N-alkylated oligoamide α-helical proteomimetics

Article abstract of DOI:10.1039/c001164a

Generic approaches for the design and synthesis of small molecule inhibitors of protein-protein interactions (PPIs) represent a key objective in modern chemical biology. Within this context, the α-helix mediated PPIs have received considerable attention as targets for inhibition using small molecules, foldamers and proteomimetics. This manuscript describes a novel N-alkylated aromatic oligoamide proteomimetic scaffold and its solid-phase synthesis - the first time such an approach has been used for proteomimetics. The utility of these scaffolds as proteomimetics is exemplified through the identification of potent μM inhibitors of the p53-hDM2 helix mediated PPI - a key oncogenic target.

Full text of DOI:10.1039/c001164a

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Organic &
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Volume 8 | Number 10 | 21 May 2010 | Pages 2269–2480
ISSN 1477-0520
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Marcel Jaspers et al.
FULL PAPER
Andrew J. Wilson et al.
N-Alkylated oligoamide -helical
proteomimetics
Dermacozines, a new phenazine family
from deep-sea dermacocci Isolated
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N-alkylated oligoamide a-helical proteomimetics†
Frederick Campbell,a Jeffrey P. Plante,a Thomas A. Edwards,*b Stuart L. Warriner*a and Andrew J. Wilson*a
Received 18th January 2010, Accepted 5th March 2010
First published as an Advance Article on the web 18th March 2010
DOI: 10.1039/c001164a
Generic approaches for the design and synthesis of small molecule inhibitors of protein–protein
interactions (PPIs) represent a key objective in modern chemical biology. Within this context, the
a-helix mediated PPIs have received considerable attention as targets for inhibition using small
molecules, foldamers and proteomimetics. This manuscript describes a novel N-alkylated aromatic
oligoamide proteomimetic scaffold and its solid-phase synthesis—the first time such an approach has
been used for proteomimetics. The utility of these scaffolds as proteomimetics is exemplified through the
identification of potent mM inhibitors of the p53–hDM2 helix mediated PPI—a key oncogenic target.
its solid phase synthesis and illustrate its use as a platform for the
discovery of potent inhibitors of the p53–hDM2 interaction.
Introduction
The elaboration of generic approaches for competitive inhibition
of protein–protein interactions (PPIs) is recognised as a major
goal of contemporary chemical biology.1–4 The challenge arises
Results and discussion
We recently described the synthesis and cyclisation of N-alkylated
aromatic oligoamides53,54 facilitated by the preferred cis geometry55
first observed by Shudo and co-workers56 for N-alkylated aromatic
oligoamides/ureas. We observed that in an extended conforma-
tion, the N-alkyl substituents mimic the side chain presentation of
functionality made by the i, i + 4 and i + 7 (8) side chains of an
a-helix. Encouraged by Rebek’s57 observations that self-assembled
capsules can revert the intrinsic preference for the cis amide
conformer through non-covalent interaction with the trans form,
we reasoned that N-alkylated trimers might similarly adopt the
desired extended conformation and inhibit a-helix mediated PPIs.
Fig. 1 shows a model of the helix mimetic in an extended confor-
mation and a comparison with the a-helix of the p53 peptide from
the crystal structure of p53–hDM2 (here the p53 peptide makes key
contacts from Phe 19, Trp 23 and Leu 26 to the cleft on hDM2).8
from the difficulty in designing ligands that can mimic a natural
protein partner; covering a large surface area of the target
protein, and matching the diverse and discontinuous non-covalent
interactions made at the protein–protein interface. For the a-
helix mediated class of PPI5 in which an a-helix from one
protein binds to a complementary cleft on its protein partner
(e.g. Bcl-2 family6,7 and p53-hDM28–10 interactions) such a general
approach is starting to emerge. In addition to the various small
molecule inhibitors of the Bcl-2 family7,11–14 and p53–hDM29,15–18
interactions, oligomers that mimic the spatial and compositional
presentation of a-amino acid side chains made by the interacting
helix have been identified. In the first approach, b-peptides,19–21
mixed a/b peptides22–24 and other foldamers25–31 closely mimic
both the core helical structure and side chain presentation found
within the helical motif whilst offering modular solid phase
syntheses. Proteomimetic32 scaffolds32–50 first introduced by the
Hamilton group, mimic only the side chain presentation that
is key to protein surface recognition; however, robust methods
for generation of screening libraries are less well defined.47,49,50
Amongst the proteomimetic scaffolds, aromatic oligoamides51 are
attractive as inhibitors34,45,47,48,52 of a-helix mediated PPIs because
they exhibit predictable folding patterns controlled largely by
the preferred conformation of the aryl–NHCO–aryl bond and
adjacent ortho interactions. Such oligomers represent a natural
bridge between b-peptides and a-helix proteomimetics but solid-
phase library syntheses for generation of screening libraries have
not been described. In the current manuscript we introduce one
of the simplest proteomimetic scaffolds described to date, outline
Fig. 1 (a) hDM2 binding p53 helix illustrating the spatial position of
key hDM2 binding side chains; (b) structure of an N-alkylated aromatic
oligoamide helix mimetic; (c) molecular model (MMFFs force field) of
an aromatic oligoamide mimic of the p53 sequence illustrating reasonable
reproduction of the spatial positioning of helix side chains in the i, i +
4 and i + 7 positions (d) overlay of the helix mimetic and p53 peptide
aSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS2
9JT, United Kingdom. E-mail: A.J.Wilson@leeds.ac.uk, S.L.Warriner@
leeds.ac.uk; Fax: +44 (0)113 3436565; Tel: +44 (0)113 3431409
˚
(RMSD is 0.493 (A) for a-carbons of helix and nitrogens of mimetic).
