The art of filling protein pockets efficiently with octahedral metal complexes

Article abstract of DOI:10.1002/anie.201108865

Better fit, less effort: An easy-to-synthesize ruthenium phthalimide complex (tan-colored carbon atoms in the picture) was designed to bind within the active site of the p21-activated kinase 1 in a novel fashion that differs from that of the previously es

Full text of DOI:10.1002/anie.201108865

Angewandte
Chemie
DOI: 10.1002/anie.201108865
Bioorganometallic Chemistry
The Art of Filling Protein Pockets Efficiently with Octahedral Metal
Complexes**
Sebastian Blanck, Jasna Maksimoska, Julia Baumeister, Klaus Harms, Ronen Marmorstein,* and
Eric Meggers*
[
3–7]
Complicated natural products whose structures and proper-
ties have evolved over millions of years frequently display
specific biological modes of action that can often be traced
back to their preorganized three-dimensional structures
which perfectly complement the shape and functional-group
scaffold.
For example, the octahedral organoruthenium
complex L-FL172 was designed as a selective inhibitor for the
p21-activated kinase 1 (PAK1), in which the bidentate
pyridocarbazole ligand of the ruthenium complex occupies
the adenine pocket (Figure 1). By interacting with the so-
called hinge region it places the ruthenium center at a defined
position within the ribose binding site, where the additional
CO, chloride, and bidentate iminopyridine ligands can form
important contacts with other parts of the active site and
thereby strongly contribute to binding the affinity and
[
8]
[4]
[1]
presentation of their target protein pockets. A recent study
analyzed the protein-binding properties of compounds from
natural as well as synthetic sources and found that the
protein-binding selectivity correlated with the shape com-
3
plexity (defined as the relative number of sp -hybridized
[
4,6]
carbon atoms) and the stereochemical complexity (defined as
selectivity.
[2]
the relative number of stereogenic carbons). Octahedral
metal complexes may offer an attractive alternative to
sophisticated globular and rigid structural templates. They
are constructed from a single metal stereocenter and chelat-
ing ligands limit the degree of conformational flexibility; thus
“
natural-product-like” structural complexities and strikingly
high target specificities are achieved, as our group has
[
3–6]
demonstrated in several previous studies.
An important aspect in the design of such metal-tem-
plated protein binders not articulated in the past is the three-
dimensional space requirement of an octahedral center. This
is a crucial aspect of the inhibitor design, mainly because the
metal must be located at a specific position within the active
site in order to be useful. For example, if the metal is located
too far within the active site or too close to the protein
backbone there will not be enough space available to
accommodate the octahedral coordination sphere. In con-
trast, if the metal is located too close to the solvent, the metal
center cannot easily impact binding affinity and selectivity. A
clear indicator for an advantageous metal position within the
protein pocket is the strong influence of the metal coordina-
tion sphere on the binding affinity and selectivity. We have
identified such a privileged position of the metal within the
ATP-binding site of protein kinases by using the well-
established staurosporine-inspired metallopyridocarbazole
Figure 1. Comparison of two strategies for the design of metal-
templated inhibitors of the protein kinase PAK1.
In order to better understand the design of metal-based
enzyme inhibitors, we wondered whether this design is unique
or whether other scaffolds with metals located at different
positions within the active site could yield similar or even
better results. To address this question, we designed new
[
*] S. Blanck, Dr. K. Harms, Prof. Dr. E. Meggers
Fachbereich Chemie, Philipps-Universitꢀt Marburg
Hans-Meerwein-Strasse, 35043 Marburg (Germany)
E-mail: meggers@chemie.uni-marburg.de
[
9]
scaffolds and discovered the simple ruthenium complex (R)-
, which places the metal at a distinct position within the ATP-
1
binding site, contains a completely different set of coordinat-
ing ligands, and yet shows an improved affinity for PAK1
compared to that of the much more complicated pyridocar-
bazole complex L-FL172.
Ruthenium complex 1 is based on a simple pyridyl-
phthalimide scaffold, which can be synthesized in just a few
steps (Scheme 1). Accordingly, a Suzuki cross-coupling of
Dr. J. Maksimoska, Prof. R. Marmorstein
The Wistar Institute
3
601 Spruce Street, Philadelphia, PA 19104 (USA)
E-mail: marmor@wistar.org
**] This work was supported by the German Research Foundation
DFG) and the U.S. National Institutes of Health (CA114046). S.B.
[
(
acknowledges a stipend from the Fonds der Chemischen Industrie.
bromophthalimide
afforded the pyridylphthalimide 3 (49%), which was sub-
2
with 2-trimethylstannylpyridine
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201108865.
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Scheme 1. Synthesis of pyridylphthalimide ruthenium complex 1.
