Harnessing Fluorine-Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue Drugs

Article abstract of DOI:10.1021/acschembio.6b00315

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a K_D = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine-protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C-PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol^-1 atom^-1). High-resolution crystal structures of the Y220C-ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein-fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells.

Full text of DOI:10.1021/acschembio.6b00315

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Article
Harnessing fluorine-sulfur contacts and multipolar
interactions for the design of p53 mutant Y220C rescue drugs
Matthias R. Bauer, Rhiannon N. Jones, Matthias G. J. Baud, Rainer Wilcken,
Frank M. Boeckler, Alan R Fersht, Andreas C. Joerger, and John Spencer
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00315 • Publication Date (Web): 08 Jun 2016
Downloaded from http://pubs.acs.org on June 9, 2016
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Harnessing FluorineꢀSulfur Contacts and Multipolar Interactions for the
Design of p53 Mutant Y220C Rescue Drugs
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#1
#2
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1
Matthias R. Bauer , Rhiannon N. Jones , Matthias G. J. Baud , Rainer Wilcken , Frank
M. Boeckler 3, Alan R. Fersht 1, Andreas C. Joerger *1,2,4,5 and John Spencer *2
1
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical
Campus, Cambridge CB2 0QH, United Kingdom.
2 Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton,
East Sussex BN1 9QJ, UK.
3
Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences,
Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
4
German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120
Heidelberg, Germany
5 Institute of Pharmaceutical Chemistry, Johann Wolfgang GoetheꢀUniversity and Buchmann
Institute for Molecular Life Sciences, MaxꢀvonꢀLaueꢀStr. 9, 60438 Frankfurt am Main,
Germany
# M.R.B and R.N.J. contributed equally to this work
* Corresponding authors
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Tel: +44ꢀ1273 877374
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eꢀmail: joerger@pharmchem.uniꢀfrankfurt.de; j.spencer@sussex.ac.uk
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ABSTRACT
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•
Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable,
including the frequently occurring Y220C mutant. We have previously developed
several smallꢀmolecule stabilizers of this mutant. One of these molecules, PhiKan083,
1ꢀ(9‐ethyl‐9H‐carbazole‐3‐yl)ꢀNꢀmethylmethanamine, binds to a mutationꢀinduced
surface crevice with a KD =150 ꢀM, thereby increasing the melting temperature of the
protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small
molecule ligands can substantially improve binding affinity to their protein targets.
We have, therefore, harnessed fluorineꢀprotein interactions to improve the affinity of
this ligand. Stepꢀwise introduction of fluorines at the carbazole ethyl anchor, which is
deeply buried within the binding site in the Y220CꢀPhiKan083 complex, led to a fiveꢀ
fold increase in affinity for a 2,2,2ꢀtrifluoroethyl anchor (ligand efficiency of 0.3 kcal
molꢀ1 atomꢀ1). Highꢀresolution crystal structures of the Y220Cꢀligand complexes
combined with quantum chemical calculations revealed favorable interactions of the
fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur
of Cys220 at the mutation site. Affinity gains were, however, only achieved upon
trifluorination, despite favorable interactions of the monoꢀ and difluorinated anchors
with the binding pocket, indicating a tradeꢀoff between energetically favorable
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proteinꢀfluorine
interactions
and
increased
desolvation penalties. Taken together, the
optimized carbazole scaffold provides a promising
starting point for the development of highꢀaffinity
ligands to reactivate the tumor suppressor function
of the p53 mutant Y220C in cancer cells.
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INTRODUCTION
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The introduction of fluorine atoms into organic small molecules has become widespread in
drug discovery.1ꢀ5 Indeed, between 20ꢀ25% of drugs on the market are estimated to contain at
least one fluorine atom.6 Apart from modulating various important compound properties such
as logP, metabolic stability, basicity, and bioavailability, fluorine substituents have been also
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successfully used for improving ligand binding affinities.
This can be achieved via
intramolecular stabilization of favorable ligand binding conformations, modulation of
polarity or basicity of neighbouring functional groups of the ligand, or direct fluorineꢀprotein
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interactions.1, These interactions can be further classified into polar interactions with
hydrogen bond donors (e.g. backbone NH, polarized CαꢀH, polar side chains, and protein
bound water), as observed in the binding of type II statins to the HMGCoA enzyme,10
hydrophobic interactions with lipophilic side chains, and orthogonal multipolar interactions,
which can be also described as n→π* interactions,11 with backbone carbonyl groups, amide
containing side chains (Asn and Gln), and guanidinium groups (Arg).