bAstbury Centre for Structural Molecular Biology, University of
Leeds, Woodhouse Lane, Leeds, LS2 9JT, United Kingdom. E-mail:
T.A.Edwards@leeds.ac.uk
Synthesis
We were keen to develop a solid phase synthesis of the aromatic
oligoamides that would facilitate library synthesis. Basing the
synthetic strategy on Fmoc protection of the aniline would also
† Electronic supplementary information (ESI) available: Synthetic pro-
cedures, protein expression, binding studies and HSQC data. See DOI:
10.1039/c001164a
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Scheme 1 (a) Synthesis of Fmoc protected N-alkylated p-aminobenzoic acid monomers 3 and 4, and (b) outline of procedure used for solid-phase
synthesis of N-alkylated aromatic oligoamide a-helix mimetics.
enable standard side chain protection from classical peptide
synthesis to be used and make the mimetic syntheses compatible
with existing solid support chemistries. Our synthetic approach
for solid-phase synthesis builds on prior studies by Kilbinger and
co-workers.58 Initial studies linked the protected aminobenzoic
acids 3a–f (Scheme 1a) directly to a Wang resin support using
1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) pro-
moted coupling.59 Although this method was effective at forming
aC-terminalester, we found itchallengingto monitor the efficiency
of subsequent coupling steps i.e. due to the water sensitivity of the
coupling chemistries explored during this study (see below) it was
challenging to ascertain if loading, coupling or resin cleavage was
the source of poor levels of dimer formation. We therefore con-
structed all the oligomers on preloaded Gly-Wang resin to remove
uncertainties associated with initial resin loading, generating a
robust amide bond in both loading and coupling reaction (Gly
was selected to avoid any side chain contribution to binding in our
subsequent analysis: see binding studies). Our earlier studies on
the coupling of amino benzoic acid derivatives48,53,54,60 relied upon
in situ generation of the acid chloride at elevated temperatures.
Such a method is not compatible with the manual solid-phase
synthesis developed in this study. Therefore, in order to effect
efficient couplings at room temperature, we reacted Ghosez’s a-
chloroenamine reagent61 with the monomer in chloroform for ~3 h
prior to addition of N-methylimidazole (MeIm) as base and then
transferred the mixture onto the resin (Scheme 1b). Due to the
water sensitivity of the coupling reagent, and low reactivity of the
aniline towards acid chlorides at room temperature, this required
significant care to exclude water from the coupling reaction.
We tested a range of other coupling reagents including PyBOP,
HCTU, TFFH and dichlorotriphenylphosphorane none of which
effected the transformation at room temperature whilst coupling
also failed when the base was diisopropylethylamine. In the small
library we constructed for this investigation, we employed a
10-fold excess of monomer, 9-fold excess of coupling reagent and
20-fold excess of base relative to the maximum resin loading. We
found coupling efficiencies under these conditions to be >95%.
