Development of a potent Bcl-xL antagonist based on α-helix mimicry

Article abstract of DOI:10.1021/ja026861k

The rational design of low-molecular weight ligands that disrupt protein-protein interactions is still a challenging goal in medicinal chemistry. Our approach to this problem involves the design of molecular scaffolds that mimic the surface functionality

Full text of DOI:10.1021/ja026861k

Published on Web 09/12/2002
Development of a Potent Bcl-xL Antagonist Based on r-Helix Mimicry
Olaf Kutzki, Hyung Soon Park, Justin T. Ernst, Brendan P. Orner, Hang Yin, and
Andrew D. Hamilton*
Department of Chemistry, Yale UniVersity, P.O. Box 208107, New HaVen, Connecticut 06520-8107
Received May 10, 2002
The design of low-molecular weight ligands that disrupt protein-
protein interactions is still a challenging goal in medicinal chemistry
due in part to the large interfacial surface areas invovled.1
Conventional methods for identifying inhibitors of protein-protein
interactions from chemical libraries have had limited success.
Therefore, new strategies for rational design of such inhibitors are
necessary.2
Our approach to this problem involves the design of molecular
scaffolds that mimic the surface functionality projected along one
face of an R-helix. We have synthesized several designed proteo-
mimetics based on a terphenyl scaffold, which in a staggered
conformation closely reproduce the projection of functionality on
the surface of an R-helix (Figure 1A).3 To further extend this
strategy we have tried to mimic parts of the R-helical, pro-apoptotic
Bak- and Bad-proteins, which interact by heterodimerization with
the anti-apoptotic protein Bcl-xL.4 Bcl-xL is overexpressed in many
types of cancer and protects transformed cells from cell death,
leading to uncontrolled cell growth even if apoptotic signals
generated by chemo- or radiotherapy are present.5 A designed Bak/
Bad-mimetic could interact with the anti-apoptotic Bcl-xL protein
and thus enable the apoptotic cascade leading to cell death. We
herein report the identification of a potent inhibitor of the Bak/
Bcl-xL interaction based on R-helix mimicry of a part of the pro-
apoptotic Bak protein.
We based our design on the crystal and solution structures of
Bak/Bcl-xL complex,6,7 which show the helical Bak-peptide binding
into a hydrophobic cleft formed by the BH1-BH3 domains of Bcl-
xL (Figure 2A). From alanine scans of the Bak-peptide it is clear
that four hydrophobic residues (Val74, Leu78, Ile81, Ile85) along one
edge of the helix are involved in binding.8 In addition, Asp83 forms
an ion pair with a lysine residue of Bcl-xL. A related 26-mer peptide
derived from the Bad-protein binds better to Bcl-xL,9 exploiting
larger hydrophobic residues (Tyr, Phe) to induce a slight structural
change in the binding region of Bcl-xL. Furthermore, it has been
shown that the R-helix propensity of these peptides is decisive for
strong binding to Bcl-xL.9a
On the basis of these structural requirements we designed a series
of terphenyl molecules (1, 3-5) containing alkyl or aryl substituents
on the three ortho positions (to mimic the key hydrophobic
substituents on the helical exterior of Bak or Bad) and carboxylic
acid substituents on either end (to mimic the additional ion pair)
(Figure 1B).
Figure 1. (A) i, i + 3, and i + 7 substituents of an R-helix. (B) Compounds
tested in the fluorescence polarization assay.
mer Bak-peptide.8 Displacement of this probe through competitive
binding of the terphenyl into the hydrophobic cleft of Bcl-xL would
lead to a decrease in its fluorescence polarization which in turn
could be related to the known affinity of the 16-mer Bak/Bcl-xL
complex. This assay showed (Figure 3) that the terphenyl molecule
with two carboxylic acids and the isobutyl, 1-naphthalenemethylene,
isobutyl sequence (4) shows the strongest binding to Bcl-xL with a
KD value of 114 nM. The less hydrophobic terphenyls (1 and 3)
show lower affinity (KD ) 2.09 and 1.89 µM, respectively),
emphasizing the importance of hydrophobic interactions for binding
to the recognition cleft in Bcl-xL.10 Scrambling the position of the
substituents, as in 5, leads to a significant loss in binding affinity
(KD ) 2.70 µM), suggesting an effective shape complementarity
for 4, as in the natural peptide. The importance of the hydrophobic
groups is further confirmed by the weak binding of an analogue of
4 lacking the naphthyl and two isobutyl substituents (73b, KD
)
27.4 µM). Finally, the role of the carboxylate groups was probed
by partial removal (as in 63a, KD ) 6.8 µM) or conversion to
positively charged groups (as in 2,3b KD ) 13.7 µM), leading in
both cases to significant loss of activity.