A number of case studies on the use of fluorine interactions in rational drug design have been
reported. Olsen et al. conducted a systematic fluorine scan with a thrombin inhibitor,
revealing favorable interactions with backbone amides and Cα protons that yielded
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significantly improved binding affinity
The introduction of fluorine substituents also
significantly increased the potency of kinesin spindle protein inhibitors, abl kinase inhibitors,
and peptidic elastase inhibitors.13ꢀ16 Vulpetti et al. identified fluorinated fragments binding to
trypsin by 19F NMR screening and crystal structure determination and described a general
approach to identify fluorophilic hotꢀspots in proteins using crystal and computational
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analysis.17 Recently, Pollock et al. investigated the impact of fluorineꢀprotein interactions on
the binding affinity of a meninꢀMLL inhibitor and introduced their computational algorithm
FMAP which aims to facilitate the rational design of fluorineꢀprotein interactions.18
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Here, we have harnessed fluorine interactions for the development of mutant p53 rescue
drugs. The tumor suppressor p53 plays a key role in regulating cellꢀcycle arrest, DNA repair,
apoptosis or cellular senescence.19ꢀ21 In virtually all human cancers, p53 is inactivated either
by mutation or overexpression of negative regulators such as MDM2 or MDMX, which leads
to proteasomal degradation of p53.22 The cancer mutation Y220C, which accounts for an
estimated 100,000 new cancer cases per year worldwide, significantly destabilizes the p53
DNAꢀbinding domain (DBD) and impairs its function via increased thermal denaturation.21, 23
We have previously developed smallꢀmolecule stabilizers of p53ꢀY220C, such as Phikan083,
PhiKan5196, and PhiKan7088 (Figure 1), which bind to a mutationꢀinduced surface crevice
on the DBD, thereby stabilizing the protein, slowing its unfolding and aggregation, and in
some cases restoring tumor suppressor activity in cancer cells harboring the p53ꢀY220C
mutation.24ꢀ28 In this study we aimed at improving the potency of the carbazoleꢀbased
compound Phikan083 and employed ab inito quantumꢀchemical calculations to probe
potential interaction energy gains upon fluorination of the ethyl anchor. We have synthesized
monoꢀ, diꢀ and, triꢀ fluorinated 9Hꢀfluoroethyl carbazoles, evaluated their binding affinities
via differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), and
determined their binding mode by Xꢀray crystallography. We found that trifluorination
significantly improved the binding affinity by approximately fiveꢀfold compared with
PhiKan083 (1), whereas both monofluoro and difluoro analogues were less potent than the
parent compound.
Insert Figure 1 here
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RESULTS AND DISCUSSION
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Quantum chemical calculations
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In the crystal structure of p53ꢀY220C in complex with PhiKan083 (pdb: 2VUK), the ethyl
moiety of PhiKan083 is in close proximity to the carbonyl groups of Leu145 and Trp146, and
the thiol group of Cys220. Given the frequent and wellꢀcharacterized interactions between
organofluorine groups and protein backbone amides, as well as the less frequently observed
interactions between fluorine and sulfur atoms,9 we investigated whether gains in binding
affinity could be achieved via fluorinated ethyl substituents using DFTꢀD calculations at
BLYPꢀD3/def2ꢀSVP level with a truncated model of PhiKan083 bound to the p53ꢀY220C
binding pocket (Figure 2B). Except for the sulfur atom of Cys220, all heavy atoms of the
Y220C binding pocket as well as the nitrogen and Cꢀ3 atom of the pyrrole ligand model were
kept frozen during the calculations.
Distances between the backbone amides of Leu145 and Trp146 and the nearest CꢀF groups
were between 3.0 Å and 3.3 Å in the optimized structures, which is in good agreement with
typical CF…C=O distances (3.0 Å – 3.7 Å) for orthogonal multipolar interactions.16 CꢀF…S
and CꢀF…HS distances ranged from 2.8 Å to 3.6 Å, which is also in good agreement with
experimentally observed distances for fluorineꢀsulfur contacts (2.8 Å – 3.4 Å) in protein
structures.9 Our DFTꢀD3 calculations indicated that the relative interaction energies (ꢁꢁE)
for all fluorinated ethyl groups improved by at least ꢀ2 kcal/mol compared with the
Nꢀethyl
reference energy ꢁE (ꢁE = Ecomplex ꢀ (Ereceptor + Eligand)) (Figure 2B). The energetically most
favorable conformation (ꢁꢁE = ꢀ6.5 kcal/mol) of the 2ꢀfluoroethyl group was orientation 1
(Figure 2B), in which the CꢀF vector points towards the backbone amides of Leu145 and
Trp146, predicting two potential orthogonal multipolar interactions between the fluorine
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atom and both carbonyl groups. Orientations 2 and 3 of the 2ꢀfluoroethyl group, where the
fluorines were oriented towards the sulfhydryl group of Cys220, were energetically less
favorable, with respective ꢁꢁE values of ꢀ2.2 kcal/mol and ꢀ3.7 kcal/mol. The relative
interaction energy of conformation 1 of the difluoro ethyl moiety (ꢁꢁE = ꢀ6.9 kcal/mol) was
similar to the most favored 2ꢀfluoroethyl conformation (see Figure S1 for difluoro ethyl
conformations 2 and 3 and their DFTꢀD energies), whereas the trifluoroꢀsubstituted ethyl
anchor was energetically less favorable with a ꢁꢁE value of ꢀ4.2 kcal/mol (Figure 2B).
However, the calculated DFTꢀD3 energies only yield an estimate of the ligandꢀprotein
interaction at the chosen computational level in a model system of small size and neglect
other contributions to the free energy of binding such as entropic changes and desolvation
penalties.
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Insert Fig 2 here
Compound synthesis
We devised PhiKan083 (
1
) analogues
2
ꢀ
4
(Figure 3) with a monoꢀ, diꢀ or trifluoroethyl
bearing a dimethylamine instead of a
anchor, in addition to their counterparts
5ꢀ6
monomethylamine group. We envisaged that having different substitution patterns (e.g.,
secondary vs tertiary amine) at the level of the pendant, solvent exposed amino group would
provide additional structureꢀactivity relationship and inform on the influence of the amino
side chain on the overall potency and binding mode of 1 and fluorinated analogues.
