A Unique Mdm2-Binding Mode of the 3-Pyrrolin-2-one- and 2-Furanone-Based Antagonists of the p53-Mdm2 Interaction

Article abstract of DOI:10.1021/acschembio.6b00596

The p53 pathway is inactivated in almost all types of cancer by mutations in the p53 encoding gene or overexpression of the p53 negative regulators, Mdm2 and/or Mdmx. Restoration of the p53 function by inhibition of the p53-Mdm2/Mdmx interaction opens up

Full text of DOI:10.1021/acschembio.6b00596

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Article
A Unique Mdm2-Binding Mode of the 3-Pyrrolin-2-one- and 2-
Furanone-Based Antagonists of the p53-Mdm2 Interaction
Ewa Surmiak, Aleksandra Twarda-Clapa, Krzysztof M. Zak, Bogdan Musielak, Marcin D.
Tomala, Katarzyna Kubica, Przemys#aw Grudnik, Mariusz Madej, Mateusz Jab#o#ski, Jan
Potempa, Justyna Kalinowska-T#u#cik, Grzegorz Dubin, Alexander Dömling, and Tad A Holak
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00596 • Publication Date (Web): 06 Oct 2016
Downloaded from http://pubs.acs.org on October 8, 2016
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Example NMR spectra for the HSQC-based titration of Mdm2 with 1a. Red: reference, Mdm2 alone; blue:
molar ratio protein/ligand 1:0.5; green: overtitrated Mdm2 (the ratio protein/ligand 1:2).
Figure 1
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Interaction of the Mdm2 protein and compound 1a (yellow sticks). (A) Stereoview comparison between the
binding modes of 1a and the p53 peptide (purple; PDB, ID: 1YCR).13 (B) Mdm2–1a complex. Left – ribbon
representation, right – surface representation.
Figure 2
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Comparison of the interactions of compounds: 1a (magenta sticks) and 2a (yellow sticks) with Mdm2
(shown in ribbon (left panel) and surface (right panel) representation.
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Key interactions that support the dimerization of Mdm2 by 1a. The Mdm2 molecules forming the dimer: grey
and magenta. Residues involved in the Mdm2 dimerization are highlighted as sticks and shown in gray. The
inhibitor molecules are shown in yellow and orange. Hydrogen bonds are depicted as blue dotted lines, the
ring stacking is depicted as red dotted lines, halogen bonds are depicted as green dotted lines. Water
molecule mediating inhibitor – adjacent Mdm2 molecule interaction is shown in blue. (A) Stereo view. (B-D)
Mixed ribbon / surface representations of Mdm2 protein molecules.
Figure 4
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Graphical Table of Contents
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A Unique Mdm2-Binding Mode of the 3-Pyrrolin-2-
one- and 2-Furanone-Based Antagonists of the p53-
Mdm2 Interaction
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Ewa Surmiak †,°, Aleksandra Twarda-Clapa ‡,#,°, Krzysztof M. Zak‡,#, Bogdan Musielak †, Marcin
D. Tomala †, Katarzyna Kubica †, Przemyslaw Grudnik‡, Mariusz Madej‡,#,Mateusz Jablonski†,
Jan Potempa‡,#, Justyna Kalinowska-Tluscik†, Alexander Dömling§, Grzegorz Dubin*,‡, # and
Tad A. Holak*,†,#
†Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30–060 Krakow, Poland
‡Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa
7, 30–387 Krakow, Poland
#Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30–387
Krakow, Poland
§Faculty of Mathematics and Natural Sciences, Department of Pharmacy, University of
Groningen, 9713AV Groningen, The Netherlands
Keywords: Cancer, Drug Discovery, Inhibitors, Mdm2-p53, Protein-Protein Interactions
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ABSTRACT
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The p53 pathway is inactivated in almost all types of cancer by mutations in the p53 encoding
gene or overexpression of the p53 negative regulators, Mdm2 and/or Mdmx. Restoration of the
p53 function by inhibition of the p53-Mdm2/Mdmx interaction opens up a prospect for a non-
genotoxic anticancer therapy. Here we present the syntheses, activities and crystal structures of
two novel classes of Mdm2-p53 inhibitors that are based on the 3-pyrrolin-2-one and 2-furanone
scaffolds. The structures of the complexes formed by these inhibitors and Mdm2 reveal the
dimeric protein molecular organization that has not been observed in the small-molecule/Mdm2
complexes described until now. In particular, the 6-chloroindole group does not occupy the usual
Trp-23 pocket of Mdm2, but instead is engaged in dimerization. This entirely unique binding
mode of the compounds opens new possibilities for optimization of the Mdm2-p53 interaction
inhibitors.