Docking studies with the AutoDock program using the Bcl-xL
conformation found in the complex with Bak showed an optimal
location for 4 in the same hydrophobic cleft as the helical peptide
but in a slightly different orientation (Figure 2B). Further support
for this binding site came from HSQC NMR experiments with 15N-
labeled Bcl-xL protein. Addition of 4 (1.2 equiv, 460 µM) led to
shifts in a number of residues on the surface of Bcl-xL (F146, L130,
I140, R139, W137, E193, S203, Y195, A104) near the predicted
binding site. These affected residues all lie in the shallow cleft on
the protein into which the Bak helix binds. Overlay of 4 and the
Bak peptide within the binding pocket suggests that the terphenyl
is indeed mimicking the cylindrical shape of the helix with the
A modular synthetic route to terphenyl derivatives 1-5 was
developed involving sequential Suzuki coupling of the correspond-
ing methoxyphenylboronate and phenyltriflate derivatives.
The binding affinity of these molecules for Bcl-xL was assessed
by a fluorescence polarization assay using fluorescein-labeled 16-
* To whom correspondence should be addressed. E-mail: andrew.hamilton@
yale.edu.
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10.1021/ja026861k CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (A) Bak-BH3-peptide/Bcl-xL complex. (B) Docking results and residues shifted in the NMR experiment of 4 and Bcl-xL. (C) Overlay of peptide
and postulated binding location for 4.
References
(1) (a) Cochran, A. G. Chem. Biol. 2000, 7, R85-R94. (b) Cochran, A. G.
Curr. Opin. Chem. Biol. 2001, 5, 654-659.
(2) (a) Andrews, M. J. I.; Tabor, A. B. Tetrahedron 1999, 55, 11711-11743.
(b) Fairlie, D. P.; West, M. L.; Wong, A. K. Curr. Med. Chem. 1998, 5,
29-62. (c) Sasaki, T.; Lieberman, M. In ComprehensiVe Supramolecular
Chemistry; Murakami, Y., Ed.; Pergamon Press: Oxford, 1996; Vol. 4,
pp 193-242.
(3) (a) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001,
123, 5382-5383. (b) Ernst, J. T.; Kutzki, O.; Debnath, A. K.; Jiang, S.;
Lu, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2002, 41, 278-281.
(4) For the role of the Bcl-2 protein family in apoptosis, see e.g.: (a) Gross,
A.; McDonnell, J. M.; Korsmeyer, S. J. Genes DeVel. 1999, 13, 1899-
1911. (b) Chao, D. T.; Korsmeyer, S. J. Annu. ReV. Immunol. 1998, 16,
395-419.
(5) For apoptosis reviews, see e.g.: (a) Reed, J. C. Trends Mol. Med. 2001,
7, 314-319. (b) Reed, J. C. Am. J. Pathol. 2000, 157, 1415-1430.
(6) Structure reviews: (a) Fesik, S. W. Cell 2000, 103, 273-282. (b) Liang,
H.; Fesik, S. W. J. Mol. Biol. 1997, 274, 291-302.
(7) Muchmore, S. W.; Sattler, M.; Liang, H.; Meadows, R. P.; Harlan, J. E.;
Figure 3. Results of the fluorescence polarization assay. Unfilled circle )
1; green circle ) 3; black circle ) 4; magenta circle ) 5; red circle ) 6;
unfilled square ) 7; blue square ) 2.
Yoon, H. S.; Nettesheim, D.; Chang, B. S.; Thompson, C. B.; Wong,
S.-L.; Ng, S.-C.; Fesik, S. W. Nature 1996, 381, 335-341.