TBS=tert-butyldimethylsilyl.
sequently reacted with the ruthenium complex [Ru(C H S )-
6
12 3
(
MeCN) ](CF SO ) under basic conditions, followed by the
3 3 3 2
addition of NaSCN to afford the racemic complex 1 (38%
over two steps). A crystal structure of the N-benzylated
derivative of 1 is shown in Figure 2 and indicates the
formation of a CÀRu bond. This cyclometalation reduces
Figure 3. IC50 curves of ligand 4, complex 1, and complexes 5–7 with
PAK1. ATP concentration was 1 mm.
the requirement for metal-coordinating heteroatoms and is
thus crucial for the short and efficient synthesis.
Figure 2. Structure of the N-benzylated derivative of complex 1.
Disordered solvent molecules and a disordered position of S101 are
not shown. Thermal ellipsoids drawn at 50% probability level. Selected
bond lengths [ꢁ]: C15–Ru1=2.033(4), N1–Ru1=2.098(3), N100–
Ru1=2.084(3), S1–Ru1=2.2770(9), S2–Ru1=2.3972(9), S3–
Ru1=2.2923(9).
Figure 4. Cocrystal structure of PAK1 (amino acids 249–545 with
mutation Lys299Arg) and (R)-1 at a resolution of 2.0 ꢁ; the van der
Waals surfaces are displayed. Coordinates of the structure have been
deposited in the Protein Data Bank (PDB ID: 4DAW).
The racemic complex 1 displays an IC50 value of (83 Æ
2
0) nm (1 mm ATP) against PAK1 (Figure 3), which is slightly
more potent than the previously reported L-FL172 (IC =
5
0
[
4]
1
30 nm, 1 mm ATP). Interestingly, the pyridylphthalimide
ligand itself is not an inhibitor of PAK1 (IC > 100 mm), thus
the entire coordination sphere is important for protein kinase
binding.
Thr406 (Figure 5). Interestingly, superimposition of this
5
0
[4]
structure with the recently disclosed PAK1/L-FL172 coc-
rystal structure demonstrates that even though both com-
pounds are ATP-competitive binders, they significantly differ
in their binding modes (Figure 6). While both form the
canonical hydrogen bonds between the maleimide moieties of
the inhibitor and the hinge region of the kinase, the aromatic
heterocycles are rotated towards each other by approximately
308 demonstrating the range that is possible for the direction-
ality of hydrogen bonds. Furthermore, the metal centers of
the two inhibitor scaffolds are 3.0 ꢀ apart from each other
and the monodentate ligands point into different areas of the
active site. Whereas the CO ligand of FL172 is located right
below the center of the glycine-rich loop (P-loop), the NCS
PAK1 contains an atypical, open ATP-binding site which
is probably responsible for the difficulties in developing high-
[
4,8,10]
affinity inhibitors of this kinase.
A cocrystal structure at
a resolution of 2.0 ꢀ reveals the binding of the R enantiomer
of 1 to the ATP binding site of PAK1 (amino acids 249–545
[11]
with mutation Lys299Arg; Figures 4 and 5). The van der
Waals surface representation shown in Figure 4 demonstrates
how nicely the ATP-binding site is filled with (R)-1 in a shape-
complementary fashion with the phthalimide moiety addi-
tionally forming two hydrogen bonds with the hinge region
(
Glu345 and Leu347) and one water-mediated contact to
2
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Chemie
complexes in an enzyme active site. Despite the brevity of the
synthesis (overall only six steps) and simplicity of the
structure (only two stereoisomers possible), complex 1 dis-
plays an IC50 value of 83 nm (1 mm ATP), which is superior to
that of FL172, a complex that is much more tedious to
synthesize (> 15 steps) and of higher stereochemical com-
plexity (20 possible stereoisomers). To date, the ruthenium
phthalimide complex 1 described is among the most potent
ATP-competitive inhibitors known for the protein kinase
[
12]
PAK1,
demonstrating the advantages of filling large or
open pockets with globular octahedral metal complexes.
Received: December 16, 2011
Published online: && &&, &&&&
Figure 5. Hydrogen bonding of (R)-1 within the ATP-binding site of
PAK1.
Keywords: bioorganometallic chemistry · enzyme inhibitors ·
protein kinases · ruthenium
.
[
[
1] E. E. Carlson, ACS Chem. Biol. 2010, 5, 639 – 653.
2] P. A. Clemons, N. E. Bodycombe, H. A. Carrinski, J. A. Wilson,
A. F. Shamji, B. K. Wagner, A. N. Koehler, S. L. Schreiber, Proc.
Natl. Acad. Sci. USA 2010, 107, 18787 – 18792.
[
3] H. Bregman, P. J. Carroll, E. Meggers, J. Am. Chem. Soc. 2006,
128, 877 – 884.