Insert Fig 3 here
1
3
and 2 were obtained from commercial sources (see Experimental Section). The synthesis of
,
4,
5, and
6
was straightforward, and is described in Scheme 1. Attempts at alkylation of
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intermediate
9
were unsuccessful. Alkylation of 9Hꢀcarbazoleꢀ3ꢀcarbaldehyde (
9
)
with the
5
6
7
8
appropriate alkyl halide or tosylate was achieved in the presence of Cs2CO3 in DMF, and
provided intermediates 7 ꢀ 8. Low yields were due to a competing fluorine elimination
pathway which has been previously reported by Suehiro et al..30 Further introduction of the
9
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amine side chain by reductive amination afforded 3, 4, 5, and 6. Two reductive amination
procedures were attempted; titanium (IV) isopropoxideꢀsodium borohydride and sodium
triacetoxyborohydride and yields in both cases were comparable.31, 32 Indeed, the titaniumꢀ
based method was used for the reductive amination of
triacetoxyborohydride would be equally effective.
7 and 8 but we anticipate that sodium
Scheme 1. Synthesis and overall yield of compounds 3, 4, 5 and 6 using the twoꢀstep synthesis
described.
O
R2
O
a
b
N
R1
N
R1
N
H
F
3, 4, 5, 6
7 =
8 =
F
c
CF3
N
N
a
N
R1
N
H
9
Synthesis of 3, 4, 5 and 6. (a) R1I (difluoroethyl) or R1OTs (trifluoroethyl), Cs2CO3, DMF, µwave, 150°C, 30 min; (b)
amine, Ti(OiPr)4, Et3N, EtOH/DCM, rt, 18 h then NaBH4, rt, 8 h; (c) NaBH(OAc)3, HNMe2, AcOH, THF, rt.
Yield
(step a)
Yield
(step b)
Compound
R1
R2
44
50
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40
3
4
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NA a
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a not applicable, precursor was bought from TCI UK.
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Biophysical evaluation
We tested PhiKan083 (
binding domain (DBD) using differential scanning fluorimetry (DSF) (Table 1). The
trifluoroethyl substituted carbazoles and increased stability of the p53ꢀY220C DBD by
1.2 K at a compound concentration of 125 ꢂM, showing a clear improvement over their
ethyl substituted counterparts PhiKan083 ( ) and ꢁTm = 0.8 K and ꢁTm =0.6 K,
1
) and analogues
2
ꢀ
6
for their stabilization of the p53ꢀY220C DNAꢀ
N
ꢀ2,2,2ꢀ
4
6
N
ꢀ
1
5 (
respectively). In contrast, the difluorinated (2) and monofluorinated (3) carbazoles were
significantly worse than the parent compound in terms of protein stabilization, with
respective thermal shifts of 0.3 K and 0.4 K. Dissociation constants (
isothermal titration calorimetry (ITC) (Table 1). The substitution pattern of the solventꢀ
exposed amine had a minor effect on affinity, with the additional methyl group in resulting
in a 30% drop in affinity. The trifluoroꢀsubstituted carbazoles and were the most potent
KD) were determined by
5
4
6
compounds and showed ITC KD values of 28 and 37 µM, respectively (Figure 4), which
corresponds to a 5ꢀfold increase in affinity compared to the nonꢀfluorinated parent
compounds (
calculations, the monoꢀ and difluorinated analogues,
affinities. They had D values of 101 ꢂM and 138 ꢂM, respectively (Figure S2).
1
) and (
5
). Despite prediction of improved interaction energies by DFTꢀD
2
and , failed to display improved
3
K
Table 1. Thermal shift data and KD values
DSF ꢀTm at
125 µM
ITC KD
(µM)
compound
1
0.8
124a
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0.4
0.3
1.2
0.6
1.2
138
101
28
2
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169
37
a data taken from Boeckler et al.24
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Insert Figure 4 here
Crystal structures
We determined highꢀresolution crystal structures of the p53ꢀY220C DBD in complex with
2,
3
, and (Table 2) in order to establish the binding modes of monoꢀ, diꢀ and trifluorinated
6
Phikan083 derivatives. All structures were determined in the same space group as for the
parent compound PhiKan083 and contained two molecules in the asymmetric unit (chains A
and B). There was excellent electron density for the ligand in chain B in all cases. For the
ligand in chain A, there was only partial occupancy for compounds
2 and 3, consistent with
the Y220CꢀPhiKan083 complex structure.24 Numbers given in the following therefore refer to
chain B, unless stated otherwise. The binding mode of the carbazoles was almost identical to
that of PhiKan083 (1). Small differences in the orientation of the central carbazole scaffold
can be attributed to the different methylation state of the amine moiety by comparison with
other structures of Y220C ligand complexes (Joerger AC unpublished data).
Upon monofluorination, the fluorine atom of
2 points towards the carbonyl groups of Leu145
and Trp146 with a F…C=O interaction distances of 3.4 Å (Figure 5A). The CꢀF vector and
the planes of the backbone carbonyl group of Leu145 and Trp146 show a nearly orthogonal
arrangement, with respective O=C…F angles of 97.5° and 80.1°, which is in good agreement
with O=C…F angles found in the PDB and CSD for orthogonal multipolar interactions.9, 16
This preferred orientation of the CꢀF vector in the crystal structure is also in good agreement
with our initial DFTꢀD predictions (Figure 5A). Similar to the Y220CꢀPhikan083 complex,
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the side chain of Cys220, in immediate vicinity to the ethyl anchor, adopts two alternative,
albeit very similar conformations.
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For the Nꢀ2ꢀdifluoroethyl anchor of 3, we observed two alternative conformations in the p53ꢀ
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Y220C binding pocket (Figure 5B and C). In both conformations, one fluorine atom interacts
with the carbonyl groups of Leu145 and Trp146 in an almost identical fashion as observed
for compound 2. The second fluorine interacts with the thiol group of Cys220, pointing either
towards Pro151 (Figure 5C) or towards Leu145 at the bottom of the binding pocket (Figure
5B), which is essentially the result of a 120° rotation around the ethyl anchor CꢀC bond.
