J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
3033
13. Apoptosis was assessed using Promega’s Caspase-GloÒ 3/7 Assay Kit
(Catalog#G8092, Madison, WI) following the manufacturer’s directions.
Protocol: RS4;11 cells were plated in NUNC white opaque 96-well plates
(Catalog#353296, Rochester, NY) at 10,000 cells per well in Sigma’s RPMI
Media (Catalog #R7509, St. Louis, Mo) containing 10% FBS (Thermofisher
Catalog#SH30396 Waltham, MA). After 16 h, cells were treated in duplicate
with compound and incubated at 37 °C for 6 h prior to caspase detection.
DMSO was used as minimum caspase activity control, while the addition of
ABT-263 was used as a measure of maximum caspase activity. An equal
volume of Caspase-Glo 3/7 reagent was added to each well and incubated for
1 h prior to reading on a Tecan Ultra luminometer (Tecan, Durham, NC) to
quantitate luminescence. EC50’s are the average of at least two experiments.
ligand–protein interaction energy for 13B is energetically more favorable
(À1053.92 kJ/mol) compared to 14B (À1002.79 kJ/mol). A similar trend was
observed for the electrostatic component of the interaction energy with 13B
being more stable (À759.05 kJ/mol) than 14B (À707.05 kJ/mol). The van der
Waals interaction energy was more comparable for 13B (À294.87 kJ/mol) and
14B (À295.74 kJ/mol).
19. ABT-737 analogs with these heteroatom modifications have been reported. In:
Bruncko, M.; Ding, H.; Elmore, S.; Kunzer, A.; Lynch, C. L.; McClellan, W.; Park,
C.-M.; Petros, A.; Song, X.; Wang, X.; Tu, N.; Wendt, M.; Shoemaker, A.; Mitten,
M. U.S. 0,072,860 A1, 2007.
20. For an example of dimethylaminoethoxy side chains applied to selective Bcl-2
inhibitors, see: Bruncko, M.; Ding, H.; Doherty, G. A.; Elmore, S. W.; Hasvold, L.;
Hexamer, L.; Kunzer, A. R.; Mantei, R. A.; McClellan, W. J.; Park, C. H.; Park, C.-
M.; Petros, A. M.; Song, X.; Souers, A. J.; Sullivan, G. M.; Tao, Z.-F.; Wang, G. T.;
Wang, L.; Wang, X.; Wendt, M. D.; Hansen, T. M. U.S. Pat. Appl. Publ. 0,298,321
A1, 2010.
16. For nitro variants of 4,4-substituted piperidines, see Ref. 4.
17. These modifications preserved the similarities and differences of these two
ligands, allowing rigid protein docking on
a
publicly available protein
21. A cell viability assay was used to determine the relative inhibition of BCL-2 and
BCL-xL test compounds. FDCP-1 cells (parental line from DSMZ) were
engineered by retroviral infection to over-express either human BCL-2 or
human BCL-xL. Both these cell lines, as well as the parental, were maintained at
37 °C in RPMI/10% FBS supplemented with 5 ng/mL IL-3. Prior to compound
treatment, cells were first washed in No IL-3 media (otherwise identical to
media above), then incubated for 48 h w/o IL3, during which time the parental
cells died out. The surviving FDCP-1 BCL-2 and FDCP-1 BCL-xL cells were then
counted, resuspended in fresh No IL-3 media, and plated at approximately 5–
7 K cells/well in 384 w microplates containing the test compounds, including
the DMSO control (untreated wells), in triplicate. The cells were then incubated
for an additional 24 h at 37 °C. After this incubation, a matching volume of Cell
Titer Glo reagent was added to each well, the plate shaken at RT for at least
15 min, and the luminescence read using a Tecan Ultra 384 (Magellan). IC50’s
structure. Method: The crystal structure of a quinazoline ligand bound to Bcl-
xL (PDB code-3QKD) was chosen for docking analysis due to the similarity of
the cognate ligand with 13 and 14. The protein complex was downloaded from
the protein data bank (www.rcsb.org) and prepared for docking using the
protein preparation workflow in Maestro (Schrodinger LLC, Portland, OR).
Ligands for docking were also prepared in Maestro using the OPLS2005/GBSA
setting for ligand minimization. Pose validation studies with the cognate
ligand were carried out in both Glide (Schrodinger LLC, Portland, OR) and Gold
(CCDC, Cambridge, UK). Default settings failed to produce desirable results and
multiple constraints were evaluated. Satisfactory results were obtained in Gold
using four hydrophobic constraints defined based on the pose of the cognate
ligand. The hydrophobic constraints were 2 A spheres defined around the
centroids of the (a) phenyl ring connected to the thioether linkage, (b) phenyl
ring with the nitro substitution, (c) piperidine ring and (d) the terminal phenyl
ring of the biphenyl moiety. The automatic GA settings were used with 200%
search efficiency and the chemscore scoring function was utilized for docking.
Energetic analysis was performed using the Embrace module of Macromodel
(Schrodinger LLC, Portland, OR) in the interaction energy mode using the
OPLS2005 potential.
were determined and reported as
a percentage of surviving cells in the
untreated control wells. Matching +IL3 controls were treated in the same
fashion, but in IL3-replete media, to verify that the compounds had minimal to
no effect under those conditions.
25. Close, J.; Grimm, J.; Heidebrecht, Jr., R. W.; Kattar, S.; Miller, T. A.; Otte, K. M.;
Peterson, S.; Siliphaivanh, P.; Tempest, P.; Wilson, K. J.; Witter, D. J. WO
010,985 A2, 2008.
18. (a) The Bcl-2 residues corresponding to Bcl-xL residues Phe97 and Ala104 are
Phe101 and Asp108, respectively. Based on the observation of available Bcl-2
structures, the Asp108 residue lies in between two helices, a region which is
highly plastic, reorganizing itself in response to ligand binding. In the apo form
(PDB code-1GJH) the residue points into the pocket but shows side chain (PDB
code-1YSW) and backbone movements (PDB code-2O2F)] away from the
ligand in response to ligand binding. The acid residue in Bcl-2 may be more
rigid and therefore causes greater energetic destabilization due to lone pair
clashes between the acid and sulfoxide with the S-sulfoxide than the
R-sulfoxide, leading to the >10 fold difference in binding affinity between
Bcl-2 and Bcl-xL.
(b) Energetic analysis of the protein–ligand complex showed that the