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INTRODUCTION
The tumor suppressor p53 protein pathway is inactivated in the majority of human cancer either
by mutations within the p53 gene or by overexpression of negative regulators of p53,
predominantly Mdm2 and/or Mdmx proteins. Restoration of active p53 has been demonstrated to
be a promising strategy against cancer.1–3 Mutations in the p53 encoding gene are found in about
50% of all human cancers.1–6 In the tumors that contain the wildtype p53, liberation of its activity
by disrupting the interaction with the Mdm2/Mdmx, using low-molecular-weight inhibitors,
should provide an efficient, non-genotoxic anticancer therapy.1 Although tremendous progress
was observed in the latter strategy4–6 since the initial demonstration of the efficacy of an early
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inhibitor nutlin-3a,7 constant development of yet novel compounds with better pharmacological
properties is sought.8–12
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The interaction of the transactivation domain of p53 with the N-terminal domain of Mdm2 is
mediated primarily by the sidechains of Phe19, Trp23 and Leu26 of p53, which occupy three
hydrophobic subpockets within Mdm2.13–14 These interactions define a three finger pharma-
cophore which characterizes the vast majority of the currently available small-molecule Mdm2
inhibitors.8–9 Since the early discovery of Garcia-Echverria et al., that substitution of the indole
moiety of Trp23 in the p53 peptide with that of 6-chloroindole significantly potentiates Mdm2
affinity,15 a large number of small-molecule inhibitors have been developed in which a major
interaction with Mdm2 is provided by a 6-chloroindole moiety occupying the Trp23
subpocket.16–18 Nevertheless, certain new approaches were recently proposed.5,6,19–23
In this study we describe novel Mdm2 inhibitors based on the 3-pyrrolin-2-one and 2-
furanone scaffolds which by design contain the 6-chloroindole structure anchoring
pharmacophore and a maximal number of halogenated phenyl groups. Crystals of the complexes
formed by these inhibitors and Mdm2 unexpectedly revealed dimeric protein arrangement
formed by the chloroindole-protein and protein-protein interactions in which the 6-chloroindole
group does not occupy the usual Trp-23 pocket of Mdm2.
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RESULTS AND DISCUSION
Design of Novel Antagonists of the Mdm2-p53 Interaction. The patent space of Mdm2
antagonists based on five-membered heterocyclic core is tightly packed nowadays. Nevertheless,
using a three finger pharmacophore model and assuming a five-membered core we have
designed two series of novel compounds, derivatives of 5-hydroxy-1,5-dihydydro-2H-pyrrol-2-
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one and 5-hydroxy-1,5-dihydro-2H-furran-2-one. The compounds selected for synthesis and
evaluation of their activity are shown in Table 1.
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Synthesis of the Substituted 3-Pyrrolin-2-ones. To prepare compounds 1a and 1b, we
devised a 6 stage synthetic route (Scheme 1). Compound 5 was easily available from N-
methylpyrrol (3), through bromination-oxidation procedure24 (obtaining 4), and subsequent
nucleophilic substitution of a single bromine with 6-chloroindole according to known
procedures.25 Then a Suzuki-Miyaura reaction allowed to substitute the second bromine with a
benzyl moiety resulting in compound 7.26 Before that, the N-H proton within 6-chloroindole was
protected with tert-butyldicarbonate, according to standard DMAP – catalyzed reaction.27
Grignard reaction was used to convert 7 into 8a and 8b28 and the protecting group was removed
in mild conditions by treatment with methylamine solution.29 Resulting compounds 1a and 1b
were separated by column chromatography.
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Scheme 1. Synthesis of compounds 1a and 1b (Numbering of atoms in the central ring of 3-
pyrrolin-2-ones are depicted in blue).a
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aReagents and conditions: (a) (i) compd 3 (1 eq.), NBS (2.5 eq.), –78ºC to RT, 16 h, anh. THF,
(ii) conc. HNO3, 0ºC, 1 h, hexane; (b) (i) 6-chloroindole (1 eq.), LiHMDS (2.5 eq.), –78°C, anh.
THF, (ii) compd 4 (1 eq.), –20ºC, anh. THF; (c) compd 5 (1 eq.), (Boc)2O (2 eq.), anh. Et3N (2
eq.), DMAP (0.1 eq.), RT, 16 hr, anh. THF; (d) (i) 4-chlorobenzyl chloride (1.5 eq.), Mg (1.5
eq.), 2 hrs, reflux, anh. Et2O, (ii) B(OMe)3 (1 eq.), –78ºC, anh. THF, (iii) KHF2 (4.5 eq.), H2O,
0ºC, 0.5 h; (e) compd 6 (1 eq.), potassium (4-chlorobenzyl)trifluoroborate (1.1 eq.), Cs2CO3 (3.5
eq.), Pd(dppf)Cl2 complex with CH2Cl2 (0.1 eq.), 80ºC, 5 h, toluene/water (5:1); (f) (i) 4-
chlorobenzyl bromide (1.5 eq.), Mg (1.5 eq.), 2 h, reflux, anh. Et2O, (ii) cmpd 7 (1 eq.), 4 h,
reflux, anh. THF; (g) compd 8a or 8b excess of MeNH2 in MeOH (2 M), 36–72 h, RT.
Compounds 1c-h were prepared using a shorter route (Scheme 2). Compound 13 was obtained
by cyclization30 of an (4-chlorophenyl)acetamide (10) and a (6-chloro-1H-indol-3-yl)oxoacetic
acid methyl ester (12) prior prepared from commercially available substrates, 9 and 11,
respectively. Compound 13 was methylated to yield monomethylated 14 and dimethylated 15
derivatives or alternatively benzylated to yield 16. This was done efficiently in mild conditions
with potassium carbonate while different evaluated bases (NaH, NaOH and Cs2CO3) led to a
number of side-products. Compounds 1c-f were obtained from 14 and 15, respectively, using
Grignard reaction as described above for 1a-b. 1g-h were obtained by reduction of 16 with either
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NaBH4 or alternatively LiAlH4 (ref. 31) with similarly high yields. Adapting the shorter synthetic
route depicted in Scheme 2 for the preparation of 1a-b proved impossible due to low yields,
multiple side products of the N-methylation step and resulting difficulties in purification.