Following cleavage from the resin using TFA, final compounds
were generally obtained by precipitation in high purity (>95%
by LC-MS). Of the monomers we attempted to couple, only
the indole functionalized monomer was problematic—we found
that the side chain was cleaved from the scaffold during resin
cleavage, presumably due to acid mediated elimination. However,
the method is clearly compatible with conventional protecting
group strategies and chemistry developed for solid phase peptide
synthesis as tBoc protected monomer 3f coupled smoothly. We
also synthesized trimers functionalized at the N-terminus with
glycine on the basis that this might improve solubility due to the
higher pKa of the primary amine as compared to the secondary
aniline. Direct coupling of Fmoc glycine to the N-terminus of our
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Table 1 IC50 values determined by fluorescence anisotropy competition
trimers failed; this was solved by use of Fmoc-Gly functionalized
monomers 4c and d (see ESI†) in the final coupling step.
assaya
Compound
IC50/mM
Binding studies
p5315-31Flu
0.074 ( 0.004)b
1.2 ( 0.04)
> 50 ( 10)
3.8 ( 0.8)
4.1 ( 0.9)
3.3 ( 0.9)
Once we established our method, a small library of compounds
(7–28) was assembled for screening against the p53–hDM2
interaction (Fig. 2). Compounds 5 and 6 from our earlier
p5315-31
7
11
12
14
15
2.8 ( 0.8)
a Conditions as indicated in Fig. 2. b Kd.
work on macrocyclizations were also added to the library. The
library compounds were screened at 10 mM in a single point
fluorescence anisotropy competition assay for inhibition of the
p53–hDM2 interaction.21,38,48 Briefly, a short p53 peptide labelled
with fluorescein undergoes an increase in anisotropy upon titration
with hDM2. Upon addition of inhibitor and perturbation of the
equilibrium, this signal then decreases again and the resultant
anisotropy values are used to calculate a % inhibition. Our small
library gave a full range of inhibition values. It was also possible
to pick out some simple structure affinity relationships from this
experiment. For instance, the most potent inhibitors had side
chain sequences similar in nature to the key Phe 19, Trp23, Leu
26, side chains of the p53 peptide (e.g. R3 = Ph, R2 = Nap,
i
i
R1 = Pr 15 and R3 = Pr, R2 = Ph, R1 = Ph 12). Oligomers
with only two side chains e.g. 7 were poor inhibitors indicating
a requirement for trimeric motifs for successful inhibition, and
suggesting that potent compounds unfold and adopt the desired
extended conformation. We also found tentative evidence to
suggest that dipole alignment is unimportant as compounds with
reversed sequences were equipotent (e.g. R1 = Ph, R2 = Ph, R3 =
i
iPr 12 and R1 = Pr, R2 = Ph, R3 = Ph 11) whilst the requirement
for hydrophobic aromatic side chains is also evidenced by the
much poorer potency of 8 and 9. Curiously, the oligomers capped
with glycine at the N-terminus (17–28) all exhibited poor potency
(and contrary to our expectations, solubility). Presumably, the
protonated N-terminus makes a repulsive electrostatic contact
with the protein reducing potency relative to those compounds
without a glycine at the N-terminus.
IC50 values for the most potent inhibitors of the p53–hDM2
interactionwere subsequentlydetermined(Table 1). Compound 15
was observed to inhibit the interaction with IC50 = 2.8 mM, which
is comparable to the native peptide IC50 = 1.2 mM. Compounds
11, 12 and 14 gave IC50 values of 3.8, 4.1 and 3.3 mM, respectively
(see ESI†). We also tested compound 7 with only two side chains—
as expected poor inhibition was observed IC50 > 50 mM. As was
the case in our recently reported article on O-alkylated aromatic
oligoamides,48,60 we were unable to use this data to extract a Kd
value because the tracer compound (p5315-29Flu) interacts with both
the helix mimetics and the positive control (unlabelled p53 peptide)
resulting in a more complicated equilibrium. A comparison with
published affinities of p53–hDM2 inhibitors15,16,18 is also difficult
to make given that various different assays have been used. The
compounds are however, equipotent to (i) the native peptide
and more significantly (ii) the rigid oligobenzamides we recently
described,48 which is impressive given the preferred cis-geometry
of the amides in the unbound ligand.
Fig. 2 Single point fluorescence anisotropy response for compounds 5–28
when tested for competitive inhibition of the p53–hDM2 interaction.
(p5315-29Flu 54 nM, hDM2 50 nM, 40 mM sodium phosphate, pH 7.4,
200 mM NaCl and 0.02 mg ml-1 of bovine serum albumin).
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Structural studies
understanding of the structural and thermodynamic basis of
this inhibition, improving potency/selectivity and targeting other
protein–protein interactions in vitro and ex vivo.