[
4] J. Maksimoska, L. Feng, K. Harms, C. Yi, J. Kissil, R.
Marmorstein, E. Meggers, J. Am. Chem. Soc. 2008, 130,
15764 – 15765.
[
[
5] A. Wilbuer, D. H. Vlecken, D. J. Schmitz, K. Krꢁling, K. Harms,
C. P. Bagowski, E. Meggers, Angew. Chem. 2010, 122, 3928 –
3932; Angew. Chem. Int. Ed. 2010, 49, 3839 – 3842.
6] L. Feng, Y. Geisselbrecht, S. Blanck, A. Wilbuer, G. E. Atilla-
Gokcumen, P. Filippakopoulos, K. Krꢁling, M. A. Celik, K.
Harms, J. Maksimoska, R. Marmorstein, G. Frenking, S. Knapp,
L.-O. Essen, E. Meggers, J. Am. Chem. Soc. 2011, 133, 5976 –
5986.
[
7] E. Meggers, G. E. Atilla-Gokcumen, H. Bregman, J. Maksi-
moska, S. P. Mulcahy, N. Pagano, D. S. Williams, Synlett 2007,
1177 – 1189.
Figure 6. Relative binding position of (R)-1 and L-FL172 (PDB ID:
3
FXZ) within the ATP-binding site of PAK1. Superimposed with the
PyMOL Molecular Graphics System, Version 1.3, Schrçdinger, LLC.
[8] The p21-activated kinases are important mediators of signaling
pathways that regulate cellular processes and are implicated in
pathological conditions including cancer. See, for example: C.
Yi, J. Maksimoska, R. Marmorstein, J. L. Kissil, Biochem.
Pharmacol. 2010, 80, 683 – 689.
ligand of 1 points towards the interface of the glycine-rich
loop (connecting strands b1 and b2) and the methylene
groups of Arg299 of the b-sheet strand b3, thereby perfectly
filling a small hydrophobic pocket as visualized in the van der
Waals representation of Figure 4. The importance of the NCS
ligand is manifested by the loss of the binding affinity of
related complexes, which carry a SeCN (IC = (0.625 Æ
[
9] Other designs of metal-based kinase inhibitors: a) J. Spencer,
A. P. Mendham, A. K. Kotha, S. C. W. Richardson, E. A. Hillard,
G. Jaouen, L. Male, M. B. Hursthouse, Dalton Trans. 2009, 918 –
921; b) B. Biersack, M. Zoldakova, K. Effenberger, R. Schobert,
Eur. J. Med. Chem. 2010, 45, 1972 – 1975; c) S. Blanck, T.
Cruchter, A. Vultur, R. Riedel, K. Harms, M. Herlyn, E.
Meggers, Organometallics 2011, 30, 4598 – 4606.
5
0
0
2
.06) mm, 7.5-fold weaker inhibitor), NCO (IC = (15.7 Æ
5
0
[
10] a) M. Lei, W. Lu, W. Meng, M.-C. Parrini, M. J. Eck, B. J. Mayer,
S. C. Harrison, Cell 2000, 102, 387 – 397; b) M. Lei, M. A.
Robinson, S. C. Harrison, Structure 2005, 13, 769 – 778.
[11] The lysine-to-arginine mutation at position 299 inactivates the
enzyme but is important for the large-scale cellular expression of
PAK1 for crystallization purposes. However, this mutation
apparently does not lead to any significant structural changes.
See Ref. [10].
) mm, 193-fold weaker inhibitor), or CO ligand instead
(
IC = (12.7 Æ 2) mm, 153-fold weaker inhibitor) (Figure 3).
5
0
This intriguing example demonstrates how a change in the
position of the metal within the active site, even if it is just
shifted by 3.0 ꢀ, requires a completely different coordination
sphere to fill the protein pocket in a comparable fashion.
In conclusion, the design of a metal-based enzyme
inhibitor described here along with the crystallographic
analysis of its binding within the enzyme active site emphasize
the broad range of possibilities for fitting octahedral metal
[
12] See the Supporting Information for a kinase selectivity profile of
1.
Angew. Chem. Int. Ed. 2012, 51, 1 – 4
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
Communications
Bioorganometallic Chemistry
Better fit, less effort: An easy-to-synthe-
size ruthenium phthalimide complex
(tan-colored carbon atoms in the picture)
was designed to bind within the active
site of the p21-activated kinase 1 in
a novel fashion that differs from that of
the previously established staurosporine-
inspired metallopyridocarbazoles (gray-
colored carbon atoms).
S. Blanck, J. Maksimoska, J. Baumeister,
K. Harms, R. Marmorstein,*
E. Meggers*
&&&& — &&&&
The Art of Filling Protein Pockets
Efficiently with Octahedral Metal
Complexes
4
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 4
These are not the final page numbers!