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Scheme 2. Synthesis of compounds 1c-h.a
aReagents and conditions: (a) (i) (9) or 9a (1 eq.) thionyl chloride (5 eq.), reflux, 3 h, (ii)
NH3(aq) (20 eq.), 0–5ºC to RT, 16 h; (b) (i) 6-chloroindole (11) (1 eq.), oxalyl chloride (1.1 eq.),
0ºC, 1 h, anh. THF, (ii) sodium methoxide in MeOH (2 eq.), 60ºC to 0ºC, 2 h; (c) (i) compd 10 or
10a (1 eq.), 12 (1 eq.), potassium tert-butoxide (4 eq.), 0ºC, 0.5 h, anh. THF, (ii) 55ºC, 5 h, (iii)
conc. HCl, RT, 0.5 h; (d) compd 13 (1 eq.), methyl iodide or benzyl bromide (1 eq.), K2CO3 (5
eq.), RT, 16 h, anh. DMF; (e) (i) 4-chlorobenzyl bromide (1.5 or 2.5 eq.), Mg (1.5 or 2.5 eq.), 2
h, reflux, anh. Et2O, (ii) compds 14 or 15 (1 eq.), 4 h, reflux, anh. THF; (f) Compd 16 (1eq.),
LiAlH4 (2.5 eq), 0ºC to RT, 1 h, anh. Et2O-THF.
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Synthesis of the Substituted 2-Furanones. Substituted 2-furanones 2a-f were prepared from
a maleimide intermediate of 3-pyrrolin-2-one by trans-cyclization (Scheme 3).32 Compound 13a
was obtained in the same manner as 13, from 3-(4-chlorophenyl)propionic amide (10a) and (6-
chloro-1H-indol-3-yl)oxoacetic acid methyl ester (12). Substituted maleimides 13 or 13a were
refluxed in basic water-ethanol solution yielding respective substituted maleic anhydrides (17
and 18, respectively). These were further modified using Grignard method as described above to
yield substituted 2-furanones 2a-d and 2e-f, respectively.
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Scheme 3. Synthesis of compounds 2a-f 1b (Numbering of atoms in the central ring of 2-
furanones are depicted in blue).a
a Reagents and conditions: (a) compd 13 or 13a (1 eq.), NaOH (15 eq.), water/ethanol (5:1),
reflux, 1 h; (b) (i) 4-chlorobenzyl bromide or 4-fluorobenzyl bromide (2.5 eq.), Mg (2.5 eq.), 2
h, reflux, anh. Et2O, (ii) compds 17 or 18 (1 eq.), 4h, reflux, anh. THF. The numbering of the
central ring is shown in blue based on formulas 2a and 2b.
Structure – Activity Relationship. To quantify the affinities of our compounds towards
Mdm2, we determined the Kd of the compound-Mdm2 interaction using NMR33 and the
microscale thermophoresis (MST). For NMR, the 15N-labeled Mdm2 was titrated with increasing
1
concentration of each evaluated compound and H-15N 2D-HSQC spectra were recorded after
each new portion of the compound has been added (Figure 1, Figure S1-S2).
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This method is based on monitoring of chemical shift changes in protein amide backbone
resonances upon protein interaction with a small molecule.34–37 It allows not only to qualitatively
evaluate the fact of interaction, but also to semiquantitatively estimate the binding affinity.
Moreover, the strong binding of small molecule to the target protein, for example, 1d to Mdm2,
results in NMR signal doubling indicating the Kd’s of less than 1 ꢀM (and a slow chemical
exchange). Assignment of the amide groups of Mdm2 was obtained after Stoll et al.38 The results
are summarized in Table 1.
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The compounds substituted with the 4-chlorophenyl group at position 3 of the central ring (1c-
h, 2e-f, numbering of atoms in the central ring is shown in Schemes 1 and 3) exhibited weak
affinity (Kd > 50 ꢀM) regardless the scaffold being that of 3-pyrrolin-2-one or 2-furanone. At the
same time the compounds substituted at position 3 with the 4-chlorobenzyl group (1a-b, 2a-d)
exhibited better affinities towards Mdm2 compared to the 4-chlorophenyl substituted
compounds, again regardless the core scaffold. It is thus clear that the extra flexibility provided
by an additional methylene group is a prerequisite of the efficient binding in these types of
compounds. For all tested structural isomers (ie. 1a vs 1b, etc.; except 1e and 1f), those
substituted at position 2 exhibited weaker activities compared to those substituted at position 5,
irrespective of the substituent at position 3 and the central ring scaffold. The N-methylated 6-
chloroindole substituent at position 4 is clearly not compatible with Mdm2 binding as evidenced
by weak affinities of compounds 1e and 1f.
The affinities of all compounds were additionally tested by microscale thermophoresis (MST;
Figure S3). The best binding compounds, 1a and 2a demonstrate affinities comparable to those
determined by NMR. The occurrence of disparities may be explained by the low solubility of
compounds, which was the major issue for NMR. For compounds with NMR Kds of up to 100
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ꢀM, which is considered an “intermediate exchange” NMR range35, the MST data demonstrate
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slightly lower affinities (below 30 ꢀM) compared to NMR data. The apparent NMR Kds
determined in the intermediate exchange range are however expected to be higher than the ones
from MST, and the relative differences in affinities of compounds within the series determined
using each method are consistent.39–40 The worst binding compounds (with NMR Kds higher than
100 ꢀM) are also characterized by highest values (>30 ꢀM) in MST.
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The two “best” identified inhibitors, 1a and 2a, show affinities in low single digit µM range.
Comparable affinities and the fact that 1a and 2a differ only within the central ring scaffold
indicate that both scaffolds identified within this study are equally suitable for further
development of Mdm2 inhibitors.
Table 1. Structures and activity of Mdm2 inhibitors reported in this study.
Kd by
MST
Kd by
NMR
[ꢀM]a
Kd by
NMR
[ꢀM]a
Kd by
MST
[ꢀM]
No
1a
Formula
No
1b
Formula
[ꢀM]
not active
27.7 0.2
<1
51
0.5 0.3
56
18.7 0.2
192
1c
1e
1d
1f
30.4 0.2
365
40.5 0.3
246
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2.1 0.1
164
24.4 0.1
40
86
1g
1h
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1.5 0.4
26.0 1.0
7.0 0.2
<1
18
3.1 04
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2c
2b
2d
no
28.7 1.0
datab
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1.3 0.2
68
2e
2f
[a] The experimental error of the measured KD values is ca. 30%.
