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binding to Bcl-2 than on binding to Mcl-1. These variations are
consistent with experimental observations of the binding of
the Bim L62A/F69A double mutant to Mcl-1 and Bcl-2.[23]
The minimal scaffold 13a (mimicking only D67 in Bim)
bound to Mcl-1 and Bcl-2 with Ki values of 0.454 and 1.350 mm,
respectively (6 and 24 times less potent than 3d, respectively).
This indicates that D67 mimicry plays a more important role in
binding to Mcl-1 than in binding to Bcl-2; affinity for Bcl-2 is
more dependent on the mimicry of hydrophobic groups. This
is also consistent with the results of alanine scanning experi-
ments: only the D67A mutant had a significant effect on Mcl-
1 binding, whereas ABT-737 (mimicking L62 and F69 but not
D67) did not bind Mcl-1.[23,25] The more minimal scaffolds syn-
thesized in this study (groups deleted individually or in combi-
nation) could mimic hotspot mutations. The data also confirm
the one-to-one correspondence between the R1, R2, and R3
groups on our scaffold and the i, i+3, and i+7 residues on
Bim.
Acknowledgements
We thank Prof. Paramjit Arora (New York University) for insightful
discussion. The work was supported by the National Natural Sci-
ence Foundation of China (21372036, 81272876, and 81273436).
Keywords: acridines
·
helical structures
·
Mcl-1
·
peptidomimetics · two-face
[3] H. Yin, G.-i. Lee, K. A. Sedey, J. M. Rodriguez, H.-G. Wang, S. M. Sebti,
[5] A. Shaginian, L. R. Whitby, S. Hong, I. Hwang, B. Farooqi, M. Searcey, J.
To further identify the direct binding ability of these a-helix
mimetics to Mcl-1, isothermal titration calorimetry (ITC) was
performed (Figure S3A–D). The most potent compound, 3d,
exhibited a Kd value of 0.107 mm with Mcl-1. The corresponding
Kd values for 11 b, 12c, and 13b were 0.342, 0.507, and
0.781 mm, respectively. Consistent with this, the Ki values
(Table 1) show that the affinity of 3d was three to seven times
improved over that of 11 b, 12c, and 13b.
[10] E. F. Lee, P. E. Czabotar, M. F. van Delft, E. M. Michalak, M. J. Boyle, S. N.
Willis, H. Puthalakath, P. Bouillet, P. M. Colman, D. C. S. Huang, W. D. Fair-
Subsequently, we tested the affinities of 3d (maximal) and
13a (minimal) with an R263A mutant of Mcl-1. ITC showed
both compounds lost most of the affinity: 3.06 mm for 3d
(nearly 30 times weaker than with wild-type Mcl-1, Figure S4),
and no appreciable binding for 13a at up to 100 mm. It further
confirmed that mimicking D67 to form a hydrogen bond with
R263 plays the most important role in binding to Mcl-1. This
explains why the naked scaffold 13a exhibits relatively good
affinity (sub-micromolar), because of capture of this very “hot”
residue. Also, the significant decrease in affinity with the one-
residue mutation rules out the possibility that the molecules
developed in this study are promiscuous binders that might
bind to many other proteins.
[11] a) M. Peters, M. Trobe, H. Tan, R. Kleineweischede, R. Breinbauer, Chem.
[13] T. L. Su, B. Kohler, T. C. Chou, M. W. Chun, K. A. Watanabe, J. Med. Chem.
[15] H. T. Nguyen Thi, C.-Y. Lee, K. Teruya, W.-Y. Ong, K. Doh-ura, M.-L. Go,
[18] a) R. d. R. Reis, E. C. Azevedo, M. C. B. V. de Souza, V. F. Ferreira, R. C.
Montenegro, A. J. Araffljo, C. Pessoa, L. V. Costa-Lotufo, M. O. de Moraes,
J. D. .B. M. Filho, A. M. T. de Souza, N. C. de Carvalho, H. C. Castro, C. R.
b) Y. F. Suen, L. Robins, B. Yang, A. Verkman, M. H. Nantz, M. J. Kurth,
[19] a) I. P. Hçglund, S. Silver, M. T. Engstrçm, H. Salo, A. Tauber, H.-K. Kyyrç-
nen, P. Saarenketo, A.-M. HoffrØn, K. Kokko, K. Pohjanoksa, J. Sallinen, J.-
b) G. Hiltensperger, N. G. Jones, S. Niedermeier, A. Stich, M. Kaiser, J.
Jung, S. Puhl, A. Damme, H. Braunschweig, L. Meinel, M. Engstler, U.
The effect of 3d on plasmid DNA pBR322 was measured, be-
cause some acridines are well known DNA intercalators (Fig-
ure S5). No significant differences in superhelical content or
band shape were found (agarose gel electrophoresis) in the
presence or absence of 3d. This implies that 3d does not in-
teract with DNA in vitro. Therefore, it seemed very likely these
a-helix mimetics are selective Bcl-2/Mcl-1 protein inhibitors.
In conclusion, we designed a cross-acridine scaffold that
projects functional groups with spatial and angular geometries
that accurately mimic the i, i+3, i+5, and i+7 side chains on
a two-turn, two-face section of an a-helix. In addition, hotspot
mutations were mimicked by different parts of the scaffold
and combinations of side chains. The affinities of these com-
pounds toward Bcl-2 and Mcl-1 were determined by FPA, and
the binding mode of the most potent compound, 3d, to Mcl-
[21] a) C. L. Day, L. Chen, S. J. Richardson, P. J. Harrison, D. C. S. Huang, M. G.
Hinds, J. Biol. Chem. 2005, 280, 4738–4744; b) Q. Liu, T. Moldoveanu, T.
Sprules, E. Matta-Camacho, N. Mansur-Azzam, K. Gehring, J. Biol. Chem.
Fletcher, M. G. Hinds, P. M. Colman, C. L. Day, J. M. Adams, D. C. S.
[22] P. E. Czabotar, E. F. Lee, M. F. van Delft, C. L. Day, B. J. Smith, D. C. S.
1
1 was confirmed by H,15N HSQC NMR.
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ChemBioChem 2014, 15, 1280 – 1285 1284