Interestingly, only one of the two Cys conformations was observed in chain A, coinciding
with a preferential orientation of the difluoroethyl anchor in the orientation highlighted in
Figure 5B whereby the fluorine forms a weak hydrogen bond with the SH group of Cys220.
In both orientations, the fluorine atom interacts with the thiol group at a distance of about 3.2
Å, which is more or less the sum of the van der Waals radii of sulfur and fluorine (rF
S = 1.80 Å).1, 33
=
1.47Å;
r
In the structure of the most potent compound
6, the CF3 group aligns well with the different
fluorine positions observed for monofluorinated
2
and difluorinated 3 (Figure 5D), i.e., it
interacts with the backbone carbonyl groups of Leu145 and Trp146 as well as with the thiol
group of Cys220. Analysis of CꢀS…F angles in the complexes with di and trifluorinated
compounds suggests that fluorine interacts with Cys220 via weak hydrogen bonding with the
polarized proton of the thiol function and via sulfur
180˚.
σ
ꢀhole bonding34ꢀ36 at an angle close to
Table 2. Xꢀray data collection and refinement statistics of Y220Cꢀcarbazole complexes
Compound
2
3
6
Data Collection
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Space Group
a (Å)
P212121
65.03
71.14
105.14
2
P212121
64.05
71.14
104.17
2
P212121
65.16
71.45
105.23
2
b (Å)
c (Å)
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53
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Molecules/AU
Resolution (Å)a
29.6ꢀ1.38
29.6ꢀ1.35
29.6ꢀ1.48
(1.45ꢀ1.38)
(1.42ꢀ1.35)
(1.56ꢀ1.48)
Unique reflections
Completeness (%)a
Multiplicitya
Rmerge (%)a
99,536
98.9 (98.6)
4.8 (4.8)
3.9 (50.6)
19.2 (3.4)
13.4
107,270
99.7 (99.9)
4.9 (4.9)
4.5 (54.9)
16.2 (3.5)
11.8
82,444
99.9 (100.0)
5.4 (5.5)
5.3 (48.7)
16.5 (3.7)
13.0
Mean I/σ(I)a
Wilson B value (Å2)
Refinement
Rwork, (%)b
14.6
17.0
14.7
16.9
14.4
17.1
Rfree, (%)b
No. of atoms
Proteinc
3187
2
3166
2
3186
2
Zinc
Water
460
430
50
466
44
Ligands
48
RMSD bonds (Å)
RMSD angles (°)
Mean B (Å2)
PDB ID
0.005
0.08
23.4
5G4M
0.005
0.8
0.005
0.8
21.2
5G4N
21.3
5G4O
aValues in parentheses are for the highest resolution shell.
bRwork and Rfree = ∑||Fobs| ꢀ |Fcalc||/∑|Fobs|, where Rfree was calculated with 5 % of the reflections chosen at random
and not used in the refinement.
cNumber includes alternative conformations.
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Discussion
Incorporation of fluorine atoms into the
PhiKan083 ( ) yielded two compounds,
stabilization (1.2 K at 125 ꢂM) and Y220C binding affinity. Compared with the parent
compounds PhiKan083 ( ) and 5, the ITC KD values improved by a factor of 5 and the free
N
ꢀethyl anchor of the p53ꢀY220C stabilizer
1
4
and , with substantially increased p53ꢀY220C
6
1
energy of binding by approximately ꢀ0.9 kcal/mol (at 293K). Typically, CꢀF…C=O
orthogonal multipolar interactions with optimal geometry raise the binding free enthalpy by
37
about ꢀ0.2 to ꢀ0.3 kcal/mol, which suggests that the observed CꢀF…C=O interactions with
the carbonyl groups of Leu145 and Trp146 are the main factor for the potency increase of the
trifluorinated PhiKan083 derivatives. The DFTꢀD calculations are also consistent with the
binding modes
2 and 3 observed in our crystal structures, as the preferred orientation of
fluorine towards the backbone carbonyl groups of Leu145 and Trp146 in these structures
indicates that interactions of the protein carbonyl groups with fluorine atoms contribute more
strongly to the affinity increase than interactions with the thiol group of Cys220 or apolar
protons of hydrophobic side chains. The preferred interaction with the protein backbone then
seems to direct the orientation of additional fluorine atoms in the binding pocket, interacting
with the thiol group of Cys220 via either a hydrogen bond or a planar sulfur
interaction.
σꢀhole
Although DFTꢀD calculations predicted improved interaction energies for monoꢀfluorinated
and diꢀfluorinated PhiKan083 derivatives, this was not reflected in the experimentally
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determined dissociation constants and stability data for compounds
2 and 3, which were close
5
6
7
8
to that of the parent compound PhiKan083. These discrepancies between theoretical and
experimental data are likely due to different desolvation penalties associated with each
fluorinated group. Fluorine can act as a weak hydrogenꢀbond acceptor in a 2,2ꢀdifluoromethyl
group and even more strongly in a 2ꢀfluoromethyl group, although hydrogenꢀbond strength
was found to be significantly weaker than for the conventional hydrogenꢀbond acceptor
acetophenone.38 For example, difluoroalkyl groups have been used as thiol surrogates in drug
discovery because of their similar steric properties and the acidity of the terminal hydrogen
resulting from the high polarization of the CꢀH bond by the geminal fluorine atoms.39 To
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estimate desolvation penalties of fluorinated
energies for ꢀethylcarbazole, ꢀ2ꢀfluoroethylcarbazole,
ꢀ2,2,2ꢀtrifluoroethylcarbazole with Jaguar using the SM8 water model at BLYPꢀD3/6ꢀ
N
ꢀethyl anchors, we calculated solvation
N
N
N
ꢀ2,2ꢀdifluoroethylcarbazole, and
N
31G** level of theory (Table 3 and Figure S3).40 Both mono and difluorinated carbazoles
showed solvation energies that were by ꢀ1.0 and ꢀ2.8 kcal/mol larger than the solvation
energy of
to ꢀethylcarbazole (ꢀ3.1 kcal/mol). Hence, the higher polarity of the 2ꢀfluoroethyl and 2,2ꢀ
difluoroethyl moieties in the PhiKan083 derivatives and and the associated desolvation
Nꢀethylcarbazole. The solvation energy of the trifluorinated carbazole was identical
N
2
3
penalties are most likely the key factors counteracting and quenching favorable fluorineꢀ
protein interactions in these compounds.
