adamantane group. Each condition was tested in triplicate. Data represent mean values ± s.d. In summary these results suggest that both adamantane and the electrophilic warhead contribute to potency of the compound.
Mentions: We next examined whether adamantane-derivatized Her3-binders could induce degradation of Her3 and thereby inhibit downstream signaling. As part of this analysis we also evaluated the importance of the electrophilic warhead by preparing TX2-135-2 (7), where the reactive acrylamide moiety is replaced with the non-reactive propyl amide (Fig. 3a). Treatment of starved PC9 GR4 cells with TX2-121-1 at concentrations of 0.5 and 2 μM for 12 hours resulted in partial degradation of Her3 and inhibition of phosphorylation of downstream Her3 effectors: Erk and Akt, after stimulation with Neuregulin (NRG) (Fig. 3b). The non-covalent analog TX2-135-2 and compounds lacking the adamantane group (e.g. TX2-120-1 (8) and TX1-85-1) were less effective at inducing Her3 degradation or blocking signaling. Growth assays also demonstrated that the acrylamide group substituted compounds possess cell growth EC50 about 7-fold lower than the propionamide containing compounds which are incapable of covalent bond formation (Fig. 3c). Analysis of the structure-activity relationships for this compound series demonstrates that degradation is dependent on the type and the length of linker.

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