We performed 1H–15N HSQC perturbation shift experiments62
to establish that the a-helix mimetics bind to hDM2 at the
peptide binding cleft. Distinct complexation induced shifts were
observed upon addition of compound 11 (used because of superior
solubility) to the apo form of the protein (see ESI†). Although
these were smaller in magnitude, they were similar in nature to
those observed upon addition of the p53 peptide. When mapped
onto the crystal structure of the hDM2–p53 structure (1YCR),8 the
shifts clearly show that throughout the protein, structural changes
occur upon formation of the hydrophobic cleft that accommodates
either p53 or compound 11 (Fig. 3). The experiment also lends
further support to the notion that the helix mimetic indeed adopts
an extended conformation to bind to the protein as shifts are
observed for residues at either end of the helix binding cleft on
hDM2.
Experimental
General considerations
All reagents were obtained from Aldrich, Alfa Aesar, Acros
or Fluka and used without further purification. All solvents
used were HPLC grade. Dry solvents were distilled from
sodium/benzophenone (THF, Et2O) or calcium hydride (CH2Cl2)
immediately prior to use. N-methylimidazole was distilled from
calcium hydride. Analytical TLC was performed using 0.2 mm sil-
ica gel 60 F254 pre-coated aluminium sheets (Merck) and visualised
using UV irradiation or, in the case of amine intermediates, by
staining with a ninhydrin solution. Flash column chromatography
was carried out on silica gel 60 (35 to 70 micron particles,
FluoroChem). Solvent ratios are described where appropriate.
Solvents were removed under reduced pressure using a Buchi
rotary evaporator at diaphragm pump pressure. Samples were
freed of remaining traces of solvents under high vacuum. 1H
and 13C NMR spectra were measured on a Bruker DPX300 or
a Bruker Avance 500 spectrometer using an internal deuterium
lock. Chemical shifts are reported in parts per million (ppm)
downfield from TMS in d units and coupling constants are given
in hertz (Hz). Coupling constants are reported to the nearest
1
0.1 Hz. TMS is defined as 0 ppm for H NMR spectra and the
centre line of the triplet of CDCl3 was defined as 77.10 ppm for
13C NMR spectra. When describing 1H NMR data the following
abbreviations are used; s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet app = apparent. Melting points were
determined using a Griffin D5 variable temperature apparatus and
are uncorrected. Microanalyses were obtained on a Carlo Erba
Elemental Analyser MOD 1106 instrument, found composition
is reported to the nearest 0.05%. Infrared spectra were recorded
on a Perkin-Elmer FTIR spectrometer and samples analysed as
solids (unless stated). Mass spectra (HRMS) were recorded in
house using a Micromass GCT Premier, using electron impact
ionisation (EI) or a Bruker Daltonics micrOTOF, using electron
spray ionisation (ES). LC-MS experiments were run on a Waters
Micromass ZQ spectrometer, samples ionised by electrospray
and analysed by a time-of-flight mass spectrometer, or a Bruker
Daltronics HCTUltraTM series spectrometer, samples ionised by
electrospray. All experiments were run through a C18 column on
an acetonitrile–water gradient (typically 0–95% acetonitrile over
3 min). The synthesis of compounds 5 and 6 was described earlier.53
A representative example of each synthetic procedure is provided
with all examples described in the ESI.†
Fig. 3 1H–15N HSQC chemical shift perturbation mapping onto the
crystal structure of p53–hDM2 (a) p53 peptide (b) helix mimetic 11—a
model of the proteomimetic has been manually docked into the protein to
indicate where we perceive binding to occur (regions that experience: large
shifts are shown in dark green, medium shifts in green and weak shifts in
light green, unassigned peaks are highlighted in grey).
Conclusions
In conclusion, we have reported the design and solid-phase library
synthesis of the simplest oligoamide a-helix mimetic scaffold yet
described and shown that such compounds act as potent inhibitors
of the p53–hDM2 interaction through interaction with hDM2.
The ability to rapidly construct focused libraries or much larger
libraries for high throughput screening using SPS offers immense
new opportunities for the discovery of inhibitors of protein–
protein interactions. Our ongoing studies will build upon these
preliminary results and are focused towards gaining a greater
Reductive amination
To a stirred solution of primary aniline (1 eq.) and aldehyde (≥1
eq.) in methanol, under an atmosphere of nitrogen, was added
borane-picoline (small excess). The reaction mixture was stirred
at 35 ◦C for 12–36 h, until TLC indicated reaction completion.
Concentration and direct purification by column chromatography
gave the target material which was dried under vacuum and fully
characterised.