[b] KD of compound 2d could not be determined by NMR.
Structural Basis of the Interaction of 1a with Mdm2. In order to better understand the
structural basis of interactions of the reported compounds at the binding pocket of Mdm2, we
have determined the crystal structure of 1a in complex with the target protein. The obtained
crystals diffracted to 2.0 Å resolution and contain four protein molecules in the asymmetric unit.
Each Mdm2 molecule contains a single inhibitor molecule at the binding pocket. No additional
inhibitor molecules were found within the structure. The inhibitors were well defined by their
electron density. The Leu26 subpocket is occupied by the 4-chlorobenzyl substituent found at
position 3 of 1a. The aromatic ring of the substituent forms a π-π stacking interaction with the
imidazole ring of His96. The interaction is further stabilized by hydrophobic contacts mediated
by the sidechains of Leu54, Ile99 and Tyr100. The second 4-chlorobenzyl substituent, located at
position 5 of 1a occupies the Trp23 subpocket of Mdm2. Its interaction is stabilized by numerous
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hydrophobic contacts mediated by the sidechains of Leu54, Leu57, Ile61, Phe86, Phe91, Ile99,
Ile103, and Cα of Gly58. The central 3-pyrrolin-2-one ring of the inhibitor molecule covers the
entrance to the binding pocket. The oxygen atom of the carbonyl group at position 2 is involved
in a water mediated interaction with Nε of His96 and the carbonyl oxygen of Val93. The N-
methyl group forms hydrophobic contact with the sidechain of Val93 and is in a relatively close
contact with the hydroxyl oxygen of Tyr67. Most interestingly, the Phe19 subpocket within
Mdm2 is not utilized by the inhibitor. Instead it is occupied by the sidechain of Tyr67 which is
oriented differently compared to what is observed in the structure of the Mdm2-p53 complex
(Figure 2, Figure S4).
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Notably, the 6-chloroindole substituent found at position 4 of 1a does not occupy the Trp23
pocket as found in a number of structures of previously reported inhibitors. Instead, it interacts
with the solvent exposed residues of Mdm2. The Nε1 of 6-chloroindole forms a water-mediated
interaction with the carbonyl oxygen of Lys51. The significance of this interaction is exemplified
by poor activity of compounds 1e and 1f which are methylated at Nε1, and as such, not capable of
forming this interaction. Another water molecule, tightly coordinated by the carbonyl oxygen of
Phe55 and Νε1 of Gln59, forms a canonical lone pair–π interaction with the 6-chloroindole.
Finally, the 6-chloroindole forms halogen-π interaction with the aromatic ring of Phe55 side
chain.
The Binding Mode of 2a in the p53 Pocket of Mdm2. We also crystallized 2a in complex
with Mdm2 using different crystallization conditions and obtaining crystals exhibiting different
arrangement of the molecules within the asymmetric unit compared to the 1a-containing
complex. Despite the differences in overall crystallographic arrangement, the structure of 2a in
complex with Mdm2 demonstrates almost identical binding mode of 2a to that observed for 1a,
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strongly suggesting that the observed binding mode is compound specific, rather than induced
within the crystal. The 4-chlorobenzyl substituents at positions 3 and 5 of 2a occupy the Leu26
and Trp23 subpockets, respectively, identically as found in the structure containing 1a. The 2-
furanone central ring is located identically to the 3-pyrrolin-2-one ring of 1a. The only difference
is that the N-methyl group is missing in the structure of 2a and an additional water mediated
interaction between oxygen within the 2-furanone ring and the sidechain hydroxyl group of
Tyr67 is observed. However, this interaction is present only in one of the molecules contained in
the asymmetric unit, which suggests its low contribution to the overall binding energy. This is
corroborated by high temperature factor characterizing the water molecule mediating the
interaction. The 6-chloroindole substituent of 2a is oriented identically to that of 1a and is
involved in analogous interactions. Overall, both compounds 2a and 1a and the fragments of
Mdm2 containing interacting residues superimpose with RMSD of 0.21 Å, demonstrating almost
identical orientation of both compounds within Mdm2 binding pocket (Figure 3, Figure S5;
Figure S6-S7).
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1a and 2a Induce Dimerization of Mdm2. The arrangement of molecules within the crystal
lattice in both structures may suggest the inhibitor-induced dimerization of Mdm2. In the 2a-
containing structure the asymmetric unit accommodates two Mdm2 molecules which face each
other via their p53 binding pocket side. This implies that the bound inhibitor molecules also
directly face each other. Such a dimer is built around a pseudo 2-fold rotation axis. Mdm2
molecules create multiple hydrogen bonds and water mediated contacts as well as a ring stacking
interaction of the Phe55 sidechains. The 6-chloroindole moiety of each inhibitor provides a
halogen bond41 with carbonyl oxygen of Lys51 of the adjacent Mdm2 molecule and water
mediated interaction between the chlorines and Nε1 atoms of neighboring inhibitor molecules.
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Identical dimers are recreated by symmetry operations in the 1a-Mdm2 structure (Figure 4,
5
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Figure S8).
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To test if the inhibitor induced Mdm2 dimerization observed in the crystal is retained in
solution, we recorded 1H NMR spectra of Mdm2 in the presence and absence of 1a. Addition of
1a to the Mdm2 containing NMR sample resulted in broadening of the NMR signals of the
protein, the NMR line widths being consistent with the induction of the oligomeric state of
Mdm2 upon its interaction with 1a (Figures S9–S10). This indicates that 1a induces Mdm2
dimerization in solution, and therefore the dimers observed in the crystal structure reflect
biological assemblies and not only crystal packing artefacts.