In conclusion, targeting Cys220 and the backbone carbonyl groups of Leu145 and Trp146 via
fluorine bonding helped to further optimize the carbazole scaffold.
Nꢀ3,3,3ꢀTrifluoroethyl
substituted carbazoles
4 and 6
exhibit a high ligand efficiency (LE = 0.3 kcal molꢀ1 atomꢀ1),
although binding is still relatively weak for these fragmentꢀlike molecules. Nevertheless, they
represent a promising starting point for the development of high affinity smallꢀmolecule
stabilizers of the oncogenic Y220C mutant.
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Table 3. Calculated solvation energies for DFTꢀD3 optimized carbazoles
3
4
5
6
7
8
solvation energy
carbazole anchor a
(kcal/mol)b
9
Nꢀethyl
ꢀ3.1
ꢀ5.9
ꢀ4.1
ꢀ3.1
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Nꢀ2ꢀfluoroethyl
Nꢀ2,2ꢀdifluoroethyl
Nꢀ2,2,2ꢀtrifluoroethyl
aSubstitution of 9Hꢀcarbazole. Structures of DFTꢀD3 optimized compounds are shown in Figure S3.
bSolvation energies were calculated from single point calculations of the global minimum conformation of each compound
in Jaguar at BLYPꢀD3/6ꢀ31G** level using the SM8 water model.
MATERIALS & METHODS
Protein expression and purification
Stabilized p53ꢀY220C DBD (residues 94ꢀ312) was expressed and purified as previously
described.26
Differential Scanning Fluorimetry (DSF)
DSF was performed as described.26 Briefly, melting temperatures (
Tm) were determined using
8 µM protein (stabilized p53ꢀY220C DBD) and 10x SYPRO orange (Life Technologies) in a
25 mM KPi pH 7.2, 150 mM NaCl, 1mM TCEP assay buffer at a final DMSO concentration
of 5% (v/v). ꢁTm values were calculated as: ꢁTm = Tm (protein+compound) ꢀ Tm (protein)).
All samples were measured in triplicate.
Isothermal Titration Calorimetry (ITC)
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ITC experiments were conducted as described.26 Samples for the cell unit contained 50 ꢂM
protein in a 25 mM KPi pH 7.2, 150 mM NaCl, 1mM TCEP, 5% (v/v) DMSO assay buffer.
The syringe contained 2 ꢀ 5 mM compound in the same assay buffer.
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Quantum chemical calculations
QM calculations were conducted with TURBOMOLE 6.4, applying a BLYP(RI)ꢀD3ꢀdef2ꢀ
SVP (DFTꢀD3) level of theory. A truncated model of the p53 Y220C binding pocket with an
Nꢀethylꢀpyrrole ligand (truncated PhiKan083 model), comprising 140 atoms, was derived
from the crystal structure (pdb code: 2VUK). For geometry optimization all heavy atoms of
the binding pocket, except the sulfur atom of Cys220, and the nitrogen atom plus one carbon
atom of the ligand pyrrole ring were frozen (see supporting information). Interaction energies
were calculated as ꢁE = Ecomplex ꢀ (Ereceptor + Eligand). Global minimum conformers, which
were used as input structures for solvation energy calculations, were determined by DFTꢀD3
calculations at BLYP(RI)ꢀD3ꢀdef2ꢀTZVP level of theory with TURBOMOLE 6.4. For Nꢀ2ꢀ
Fluoroethylcarbazole and Nꢀ2,2ꢀDifluoroethylcarbazole, we rotated the CꢀC axis of the ethyl
moiety stepꢀwise by 60˚ to obtain six different conformers that were used as input structures
for the DFTꢀD3 calculations. Solvation energies were calculated with Jaguar 8.9
(Schrödinger, USA, NY) as single point calculations using the SM8 water model at BLYPꢀ
D3/6ꢀ31G** level of theory.
Xꢀray crystallography
Crystals of the p53ꢀY220C DBD were grown at 18
°
C as described.41 Crystals were soaked
for either 90 min ( ) or 4 h ( and ) in a 20 mM solution of compound in cyro buffer (19%
6
2
3
polyethylene glycol 4000, 20% glycerol, 10 mM sodium phosphate, pH 7.2, 100 mM Hepes,
pH 7.2, 150 mM NaCl) and flash frozen in liquid nitrogen. Xꢀray data sets were collected at
100 K at beamlines I03 and I04 of the Diamond Light Source, Oxford, UK. Indexing of the
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datasets was performed using XDS and scaling using the program SCALA within the
CCP4 software suite 44. The structures of the Y220Cꢀcarbazole complexes were then solved
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using the program PHENIX:
initial rigid body refinement using PDB entry 2J1X as a
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starting model. The models were further refined using iterative cycles of manual model
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building in COOT and refinement with PHENIX. While there was clear electron density
for the ligands in chain B, there was only weak ligand density in chain A of the Y220C
complex with
2 and 3, indicating partial occupancy. The same observation had been made for
the parent compound PhiKan083.24 Accordingly, alternative states of the binding pocket were
refined: bound ligand and water network of the ligandꢀfree structure. Data collection and
refinement statistics are given in Table 2. Structural figures were prepared using PyMOL
(www.pymol.org). The coordinates and structure factors of the complexes are deposited in
the Protein Data Bank (PDB ID: 5G4M, 5G4N, 5G4O).