(8) (a) Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.;
Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.;
Thompson, C. B.; Fesik, S. W. Science 1997, 275, 983-986. (b) Wang,
J.-L.; Liu, D.; Zhang, Z.-J.; Shan, S.; Han, X.; Srinivasula, S. M.; Croce,
C. M.; Alnemri, E. S.; Huang, Z. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
7124-7129.
substituents making a series of hydrophobic contacts with the
protein (Figure 2C).
In conclusion, a strategy of helix mimicry based on a substituted
terphenyl scaffold was successfully applied to the design of a Bcl-
xL antagonist with binding affinity in the lower nM region. Previous
small-molecule inhibitors of Bcl-xL that were discovered by
screening of large libraries,8,12-15 or by serendipity,16 only have
KD values in the µM range.11 On the basis of the promising in vitro
results for the inhibition of Bcl-xL by 4, preliminary cell studies
on breast cancer cells are currently under investigation.
(9) (a) Petros, A. M.; Nettesheim, D. G.; Wang, Y.; Olejniczak, E. T.;
Meadows, R. P.; Mack, J.; Swift, K.; Matayoshi, E. D.; Zjang, H.;
Thompson, C. B.; Fesik, S. W. Protein Sci. 2000, 9, 2528-2534. (b)
Kelekar, A.; Chang, B. S.; Harlan, J. E.; Fesik, S. W.; Thompson, C. B.
Mol. Cell Biol. 1997, 17, 7040-7046.
(10) Under these assay conditions, the unlabeled 16-mer Bak peptide gave a
KD of 120 nM.
(11) Review: (a) Huang, Z. Oncogene 2000, 19, 6627-6631. (b) Zheng, T.
S. Nature Cell Biol. 2001, 3, E43-E46.
(12) Yu, N.; Aramini, J. M.; Germann, M. W.; Huang, Z. Tetrahedron Lett.
2000, 41, 6993-6996. See also ref 8b.
(13) (a) Degterev, A.; Lugovskoy, A.; Cardone, M.; Mulley, B.; Wagner, G.;
Mitchison, T.; Yuan, J. Nat. Cell Biol. 2001, 3, 173-182. (b) Lugovskoy,
A. A.; Degterev, A. I.; Fahmy, A. F.; Zhou, P.; Gross, J. D.; Yuan, J.;
Wagner, G. J. Am. Chem. Soc. 2002, 124, 1234-1240.
Acknowledgment. We thank the NIH for partial support of this
work, and O.K. is grateful for a research fellowship of the Deutsche
Forschungsgemeinschaft (DFG). We also thank Professor Gerhard
Wagner and Dr. Alexey A. Lugovskoy (Harvard Medical School)
for providing HSQC assignments of Bcl-xL before publication,
Sang-gyu Lee for assistance with the NMR experiments, and
Professor J. Yuan for help with protein expression.
Supporting Information Available: Experimental details, char-
acterization data (NMR, MS), docking parameters, and results (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Nakashima, T.; Miura, M.; Hara, M. Cancer Res. 2000, 60, 1229-1235.
(15) Enyedy, I. J.; Ling, Y.; Nacro, K.; Tomita, Y.; Wu, X.; Cao, Y.; Guo, R.;
Li, B.; Zhu, X.; Huang, Y.; Long, Y.-Q.; Roller, P. P.; Yang, D.; Wang,
S. J. Med. Chem. 2001, 44, 4313-4324.
(16) (a) Tzung, S.-P.; Kim, K. M.; Basan˜ez, G.; Giedt, C. D.; Simon, J.;
Zimmerberg, J.; Zhang, K. Y. J.; Hockenbery, D. M. Nat. Cell Biol. 2001,
3, 183-191. (b) Kim, K. M.; Giedt, C. D.; Basan˜ez, G.; O’Neill, J. W.;
Hill, J. J.; Han, Y.-H.; Tzung, S.-P.; Zimmerberg, J.; Hockenbery, D. M.;
Zhang, K. Y. J. Biochemistry 2001, 40, 4911-4922.
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