To further confirm the presence of the dimer in solution, we performed DOSY NMR
experiment. The comparison of logD of the complex of Mdm2 with our compound
(1a, logD = –9.82) and with RG7388(ref. 42) (used as a reference compound which does not
induce the dimerization of Mdm2, logD = –9.72) clearly indicates that compound 1a induces
Mdm2 dimerization in solution (Figure S11). This is also confirmed by the hydrodynamic radii
calculated from the Stokes-Einstein equation from the DOSY experiments, which were equal to
12.6 Å and 16.9 Å for the monomer and dimer, respectively.
Discussion and conclusions. Since the early discovery of Garcia-Echverria et al.15 that
substitution of the indole moiety of Trp23 in the p53 peptide with that of 6-chloroindole
significantly potentiates Mdm2 affinity,15 a large number of small-molecule inhibitors have been
developed in which a major interaction with Mdm2 is provided by a 6-chloroindole moiety
occupying the Trp23 subpocket. These include, among others, imidazoles,16 pyrazoles,43
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pyrrolidines,17 and spirooxindoles.18 In fact, 6-chloroindole itself binds weakly to Mdm2 as can
be appreciated from our NMR titration experiments (Figure S12). The moieties filling the other
two subpockets (Phe19 and Leu26) belong to many different classes in previously published
inhibitors, however, substituted phenyls and benzyls predominate. Our design approach was
initially based on the same premises. Surprisingly, the binding modes of both the 3-pyrrolin-2-
one and 2-furanone based inhibitors described in this study differ from those anticipated in
modelling. In our structures the 6-chloroindole moiety does not occupy the Trp23 binding
pocket, but instead provides a number of interactions at the surface of the Mdm2 molecule and
with the adjacent Mdm2 and inhibitor molecules thereby inducing dimerization.
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Graves et al.44 recently reported a class of Mdm2 inhibitors, exemplified by RO-2443, which
induce Mdm2 dimerization by inserting difluorobenzyl moiety into the Trp23 binding pocket of
one Mdm2 molecule and the 6-chloro-7-methyl-indole moiety into the extended Phe19 pocket of
the other Mdm2 molecule within the dimer. The dimeric protein complex is kept together by a
dimeric small-molecule core. Therefore the interaction of RO-2443 with Mdm2 is comparable to
that of the inhibitors described within this study only in the fact that the 6-chloroindole moiety is
located outside the Trp23 pocket and that the dimers are stable in solution. The modes by which
the inhibitors induce dimerization are, however, completely different, including the fact that the
6-chloroindole moieties of adjacent inhibitors within the dimer do not directly interact in the 1a-
or 2a-containing structures contrary to that containing RO-2443. Consistently with the different
mode of interaction and mechanism of dimerization, the relative orientation of Mdm2 monomers
within the dimer is utterly different in the RO-2443 containing structure compared to the 1a- or
2a-containing structures. (Supporting Text S1, Figure S13).
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The SAR for the series of compounds reported in this study is clearly explained by provided
crystal structures. Methyl substituent at position 1 of the 3-pyrrolin-2-one ring provides
additional hydrophobic contact with Val99 compared to the 2-furanone scaffold, but the absence
of a hydrophobic interaction is compensated in the latter scaffold by a weak, water mediated
interaction of the oxygen within the 2-furanone ring and the sidechain hydroxyl group of Tyr67.
The geometry of 4-chlorophenyl group at positon 3 of the central ring (1c-h, 2e-f) is not
compatible with the π-π interaction with the sidechain of His96, one of the prominent
interactions observed in the structure of 1a and 2a containing a more flexible 4-chlorobenzyl at
position 3. This explains higher affinities of the 4-chlorobenzyl substituted compounds.
Inhibitors substituted at position 5 exhibit higher affinities compared to those substituted at
position 2 most probably because the latter are not capable of forming a water mediated
interaction of the central ring with the sidechain of His96 due to different orientation of oxygen
atom at position 2. The methyl group at the Nε atom of the 6-chloroindole substituent prevents its
water mediated interaction with the carbonyl oxygen of Lys51, thereby negatively affecting the
affinity of such compounds.
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Despite the unexpected localization of the 6-chloroindole group in the Mdm2 complexes of the
inhibitors described in this study, certain other features describing the inhibitor-Mdm2
interaction are comparable to those previously observed for other Mdm2 inhibitors, rationalizing
further optimization of 1a and 2a (Supporting Text S2, Figure S14)46. The π-stacking interaction
of 1a/2a and His96 of Mdm2 resembles a similar interaction of YH239 (Figure S15.B,C).46
Hydrophobic interactions of chlorobenzyl moieties of 1a and 2a at the Leu26 and Trp23
subpockets of Mdm2 binding site are reminiscent to those of nutlin (Figure S15.A) and its
derivatives.47
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In conclusion, we have developed two novel classes of Mdm2 inhibitors. The fact that the
inhibitors based on two different scaffolds both interact with Mdm2 in an unexpected, non-
canonical fashion opens multiple avenues for further rational optimization.
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METHODS
Synthesis. Compounds were synthesized as depicted in Schemes 1–3. For details of the
reported syntheses and compound analysis see the Supporting Information.