General Procedures
All reactions were carried out in air unless otherwise stated, using commercial grade starting
materials, solvents and reagents. The progress of all reactions was monitored by thin layer
chromatography (TLC) using commercially available glass silica gel plates (60 Å, F254). The
mobile phase was generally a solvent mixture and the visualisation was undertaken using UV
light. Microwave reactions were conducted in a CEM Discovery microwave reactor. All
NMR spectra were measured on a Varian NMR 500 spectrometer at either 500 MHz (1H) or
126 MHz (13C). Chemical shifts are quoted in parts per million (ppm) (δ relative to a residual
solvent peak for 1H and 13C). Chromatographic purifications were undertaken using an ISCO
purification unit, Combi Flash RF 75 PSI, using Biotage silica gel columns. LCꢀMS purity
analyses were undertaken using a 5 ꢂm C18 110 Å column. Percentage purities were
performed using a 30 min method in water/acetonitrile with 0.1% formic acid (5 min at 5%,
5–95% over 20 min, 5 min at 95%) with the UV set to 254 nm. All highꢀresolution mass
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spectrometry was carried out at the EPSRC UK National Mass Spectrometry Facility
(NMSF), Swansea University, using a Thermo Scientific LTQ Orbitrap XL spectrometer.
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Materials: 1ꢀ(9ꢀ(2ꢀFluoroethyl)ꢀ9Hꢀcarbazolꢀ3ꢀyl)ꢀNꢀmethylmethanamine (
from Enamine; its purity was assessed by LCꢀMS and found to be higher than 95% (LCꢀMS
purity > 95% (UV), ret. time = 10.83 min). 9ꢀEthylꢀ9 ꢀcarbazoleꢀ3ꢀcarbaldehyde was
2) was purchased
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H
purchased from TCI UK (>98% purity by manufacturer). 1ꢀIodoꢀ2,2ꢀdifluoroethane and
2,2,2ꢀtrifluoroethyl pꢀtoluenesulfonate were purchased from fluorochem. 9HꢀCarbazoleꢀ3ꢀ
carbaldehyde was synthesized according to a known procedure.29 Final compounds that were
tested had an LC purity of >95%.
Representative procedure for the synthesis of intermediates 7ꢀ8
9ꢀ(2,2ꢀDifluoroethyl)9Hꢀcarbazoleꢀ3ꢀcarbaldehyde (7). To a suspension of 9Hꢀcarbazoleꢀ3ꢀ
carbaldehyde (196 mg, 1 mmol) and Cs2CO3 (652 mg, 2 mmol) in anhydrous DMF (5 mL)
was added 1ꢀiodoꢀ2,2ꢀdifluoroethane (172 mg, 2 mmol). The resulting suspension was stirred
under microwave irradiation at 150 °C for 30 minutes. The suspension was cooled and
diluted with H2O (20 mL). Crude product was extracted with ethyl acetate (EtOAc) (3 X 20
mL). The organic extracts were washed with brine (3 X 20 mL) and dried over anhydrous
MgSO4. The suspension was filtered and was concentrated to dryness in vacuo. Crude
product was purified by chromatography on silica gel using dichloromethane (DCM) as an
eluent to yield product as a pale yellow solid, yield 44% (115 mg, 0.44 mmol). 1H NMR (500
MHz, DMSOꢀd6) δ 10.06 (s, 1H), 8.73 (s, 1H), 8.33 – 8.17 (m, 1H), 7.99 (d, J = 8.4 Hz, 1H),
7.81 (d, J = 8.4 Hz, 1H), 7.76 – 7.66 (m, 1H), 7.60 – 7.47 (m, 1H), 7.36 – 7.24 (m, 1H), 6.50
(tt, 2JFH = 54.5 Hz, 3JHH = 3.2 Hz, 1H), 4.97 (td, 3JFH = 16.1 Hz, 3JHH = 3.2 Hz, 2H). 13C NMR
(126 MHz, DMSOꢀd6) δ 192.3, 144.6, 141.7, 129.4, 127.3, 127.2, 124.0, 123.0, 122.8, 121.1,
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121.0, 115.1 (t, JFC = 241.9 Hz), 110.9, 110.8, 45.0 (t, JFC = 24.5 Hz). HRMSꢀESI (m/z)
found 260.0884, calcd 260.0881 for [C15H11F2NO+H]+.
