Novel covalent modification of human anaplastic lymphoma kinase (ALK) and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787

Figures & data

Table 1 Crystal characteristics and data collection statistics

Unit cell (Å, °)	51.535 57.157 104.216 90.000 90.000 90.000
Space group	P212121
Resolution range (Å)	46.20–2.10 (2.21–2.10)
Number of observations	102,983
Number of unique reflections	18,384
Redundancy	5.6 (5.7)
Completeness (%)	98.8 (97.5)
Mean I/sigma(I)	10.1 (2.8)
Rmerge	0.135 (0.621)

Note: Outer shell statistics are in parenthesis.

Table 2 Crystallographic data and refinement statistics

Resolution range (Å)	46.196–2.100
Number of reflections	18,340 (17,402 working set, 938 test set)
Number of protein chains	1 (chain A)
Number of protein residues	293
Number of ligands	5
Number of waters	159
Number of atoms	2,452
Mean B-factor	21.256
Rwork	0.1927
Rfree	0.2448
RMSD bond lengths (Å)	0.010
RMSD bond angles (°)	1.196
Number of disallowed φψ angles	1

Abbreviation: RMSD, root-mean-square deviation.

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.