Protein Expression and Purification. Variants of the N-terminal domain of human Mdm2
were cloned into pET-20 vector (Novagen) and expressed in the Escherichia coli BL21(DE3) as
described previously.48 In brief, cells were grown at 37°C and induced with 1 mM IPTG at
OD600 of 0.8 and grown for additional 5 h at 37°C. Cells were collected by centrifugation and
lysed by sonication. Inclusion bodies were collected by centrifugation, washed with PBS
containing 0.05% Triton-X100 and solubilized in 6 M guanidine hydrochloride in 100 mM Tris-
HCl, pH 8.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol. The protein was dialyzed
against 4 M guanidine hydrochloride, pH 3.5 with 10 mM β-mercaptoethanol. The protein was
refolded by dropwise addition into 10 mM Tris-HCl, pH 7.0, containing 1 mM EDTA and 10
mM β-mercaptoethanol and slow mixing overnight at 4°C. Ammonium sulphate was added to
the final concentration of 1.5 M and the refolded protein was recovered on Butyl Sepharose 4
Fast Flow (GE Healthcare). The protein was eluted using 100 mM Tris-HCl, pH 7.2, containing
5 mM β-mercaptoethanol and further purified by gel filtration using HiLoad 16/600 Superdex75
(GE Healthcare).
Binding Analysis by Microscale Thermophoresis (MST). MST (NanoTemper Technologies
GmbH) was used to determine the binding affinities between Mdm2 (residues 1–118 T47W; 500
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nM) and inhibitors. T47W mutation was introduced to facilitate label-free measurements and did
not interfere with small molecule binding as evidenced by unaffected affinity towards a reference
compound, nutlin-3. Experiments were performed in 50 mM phosphate buffer pH 7.4 containing
150 mM NaCl, 5 mM DTT and 5% DMSO. Inhibitors at increasing concentration (0.763ꢀnM to
25ꢀꢀM; highest concentration was limited by solubility) were incubated with the protein for 5
minutes prior to measurement at 25°C (excitation 280 nm, emission 350 nm). An inhibitor
concentration-dependent decrease in tryptophan fluorescence was observed. Inhibitor binding-
specific fluorescence quenching was evidenced by loss of the effect in samples containing the
inhibitor, but denatured by heating (95°C for 5 min) in presence of 2% SDS and 20 mM DTT. Kd
values and uncertainties were calculated using the MO. Affinity Analysis software.
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NMR Measurements. Uniform 15N isotope labeling was achieved by expression of the protein
in the M9 minimal media containing 15NH4Cl as the sole nitrogen source. Final step of
purification of Mdm2 (residues 1-118, chosen to enable interactions of N-terminal Mdm2 part49)
for NMR consisted of gel filtration into the NMR buffer (50 mM phosphate buffer pH 7.4
containing 150 mM NaCl, 5 mM DTT). 10% (v/v) of D2O was added to the samples to provide
lock signal. All the spectra were recorded at 300K using a Bruker Avance 600 MHz
1
spectrometer. H-15N heteronuclear correlations were obtained using the fast HSQC pulse
sequence.50 Assignment of the amide groups of Mdm2 was obtained after Stoll et al.38
Crystal Structure Determination. Human Mdm2 (18–125) was prepared in 5 mM Tris-HCl
pH 8.0 containing 50 mM NaCl and 10 mM β-mercaptoethanol. Molar excess (3x) of 1a or 2a
was added, the Mdm2 inhibitor complex was concentrated to 20 mgml–1 and screening for
crystallization conditions was performed using a sitting drop vapor diffusion method and
commercially available buffer sets. Crystals of the Mdm2(18–125)-1a complex appeared after
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few days at 4°C in 0.1 M HEPES pH 7.5 containing 0.2 M sodium chloride and 25% (w/v) PEG
3350. The crystals of Mdm2(18–111)-2a complex were obtained from 0.1 M HEPES pH
7.5 containing 30% (w/v) PEG 1000. Crystals were flash-cooled in liquid nitrogen without
further cryoprotection. The data collection and refinement description and statistics (Table S2)
are summarized in the Supporting Information.
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The structure factors and final models were deposited into Protein Data Bank with accession
numbers: 4ZFI and 4ZGK, for the structure with inhibitor 1a and 2a, respectively.
Small-Molecule Crystal Structure Determination. For details of small molecule
crystallography experiments, data collection and compound structures see the Supporting
Information (Figure S16, Table S1).
Accession Codes. The structure factors and final models of Mdm2 complexes with inhibitors
were deposited into Protein Data Bank with the following accession numbers: 4ZFI and 4ZGK,
for the structure containing inhibitor 1a and 2a respectively.
All crystallographic data for the small molecule structures have been deposited at the
Cambridge Crystallographic Data Centre under accession numbers: CCDC 1054955 1e,
CCDC 1054956 1g and CCDC 1054957 1d.
ASSOCIATED CONTENT
Supporting Information
Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: grzegorz.dubin@uj.edu.pl; holak@biochem.mpg.de.
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Authors Contribution
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° These authors contributed equally to this work.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research has been supported by a Marie Curie FP7-Reintegration-Grants within the 7th
European Community Framework Programme, by the project operated within the Foundation for
Polish Science TEAM Programme, co-financed by the EU European Regional Development
Fund, and by the Polish National Centre of Science, grant Symphony-2-UMO-
2014/12/W/NZ1/00457 (to T.A.H.); by the Polish National Centre of Science, grant UMO-
2011/01/D/NZ1/01169 (to G.D.). A.D. was supported by the NIH (1R21GM087617,
1R01GM097082, and 1P41GM094055). E.S. and K.Z. received the scholarship funding for the
doctoral thesis preparation from the National Centre of Science, (grants UMO-
2014/12/T/ST5/00684 to E.S., UMO-2016/20/T/NZ1/00519 to K.Z.). P.G. was supported by the
European Union within the SET project and the National Centre of Science (grant UMO-
2015/19/D/NZ1/02009). The research was carried out with the equipment purchased thanks to
the financial support of the European Union structural funds (contract no. POIG.02.01.00-12-
064/08 and POIG.02.01.00-12-167/08) and European Regional Development Fund in the
framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-
12-023/08). A.T-C. has received the financial support from the Faculty of Biochemistry,
Biophysics and Biotechnology of Jagiellonian University which is a partner of the Leading
National Research Centre (KNOW) supported by the Ministry of Science and Higher Education.