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9
9ꢀ(2,2,2ꢀTrifluoroethyl)ꢀ9Hꢀcarbazoleꢀ3ꢀcarbaldehyde (8). Using 2,2,2ꢀtrifluoroethyl pꢀ
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toluenesulfonate as an electrophile. Reaction carried out on a 0.48 mmol scale. Pale yellow
1
solid, yield 50% (68 mg, 0.24 mmol). H NMR (500 MHz, DMSOꢀd6) δ 10.07 (s, 1H), 8.75
(s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.80 (d, J
3
= 8.3 Hz, 1H), 7.61 – 7.49 (m, 1H), 7.40 – 7.25 (m, 1H), 5.51 (q, JFH = 9.3 Hz, 2H).13C
1
NMR (126 MHz, DMSOꢀd6) δ 192.3, 144.2, 141.3, 129.8, 127.5, 127.4, 125.4 (q, JFC
=
2
281.8 Hz), 124.0 123.3, 122.9, 121.5, 121.2, 110.9, 110.8, 44.1 (q, JFC = 33.5 Hz). HRMSꢀ
ESI (m/z) found 278.0789, calcd 278.0787 for [C15H10F3NO+H]+.
Representative procedure for the synthesis of compounds 3, 4, 5 and 6
1ꢀ(9ꢀ(2,2ꢀDifluoroethyl)ꢀ9Hꢀcarbazolꢀ3ꢀyl)ꢀNꢀmethylmethanamine (3). To a solution of 7 (75
mg, 0.29 mmol) in anhydrous EtOH/DCM (10 mL) was added methylamine hydrochloride
(39 mg, 0.58 mmol), triethylamine (61 ꢂL, 0.44 mmol) and titanium (IV) isopropoxide (172
ꢂL, 0.58 mmol). The resulting solution was stirred at room temperature for 18 h before the
addition of sodium borohydride (22 mg, 0.58 mmol). The solution was stirred at room
temperature for 8 hours before pouring into 2M aqueous ammonia (25 mL). The suspension
was filtered through celite and to the filtrate was added H2O. Crude product was extracted
with DCM, dried over anhydrous K2CO3. The suspension was filtered and the filtrate was
concentrated in vacuo to yield crude product that was purified by chromatography on silica
gel using DCM/MeOH 9/1 as an eluent to yield product as a pale yellow oil, yield 58% (46
1
mg, 0.16 mmol). H NMR (500 MHz, Acetonitrileꢀd3) δ 8.19 – 8.13 (m, 1H), 8.10 (s, 1H),
7.62 – 7.56 (m, 1H), 7.56 – 7.45 (m, 3H), 7.32 – 7.25 (m, 1H), 6.31 (tt,2JFH = 55.0 Hz, 3JHH
=
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3.2 Hz, 1H), 4.76 (td, JFH = 15.6 Hz, JHH = 3.2 Hz, 2H), 3.89 (s, 2H), 2.43 (s, 3H) (NH
missing due to DꢀH exchange). 13C NMR (126 MHz, Acetonitrileꢀd3) δ 141.1, 139.9, 132.5,
126.6, 125.9, 122.9, 122.8, 120.1, 119.7, 119.6, 114.8 (t, 1JFC = 241.9 Hz), 109.3, 109.0, 55.7,
44.9 (t, 2JFC = 25.6 Hz), 35.1. LCꢀMS purity = 96% (UV), ret. time = 11.01 min. HRMSꢀESI
(m/z) found 275.1353, calcd 275.1354 for [C16H16F2N2+H]+.
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1ꢀ(9ꢀ(2,2,2ꢀTrifluoroethyl)ꢀ9Hꢀcarbazolꢀ3ꢀyl)ꢀNꢀmethylmethanamine (4). Reaction carried
out on a 0.31 mmol scale. Product isolated as a pale yellow solid, yield 40% (37 mg, 0.12
1
mmol). H NMR (500 MHz, acetonitrileꢀd3) δ 8.20 – 8.14 (m, 1H), 8.12 (s, 1H), 7.64 – 7.58
(m, 1H), 7.58 – 7.47 (m, 3H), 7.36 – 7.28 (m, 1H), 5.07 (q, 3JFH = 9.1 Hz, 2H), 3.89 (s, 2H),
2.43 (s, 3H) (NH missing due to DꢀH exchange). 13C NMR (126 MHz, DMSOꢀd6) 140.9,
1
140.2, 128.3, 127.7, 126.6, 126.1 (q, JFC = 281.2 Hz), 122.9 122.8, 121.3, 120.6, 120.5,
2
110.4, 110.1, 53.9, 44.0 (q, JFC = 33.4 Hz), 34.2. LCꢀMS purity = 97% (UV), ret. time =
11.55 min. HRMSꢀESI (m/z) found 293.1259, calculated 293.1260 for [C16H15F3N2+H]+.
1ꢀ(9ꢀEthylꢀ9
H
ꢀcarbazolꢀ3ꢀyl)ꢀN,Nꢀdimethylmethanamine (
5
). Reaction carried out on a 0.44
1
mmol scale. Product isolated as a white solid, yield 82% (91 mg, 0.36 mmol). H NMR (500
MHz, Chloroformꢀ
(m, 1H), 4.37 (q,
d
) δ 8.14 – 8.10 (m, 1H), 8.06 (s, 1H), 7.51 – 7.34 (m, 4H), 7.26 – 7.20
J
= 7.2 Hz, 2H), 3.64 (s, 2H), 2.33 (s, 5H), 1.45 (t, J
= 7.2 Hz, 3H). 13C
NMR (126 MHz, Chloroformꢀd) δ 140.28, 139.3, 129.3, 127.1, 125.5, 122.9 (2C –
quaternary), 121.0, 120.4, 118.7, 108.4, 108.1, 64.7, 45.3 (2C), 37.5, 13.8. LCꢀMS purity
97% (UV), ret. time = 11.36 min. HRMSꢀESI (m/z) found 208.1120, calculated 208.1121 for
[C15H14N]+ (loss of NMe2).
1ꢀ(9ꢀ(2,2,2ꢀTrifluoroethyl)ꢀ9Hꢀcarbazolꢀ3ꢀyl)ꢀN,Nꢀdimethylmethanamine
(6
).
