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					 1   JPET           Manuscript #

 2

 3   Journal:       The Journal of Pharmacology and Experimental Therapeutics

 4   Title:         Target Binding Properties and Cellular Activity of Afatinib (BIBW 2992), an
 5                  Irreversible ErbB Family Blocker

 6   Authors:       Flavio Solca, Goeran Dahl, Andreas Zoephel, Gerd Bader, Michael Sanderson,
 7                  Christian Klein, Oliver Kraemer, Frank Himmelsbach, Eric Haaksma, Guenther R.
 8                  Adolf

 9




10
11   Supplementary Figure 3S1. X-ray structure of BIBW 2992 covalently bound to EGFR. The electron
12   density (a 2FoFc-map contoured ar 1 σ level) depicted as a blue mesh around the ligand and Cys797
13   clearly indicates the presence of the covalent bond.
           Statistics of data collection and processing (T790M)              Refinement statistics1
           Ligand                                           Afatinib         Ligand                                                   Afatinib
           X-ray source                                     PX (SLS1)        Resolution [Å]                                          82.48-3.17
           Wavelength [Å]                                     1.000          Number of reflections (working / test)                 11432 / 472
           Detector                                       PILATUS 6M         Rcryst [%]                                                 24.4
           Temperature [K]                                     100           Rfree2 [%]                                                 32.3
           Space group                                     P 21 21 21        Total number of atoms:
           Cell: a; b; c [Å]                          51.23; 89.43; 164.68      Protein                                                    4830
                   ; ;  [ ]                           90.0; 90.0; 90.0       Water                                                       38
           Resolution [Å] 1                              3.17 (3.43-3.17)       Ligand                                                      34
           Unique reflections 1                           11931 (2384)       Deviation from ideal geometry: 3
           Multiplicity 1                                    4.3 (4.1)          Bond lengths [Å]                                       0.008
           Completeness [%] 1                               89.9 (88.8)         Bond angles [ ]                                         1.06
           Rsym[%] 1                                        13.0 (35.5)         Bonded B s 4 [Å2]                                       1.6
           Rmeas [%] 1, 2                                   16.6 (45.5)      Ramachandran Plot: 5
           mean(I)/sigma 1,2                                10.2 (3.9)          Most favoured regions                                      83.6
                                                                                Additional allowed regions                                 15.3
           1 Brackets   correspond to the highest resolution bin
           2 Calculated from independent reflections
                                                                                Generously allowed regions                                 0.8
                                                                                Disallowed regions                                         0.4


                                                                             1 Values asdefined in REFMAC5 (CCP4), without sigma cut-off
                                                                             2 Test-setcontains 5 % of measured reflections
                                                                             3 Root mean square deviations from geometric target values
                                                                             4 Calculated with MOLEMAN
                                                                             5 Calculated with PROCHECK




14
15   Supplementary Figure 3S2. Data collection and refinement statistics of the EGFR T790M mutant in
16   complex with BIBW 2992.

17
18

19

20   Supplementary Figure 4S1. Comparison of fragmentation mass spectra (MS/MS) demonstrates
21   covalent binding of afatinib to HER2. (A) Chemically synthesized HER2 peptide amino acids 801-
22   808, covalently modified with afatinib and (B) the corresponding peptide, isolated from the HER2
23   kinase domain after incubation with afatinib were resolved by liquid chromatography/tandem mass
24   spectrometry. The peptide sequence was derived from information of b- and y-ion fragmentation
25   series, isotopic distribution patterns of fragments as well as the mass of the parent ion. Identified ions
26   of the b- and y-ion fragmentation series have been annotated.
27

28

29   Supplementary Figure 4S2. Comparison of fragmentation mass spectra (MS/MS) demonstrates
30   covalent binding of afatinib to ErbB-4. (A) Chemically synthesized HER2 peptide amino acids 795-
31   806, covalently modified with afatinib and (B) the corresponding peptide, isolated from the ErbB-4
32   kinase domain after incubation with afatinib were resolved by liquid chromatography/tandem mass
33   spectrometry. The peptide sequence was derived from information of b- and y-ion fragmentation
34   series, isotopic distribution patterns of fragments as well as the mass of the parent ion. Identified ions
35   of the b- and y-ion fragmentation series have been annotated.

				
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posted:11/6/2012
language:English
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