REFERENCES
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(1) Brown, C. J., Lain, S., Verma, C. S., Fersht, A. R., and Lane, D. P. (2009) Awakening
guardian angels: drugging the p53 pathway. Nat. Rev. Cancer 9, 862–873.
(2) Cheok, C. F., Verma, C. S., Baselga, J., and Lane, D. P. Translating p53 into the clinic.
(2011) Nat. Rev. Clin. Oncol. 8, 25–37.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) Hock, A. K., and Vousden, K. H. Biochim. (2014) The role of ubiquitin modification in the
regulation of p53. Biophys. Acta 1843, 137–149.
(4) Wade, M., Li, Y. C., and Wahl, G. M. (2013) MDM2, MDMX and p53 in oncogenesis and
cancer therapy. Nat. Rev. Cancer, 13, 83–96.
(5) Zhao, Y., Aguilar, A., Bernard, D., and Wang, S. (2015) A potent small-molecule
inhibitor of the MDM2-p53 interaction (MI-888) achieved complete and durable tumor
regression in mice. J. Med. Chem. 58, 1038–1052.
(6) Hoe, K. K., Verma, C. S., and Lane, D. P. (2014) Drugging the p53 pathway:
understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 13, 217–236.
(7) Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N.,
Kammlott, U., Lukacs, C., Klein, C., Fotouhi, N., and Liu, E. A. (2004) In vivo activation of the
p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848.
(8) Popowicz, G. M., Dömling, A., and Holak, T. A., (2011) The structure-based design of
Mdm2/Mdmx-p53 inhibitors gets serious. Angew. Chem., Int. Ed. 50, 2680−2688;
(9) Zak, K., Pecak, A., Rys, B., Wladyka, B., Dömling, A., Weber, L., Holak, T. A., Dubin, G.
(2013) Mdm2 and MdmX inhibitors for the treatment of cancer: a patent review (2011-present).
Expert Opin. Ther. Pat. 23, 425–448.
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5
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7
8
9
(10) Uversky, V. N., Dave, V., Iakoucheva, L. M., Malaney, P. Metallo, S. J., Pathak, R. R.,
amd Joerger, A. C. (2014) Pathological unfoldomics of uncontrolled chaos: intrinsically
disordered proteins and human diseases. Chem. Rev. 114, 6844–6879.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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41
42
43
44
45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
(11) Milroy, L. G., Grossmann, T. N., Hennig, S., Brunsveld, L., and Ottmann, C., (2014)
Modulators of protein–protein interactions. Chem. Rev. 114, 4695-4748.
(12) Pelay-Gimeno, M., Glas, A., Koch, O., and Grossmann, T. N., Structure-based design of
inhibitors of protein-protein interactions: mimicking peptide binding epitopes. (2015) Angew.
Chem. Int. Ed. 54, 8896–8927.
(13) Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau J., Levine, A. J., and
Pavletich, N. P., (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor
transactivation domain. Science 274, 948–953.
(14) Joerger, A. C., and Fersht, A. R. (2008) Structural biology of the tumor suppressor p53.
Annu. Rev. Biochem. 77, 557–582.
(15) García-Echeverría, C., Chène, P., Blommers, M. J., and Furet, P. (2000) Discovery of
potent antagonists of the interaction between human double minute 2 and tumor suppressor p53.
J. Med. Chem. 43, 3205–3208.
(16) Popowicz, G. M., Czarna, A., Wolf S., K. Wang, W. Wang, A. Dömling, and Holak, T.
A. (2010) Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal
new approaches for p53-MDMX/MDM2 antagonist drug discovery. Cell Cycle 15, 1104–1111.
(17) Ding, K. M., Lum, Y., Nikolovska-Coleska, Z., Wang, G., Qiu, S., Shangary, S., Gao,
W., Qin, D., Stuckey, J., Krajewski, K., Roller, P. P., and Wang, S. (2006) Structure-based
design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J. Med. Chem. 15,
3432–3435.
ACS Paragon Plus Environment
21

Page 27 of 31
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
(18) Ding, K., Lu, Y., Nikolovska-Coleska, Z., Qiu, S., Ding, Y., Gao, W., Stuckey, J.,
Krajewski, K., Roller, P. P., Tomita, Y., Parrish, D. A., Deschamps, J. R., and Wang, S. (2005)
Structure-based design of potent non-peptide MDM2 inhibitors. J. Am. Chem. Soc. 127, 10130–
10131.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Michelsen, K., Jordan, J. B., Lewis, J., Long, A. M., Yang, E., Rew, Y., Zhou, J.,
Yakowec, P., Schnier, P. D., Huang, X., Poppe, L., (2012) Ordering of the N-terminus of human
MDM2 by small molecule inhibitors. J. Am. Chem. Soc. 134, 17059–17067.
(20) Bista, M., Wolf, S., Khoury, K., Kowalska, K., Huang, Y., Wrona, E., Arciniega, M.,
Popowicz, G. M., Holak, T. A., and Dömling A. (2013) Transient protein states in designing
inhibitors of the MDM2-p53 interaction. Structure 21, 2143–2151.
(21) Baek, S., Kutchukian, P.S., Verdine, G. L., Huber, R., Holak, T. A., Lee, K. W., and
Popowicz, G. M. (2012) Structure of the stapled p53 peptide bound to Mdm2. J. Amer. Chem.