Reaction
carried out on a 0.10 mmol scale. Product isolated as a white solid, yield 56% (17 mg, 0.05
1
mmol). H NMR (500 MHz, chloroformꢀd) δ 8.04 – 8.00 (m, 1H), 7.99 (s, 1H), 7.45 – 7.39
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(m, 2H), 7.37 – 7.29 (m, 2H), 7.27 – 7.20 (m, 1H), 4.73 (q, 3JFH = 8.7 Hz, 2H), 3.62 (s, 2H),
2.28 (s, 6H). 13C NMR (126 MHz, chloroformꢀd) δ 140.9, 140.1, 129.8, 127.8, 126.4, 124.3
(q, 1JFC = 281.5 Hz), 123.6, 123.4, 121.3, 120.6, 120.4, 108.7, 108.5, 45.3 (q, 2JFC = 35.7 Hz),
44.9 (2C). LCꢀMS purity > 99% (UV), ret. time = 11.68 min. HRMSꢀESI (m/z) found
307.1419, calcd 307.1417 for [C16H17F3N2+H]+.
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1ꢀ(9ꢀCarbazolꢀ3ꢀyl)ꢀN,Nꢀdimethylmethanamine (9). To a solution of 9Hꢀcarbazolꢀ3ꢀ
carbaldehyde (45 mg, 0.26 mmol) in anhydrous THF (5 mL) was added dimethylamine (130
µL, 2.0 M solution in THF, 0.26 mmol) and acetic acid (12 µL, 0.26 mmol). Sodium
triacetoxyborohydride (80 mg, 0.39 mmol) was added and the resulting solution was stirred
for 18 h. Solvent was removed under a reduced pressure and to the residue was added DCM
and H2O. The solution was filtered through a hydrophobic frit and the filtrate was
concentrated in vacuo to yield crude product which was purified by chromatography on silica
gel (using DCM/MeOH 9/1 as an eluent) to yield product as a pale yellow solid, yield 88%
1
(51 mg, 0.22 mmol). H NMR (500 MHz, chloroformꢀ
d
) δ 8.59 (s, 1H), 8.09 – 8.03 (m, 1H),
8.02 (s, 1H), 7.46 – 7.38 (m, 3H), 7.38 – 7.33 (m, 1H), 7.25 – 7.18 (m, 1H), 3.77 (s, 2H), 2.40
(s, 6H).13C NMR (126 MHz, chloroformꢀ
d
) δ 139.9, 139.2, 127.5, 126.8, 125.9, 123.3, 123.0,
121.4, 120.3, 119.4, 110.7, 110.6, 63.9, 44.2 (2C). HRMSꢀESI (m/z) found 180.0805,
calculated 180.0808 for [C13H10N]+ (loss of NMe2).
ACKNOWLEDGEMENTS
This work was funded by Worldwide Cancer Research Grant 14ꢀ1002 ’Rescuing thermally
unstable p53 mutants with small molecule stabilisers; new targeted cancer therapies’ and
ERC Advanced Grant 268506. RJ is funded by the University of Sussex (PhD studentship).
We thank the staff at Diamond beamlines I03 and I04 for technical assistance during data
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collection. Access was supported in part by the EU FP7 infrastructure grant BIOSTRUCTꢀX
(contract no. 283570).
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Supporting Information
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Additional DFTꢀD data and coordinates used for calculations; ITC data for 2, 3, and 5; global
minimum conformers of fluorinated carbazole analogues and their calculated solvation
energies; scans of NMRs; LC purity.
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Harnessing Fluorine-Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue
Drugs
Matthias R. Bauer, Rhiannon N. Jones, Matthias G. J. Baud, Rainer Wilcken, Frank M. Boeckler, Alan R.
Fersht, Andreas C. Joerger, and John Spencer
81x81mm (600 x 600 DPI)
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H
N
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PhiKan083 (1)
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N
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OH
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HN
PhiKan5196
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A
Structure of p53-
Y220C binding pocket
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T150
PhiKan083
P151
V147
1-(9-ethyl-9H-carbazol-3-yl)
-N-methylmethanamine
W146
L145
10
C220
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B
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DFT-D optimized models of fluorinated N-ethyl anchors
P151
V147
C220
L257
W146
L145
N-ethyl
N-2-fluoroethyl c. 1
N-2-fluoroethyl c. 2
N-2-fluoroethyl c. 3
N-2,2-difluoroethyl c. 1
N-2,2,2-trifluoroethyl
N-2,2-
N-2-
N-2-
N-2-
9H-carbazole
substitution
N-2,2,2-
N-ethyl
fluoroethyl fluoroethyl fluoroethyl difluoroethyl
trifluoroethyl
conf. 1
conf. 2
conf. 3
conf. 1
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DFT-D ΔΔE
0.0
-6.5 -2.2 -3.7
-6.9
-4.2
(kcal/mol)
48

HN
HN
HN
HN
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N
N
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F
F
F
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F
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PK083 (1)
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Time (min)
Time (min)
A
B
0
10
20
30
40
0
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20
30
40
1
2
0.00
0.00
-0.10
-0.20
-0.30
3
4
5
6
-0.10
-0.20
7
-0.30
8
9
-0.40
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13
0.0
14
-0.50
-0.40
0.0
15
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17
-0.5
-0.5
-1.0
18
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26
-1.0
KD (ITC) = 28.3 µM
KD (ITC) = 37.2 µM
-1.5
-1.5
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[compound 4]T/[p53]T
[compound 6]T/[p53]T