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(Note: borane-picoline—a white solid with greater stability than
borane-pyridine can also be used for this reaction.)
Coupling
Prior to the reaction, each fully protected monomer was dissolved
in anhydrous chloroform and concentrated before final drying un-
der high vacuum. The monomer was then dissolved in anhydrous
chloroform (~450 mL) and Ghosez reagent added. This mixture
was then allowed to incubate, prior to addition to the resin, for 3 h
at room temperature in a sealed, flame dried flask. (Due to issues
with solubility, it was necessary to incubate the Fmoc-glycine-4-
naphthylaminobenzoic acid monomer with Ghosez in anhydrous
chloroform reagent at 50 ◦C for 3 h.)
After three hours incubation, methyl imidazole was added and
the reaction mixture added to the deprotected resin that had been
washed several times with anhydrous chloroform. Each coupling
reaction was allowed to stir for approximately 20 h.
4-(Propylamino)benzoic acid 2a
4-Aminobenzoic acid (2.50 g, 18.2 mmol), propionaldehyde
(1.70 mL, 23.6 mmol) and picoline-borane complex (2.00 g,
18.7 mmol) were stirred at room temperature in methanol
(25 mL) for 14 h. The reaction mixture was then concentrated
and triturated with the minimum amount of hot chloroform
then allowed to cool. The resultant solid was crystallised from
chloroform to yield target material (2.28 g, 12.7 mmol, 70%)
as a pale cream solid; m.p. 163–164 ◦C; RF 0.19 (10% MeOH
in CH2Cl2); dH (300 MHz, MeOD) 0.89 (t, 3H, J = 7.4 Hz,
CH3CH2CH2), 1.53 (m, 2H, CH3CH2CH2), 2.98 (t, 2H, J =
7.2 Hz, CH3CH2CH2), 6.46 (d, 2H, J = 8.9 Hz, ArH), 7.66 (d,
2H, J = 8.9 Hz, ArH); dC (75 MHz, MeOD) 12.2, 23.6, 46.1,
112.3, 118.1, 133.1, 155.0, 171.2; nmax/cm-1 (solid state) = ~3000
(COOH), 1657 (CO); ESI-HRMS found m/z 180.1021 [M+H]+
C10H14NO2 requires 180.1019.
Fmoc deprotection
After each coupling reaction, the contents of the reservoir were
drained and the resin washed three times with CH2Cl2 and
three times with DMF (750 mL each). Following the final DMF
wash, the resin was then consecutively washed with 25% piperidine
in DMF then neat DMF four times (750 mL each). Finally, the
deprotected resin was washed twice with CH2Cl2 (750 mL each).
4-((((9H-Fluoren-9-yl)methoxy)carbonyl)(propyl)amino)benzoic
acid 3a
Cleavage and recrystallisation
To a refluxing solution of 4-(propylamino)benzoic acid (500 mg,
2.8 mmol) in chloroform (15 mL) was added dropwise a solution
of Fmoc chloride (865 mg, 3.3 mmol) in chloroform (10 mL) over
a period of 1 h. The reaction mixture was then stirred at reflux for
an additional 16 h. Column chromatography (Stationary Phase:
Silica, 90 g; Mobile Phase: CH2Cl2 to 15% EtOAc in CH2Cl2)
yielded target material (939 mg, 2.3 mmol, 88%) as an amorphous
solid; (Found: C, 74.50; H, 5.85; N, 3.45; requires: C, 74.79; H,
5.77; N, 3.49); RF 0.36 (15% EtOAc in CH2Cl2); dH (300 MHz,
CDCl3) 0.81 (t, 3H, J = 7.4 Hz, CH3CH2CH2), 1.46 (m, 2H,
CH3CH2CH2), 3.58 (t, 2H, J = 7.4 Hz, CH3CH2CH2), 4.13 (t,
1H, J = 6.0 Hz, CHCH2), 4.52 (d, 2H, J = 6.1 Hz, CHCH2), 7.16-
7.39 (m, 8H, ArH), 7.71 (d, 2H, J = 7.5 Hz, ArH), 8.04 (d, 2H,
J = 8.6 Hz, ArH); dC (75 MHz, CDCl3) 11.0, 21.5, 47.2, 51.7, 64.2,
67.3, 119.9, 120.1, 124.8, 126.8, 127.0, 127.7, 131.0, 141.4, 143.7,
146.8, 154.9, 170.9; nmax/cm-1 (solid state) = ~3000 (COOH), 1720,
1673 (CO); ESI-MS m/z 402 [M+H]+.
