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Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

Moccia M, Liu Q, Guida T, Federico G, Brescia A, Zhao Z, Choi HG, Deng X, Tan L, Wang J, Billaud M, Gray NS, Carlomagno F, Santoro M - PLoS ONE (2015)

Bottom Line: They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes.They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes.The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", Naples, Italy; Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Naples, Italy.

ABSTRACT
Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

No MeSH data available.


Related in: MedlinePlus

Inhibition of RET phosphorylation and signaling by ALW-II-41-27, XMD15-44 and HG-6-63-01 in RET mutant thyroid carcinoma cell lines.Serum-starved human TT (A) and MZ-CRC-1 (B) (MTC), TPC1 (PTC) (C), and non-transformed Nthy-ory-3-1 (NTHY) (D) cell lines were treated for 2 hr with indicated concentrations of ALW-II-41-27, HG-6-63-01 and XMD15-44. 50 μg of total cell lysates were subjected to immunoblotting with αp1062 RET antibodies, phospho-MAPK (αpMAPK) and phospho-SHC (αpSHC) antibodies. The blots were normalized using anti-RET (αRET), anti-MAPK (αMAPK) and anti-SHC (αSHC).
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pone.0128364.g004: Inhibition of RET phosphorylation and signaling by ALW-II-41-27, XMD15-44 and HG-6-63-01 in RET mutant thyroid carcinoma cell lines.Serum-starved human TT (A) and MZ-CRC-1 (B) (MTC), TPC1 (PTC) (C), and non-transformed Nthy-ory-3-1 (NTHY) (D) cell lines were treated for 2 hr with indicated concentrations of ALW-II-41-27, HG-6-63-01 and XMD15-44. 50 μg of total cell lysates were subjected to immunoblotting with αp1062 RET antibodies, phospho-MAPK (αpMAPK) and phospho-SHC (αpSHC) antibodies. The blots were normalized using anti-RET (αRET), anti-MAPK (αMAPK) and anti-SHC (αSHC).

Mentions: We selected TT and MZ-CRC-1 cells, that derive from human MTC harbouring the RET/C634W or RET/M918T mutation, respectively, and TPC1 cells, that derive from human PTC bearing the RET/PTC1 (CCDC6-RET) rearrangement [30–32]. Cells were treated with ALW-II-41-27, XMD15-44 and HG-6-63-01 at 10, 100 and 1,000 nM concentration and RET, SHC and MAPK phosphorylation was analysed. Concentrations as low at 10 nM of all three drugs inhibited RET autophosphorylation as well as SHC and MAPK activation by 20–40% of control (Fig 4A–4C). Importantly, at the used doses, ALW-II-41-27, XMD15-44 and HG-6-63-01 did not exert any detectable effects on SHC and MAPK phosphorylation in non-transformed thyroid follicular Nthy-ori-3-1 cells, used as control (Fig 4D).


Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

Moccia M, Liu Q, Guida T, Federico G, Brescia A, Zhao Z, Choi HG, Deng X, Tan L, Wang J, Billaud M, Gray NS, Carlomagno F, Santoro M - PLoS ONE (2015)

Inhibition of RET phosphorylation and signaling by ALW-II-41-27, XMD15-44 and HG-6-63-01 in RET mutant thyroid carcinoma cell lines.Serum-starved human TT (A) and MZ-CRC-1 (B) (MTC), TPC1 (PTC) (C), and non-transformed Nthy-ory-3-1 (NTHY) (D) cell lines were treated for 2 hr with indicated concentrations of ALW-II-41-27, HG-6-63-01 and XMD15-44. 50 μg of total cell lysates were subjected to immunoblotting with αp1062 RET antibodies, phospho-MAPK (αpMAPK) and phospho-SHC (αpSHC) antibodies. The blots were normalized using anti-RET (αRET), anti-MAPK (αMAPK) and anti-SHC (αSHC).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457528&req=5

pone.0128364.g004: Inhibition of RET phosphorylation and signaling by ALW-II-41-27, XMD15-44 and HG-6-63-01 in RET mutant thyroid carcinoma cell lines.Serum-starved human TT (A) and MZ-CRC-1 (B) (MTC), TPC1 (PTC) (C), and non-transformed Nthy-ory-3-1 (NTHY) (D) cell lines were treated for 2 hr with indicated concentrations of ALW-II-41-27, HG-6-63-01 and XMD15-44. 50 μg of total cell lysates were subjected to immunoblotting with αp1062 RET antibodies, phospho-MAPK (αpMAPK) and phospho-SHC (αpSHC) antibodies. The blots were normalized using anti-RET (αRET), anti-MAPK (αMAPK) and anti-SHC (αSHC).
Mentions: We selected TT and MZ-CRC-1 cells, that derive from human MTC harbouring the RET/C634W or RET/M918T mutation, respectively, and TPC1 cells, that derive from human PTC bearing the RET/PTC1 (CCDC6-RET) rearrangement [30–32]. Cells were treated with ALW-II-41-27, XMD15-44 and HG-6-63-01 at 10, 100 and 1,000 nM concentration and RET, SHC and MAPK phosphorylation was analysed. Concentrations as low at 10 nM of all three drugs inhibited RET autophosphorylation as well as SHC and MAPK activation by 20–40% of control (Fig 4A–4C). Importantly, at the used doses, ALW-II-41-27, XMD15-44 and HG-6-63-01 did not exert any detectable effects on SHC and MAPK phosphorylation in non-transformed thyroid follicular Nthy-ori-3-1 cells, used as control (Fig 4D).

Bottom Line: They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes.They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes.The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", Naples, Italy; Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Naples, Italy.

ABSTRACT
Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

No MeSH data available.


Related in: MedlinePlus