Soc. 134, 103–106.
(22) Chang, Y. S., Graves, B., Guerlavais V., Tovar, C., Packman, K., To, K. H., Olson, K.
A., Kesavan, K., Gangurde, P., Mukherjee, A., Baker, T., Darlak, K., Elkin, C., Filipovic, Z.,
Qureshi, F. Z., Cai, H., Berry, P., Feyfant, E., Shi, X. E., Horstick, J., Annis, D. A., Manning, A.
M., Fotouhi, N., Nash, H., Vassilev, L. T., and Sawyer, T. K. (2013) Stapled α-helical peptide
drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer
therapy. Proc. Natl. Acad. Sci. U.S.A. 110, E3445–E3454.
(23) Vogel, S. M., Bauer, M. R., Joerger, A. C., Wilcken, R., Brandt, T., Veprintsev, D. B.,
Rutherford, T. J., Fersht, A. R., and Boeckler, F. M. (2012) Lithocholic acid is an endogenous
inhibitor of MDM4 and MDM2. Proc. Natl. Acad. Sci. USA 109, 16906−16910.
ACS Paragon Plus Environment
22

ACS Chemical Biology
Page 28 of 31
1
2
3
4
5
6
7
8
9
(24) Choi, D. S., Huang, S., Huang, M., Barnard, T. S., Adams, R. D., Seminario, J. M., and
Tour, J. M. (1998) Revised structures of N-substituted dibrominated pyrrole derivatives and their
polymeric products. Termaleimide models with low optical band gaps. J. Org. Chem. 48, 2646–
2655.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(25) Bourderioux, A., Routier, S., Bénéteau, V., Mérour, J. Y. (2007) Synthesis of benzo
analogs of oxoarcyriaflavins and caulersine. Tetrahedron 63, 9465–9475.
(26) Molander, G. A., Ham, J., and Seapy, (2007) D. G. Palladium-catalyzed cross-coupling
reaction of alkenyl bromides with potassium alkyltrifluoroborates. Tetrahedron 63, 768–775.
(27) Wang, K., and Liu.; Z.-Z. (2010) Synthesis of N-methyl-bisindolylmaleimide amino acid
methyl ester conjugates and cytotoxicity study. Eur. J. Med. Chem. 45, 4175–4179.
(28) Watson, A. F., Liu, J., Bennaceur, K., Drummond, C. J., Endicott, J. A., Folding, B. T.,
Griffin R. J., Haggerty, K., Lu, X., McDonnell, J. M., Newell, D. R., Noble, M. E. M., Revill, C.
H., Riedinger, C., Xu, Q., Zhao, Y., Lunec, J., and Hardcastle, I. R. (2011) MDM2-p53 protein–
protein interaction inhibitors: A-ring substituted isoindolinones. Bioorg. Med. Chem. Lett. 21,
5916–5919.
(29) Ohkubo, M., Nishimura, T., Jona, H., Honma, T., and Morishima, H. (1996) Practical
synthesis of indolopyrrolocarbazoles. Tetrahedron 52, 8099–8112.
(30) Faul, M. M., Winneroski, L. L., and Krumrich, C. A. (1998) A new, efficient method for
the synthesis of bisindolylmaleimides. J. Org. Chem. 63, 6053–6058.
(31) Mahboobi, S., Dove, S., Kuhr, S., and Pongratz, H. (1999) Synthesis of arcyriarubine
regioisomersi by Pd(0) catalysis or via lithiated indol derivatives – conformational analysis by
semiempirical and X-ray methods. Pharmazie 54, 820–827.
ACS Paragon Plus Environment
23

Page 29 of 31
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
(32) Davis, P. D., Hill, C. H., Lawton, G., Nixon, J. S., Wilinson, S. E., Hurst, S. A., and
Keech, E. (1992) Inhibitors of protein kinase C. 1. 2,3-bisarylmaleimides. J. Med. Chem. 35,
177–184.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(33) Markin, C.J., Spyracopoulos, L. (2012) Increased precision for analysis of protein–ligand
dissociation constants determined from chemical shift titrations. J Biomol NMR 53,125–138.(34)
Powers, R. Advances in nuclear magnetic resonance for drug discovery. (2009) Exp.
Opin. Drug Discv. 4, 1077–1098.
(35) Shuker, S. B., Hajduk, P. J., Meadows, R. P., and Fesik, S. W. (1996) Discovering high-
affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534.
(36) Barile, E., and Pellecchia, M. (2014) NMR-based approaches for the identification and
optimization of inhibitors of protein–protein interactions. Chem. Rev. 114, 4749–4763.
(37) Skinner, A. L., and Laurence, J. S. (2008) High-field solution NMR spectroscopy as a
tool for assessing protein interactions with small molecule ligands. J. Pharm. Sci. 97, 4670–
4695.
(38) Stoll, R., Renner, C., Hansen, S., Palme, S., Klein, C., Belling, A., Zeslawski, W.,
Kamionka, M., Rehm, T., Muhlhahn, P., Schumacher, R., Hesse, F., Kaluza, B., Voelter, W.,
Engh, R. A., and Holak, T. A. (2001) Chalcone derivatives antagonize interactions between the
human oncoprotein MDM2 and p53. Biochemistry 40, 336–344.
(39) Wüthrich K. (1986) NMR of proteins and nucleic acids. Viley-VCH, Weinheim, ISBN:
978–0–471–82893–8.
(40) Meyer, B., and Peters, T. (2003) NMR spectroscopy techniques for screening and
identifying ligand binding to protein receptors. Angew. Chem. Int. Ed. 42, 864–890.
ACS Paragon Plus Environment
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