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Onco Targets Ther
2015 Jan 05;8:375-83. doi: 10.2147/OTT.S73690.
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Novel covalent modification of human anaplastic lymphoma kinase (ALK) and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787.
Parker AR
,
Petluru PN
,
Nienaber VL
,
Zhao M
,
Ayala PY
,
Badger J
,
Chie-Leon B
,
Sridhar V
,
Logan C
,
Kochat H
,
Hausheer FH
.
Abstract
BNP7787 (Tavocept, disodium 2,2'-dithio-bis-ethanesulfonate) is a novel, investigational, water-soluble disulfide that is well-tolerated and nontoxic. In separate randomized multicenter Phase II and Phase III clinical trials in non-small-cell lung cancer (NSCLC) patients, treatment with BNP7787 in combination with standard chemotherapy resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma of the lung in the first-line treatment setting. We hypothesized that BNP7787 might interact with and modify human anaplastic lymphoma kinase (ALK). At least seven different variants of ALK fusions with the gene encoding the echinoderm microtubule-associated protein-like 4 (EML4) are known to occur in NSCLC. EML4-ALK fusions are thought to account for approximately 3% of NSCLC cases. Herein, we report the covalent modification of the kinase domain of human ALK by a BNP7787-derived mesna moiety and the functional consequences of this modification in ALK assays evaluating kinase activity. The kinase domain of the ALK protein crystallizes as a monomer, and BNP7787-derived mesna-cysteine adducts were observed at Cys 1235 and Cys 1156. The BNP7787-derived mesna adduct at Cys 1156 is located in close proximity to the active site and results in substantial disorder of the P-loop and activation loop (A-loop). Comparison with the P-loop of apo-ALK suggests that the BNP7787-derived mesna adduct at Cys 1156 interferes with the positioning of Phe 1127 into a small pocket now occupied by mesna, resulting in a destabilization of the loop's binding orientation. Additionally, in vitro kinase activity assays indicate that BNP7787 inhibits ALK catalytic activity and potentiates the activity of the ALK-targeted drug crizotinib.
Figure 1. Ribbon diagrams of ALK with covalently bound BNP7787-derived mesna adducts (PDB IB code 4TT7).Notes: (A) BNP7787-derived mesna adducts observed at Cys 1235 and Cys 1156. (B) Overlay of region of apo-ALK (pink) with BNP7787-xenobiotically modified ALK (lavender) that has a Cys 1156-mesna adduct. The BNP7787-derived mesna adduct occupies the same pocket as Phe 1127 of the P-loop.Abbreviation: ALK, anaplastic lymphoma kinase.
Figure 2. Electron density and binding site maps showing BNP7787-derived mesna-cysteine adducts on ALK.Notes: 2Fo-Fc electron density map contoured at 1 sigma showing BNP7787-derived mesna-cysteine adducts on ALK at (A) Cys 1235 and (B) Cys 1156. (C) Binding site of the BNP7787-derived mesna-cysteine adduct at Cys 1235. There are no obvious interactions of the BNP7787-derived mesna with the protein other than the covalent bond with Cys 1235. (D) Molecular surface of ALK with the BNP7787-derived mesna at Cys 1156 removed to show the interaction of the adduct with the protein. A water-mediated hydrogen bond is present between the BNP7787-derived mesna sulfonate and Asp 1160 carbonyl.Abbreviation: ALK, anaplastic lymphoma kinase.
Figure 3. Concentration-dependent inhibition of ALK activity by BNP7787 in presence of ATP. Notes: (A) 100 μM ATP; (B) 500 μM ATP.Abbreviation: ALK, anaplastic lymphoma kinase.
Figure 4. BNP7787 stimulates crizotinib-mediated inhibition of ALK activity under varying ATP and crizotinib concentration combinations.Notes: (A) 15 nM (IC25) crizotinib and 100 μM ATP; (B) 30 nM (IC50) crizotinib and 100 μM ATP; (C) 30 nM (IC25) crizotinib and 500 μM ATP; and (D) 65 nM (IC50) crizotinib and 500 μM ATP. Analysis of variance P-values are indicated above bracketed experimental conditions.Abbreviations: ALK, anaplastic lymphoma kinase; IC25, one-fourth maximal inhibitory concentration; IC50, half maximal inhibitory concentration.
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