Each reservoir was filled with neat TFA (750 mL) and allowed to
stir for 2 h. After 2 h the contents of the reservoir were collected,
concentrated and the target compound purified as detailed below
2-(4-(N-Isobutyl-4-(N-isobutyl-4-(isobutylamino)benzamido)-
benzamido)benzamido)acetic acid 8. Purified by column chro-
matography (Stationary Phase: Silica, 15 g; Mobile Phase: CH2Cl2
to 15% MeOH in CH2Cl2). Precipitated from CHCl3–hexane.
Isolated yield: 4 mg. dH (500 MHz, MeOD) 0.78 (d, 6H, J =
6.7 Hz, (CH3)2), 0.86 (d, 12H, J = 6.8 Hz, 2 ¥ (CH3)2), 1.76 (m,
3H, 3 ¥ CH), 2.79 (d, 2H, J = 6.8 Hz, CH2), 3.61 (d, 2H, J =
6.9 Hz, CH2), 3.74 (d, 2H, J = 7.0 Hz, CH2), 3.99 (s, 2H, CH2),
6.21 (d, 2H, J = 9.6 Hz, ArH), 6.83 (d, 2H, J = 8.5 Hz, ArH), 6.88
(d, 2H, J = 8.8 Hz, ArH), 7.12 (m, 4H, ArH), 7.68 (d, 2H, J =
8.7 Hz, ArH); nmax/cm-1 (solid state) = 3359 (COOH), 1734, 1635,
1602 (CO); ESI-HRMS found m/z 601.3380 [M+H]+ C35H45N4O5
requires 601.3384.
Expression and purification of hDM2 17-126 L33E. hDM2
(17–126) construct was kindly provided by John Robinson at
University of Zu¨rich. The pET14b plasmid (Novagen) containing
cDNA encoding hDM2 residues Ser17 to Asn126, with a single
mutation L33E, was introduced into E. coli BL21 (DE3) GOLD.
Protein production in 2xYT medium with ampicillin (100 mg mL-1)
was induced with IPTG (0.8 mM) when the optical density of the
cell suspension reached OD600 = 0.8. Induced cells were grown at
Solid phase synthesis experimental
25–30 mg of Fmoc-Gly-Wang resin (0.4–0.8 mmol g-1, 100-
200 mesh; carrier: polystyrene, crosslinked with 1% DVB) from
Fluka, was used throughout. All solvents used were HPLC grade.
Anhydrous chloroform was freshly distilled over calcium chloride
prior to every use. 1-Chloro-N,N,2-trimethyl-1-propenylamine
(Ghosez reagent) was purchased from Sigma-Aldrich and used as a
10% stock solution in anhydrous CH2Cl2. Freshly distilled methyl
imidazole was used throughout. The reactions were all carried
out in 1.5 mL ‘Extract-Clean’ polypropylene reservoirs fitted with
20 mm polyethylene frits, both available from Alltech. For each
coupling reaction, 10 equivalents of fully protected monomer, 9
equivalents of Ghosez reagent (~270 mL of 10% stock solution) and
20 equivalents of methyl imidazole were used. The total volume in
each reservoir was 750 mL.
◦
18 C for 12 h and harvested by centrifugation at 6000 rpm for
10 min. Cells were resuspended in buffer A (20 mM Tris, 500 mM
NaCl, pH 7.9) with 0.1% Triton X-100, disrupted by 20 kPsi on
a Cell Disruptor (Constant System Ltd.) and sonicated in the
presence of DNaseI, 1 U mL-1 (EPICENTRE biotechnologies)
and 5 mM MgCl2. Cell lysate was centrifuged at 17 000 rpm for
30 min. Supernatant was loaded onto a Ni2+-nitriloacetic acid
(NTA) column equilibrated with buffer A. His6-tagged hDM2
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recombinant fragment was washed with 50 mL buffer A and
buffer A with 60 mM imidazole added. The protein was eluted
with 120 mM and 300 mM imidazole and the column was washed
with 1 M imidazole in buffer A. Fractions containing protein
were detected by 16% SDS-PAGE and concentrated to 5 mL then
loaded onto a SuperdexTM 75 column and washer with buffer B
(25 mM Tris, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM
DTT, pH 7.3). The purified protein was concentrated to a final
concentration of 83 mM and stored at -70 ◦C until use.
(4)
r = anisotropy, I = total intensity, P = perpendicular intensity, S =
parallel intensity, Lb = fraction ligand bound, l = Ibound/Iunbound
=
1, [FL] = concentration of fluorescent ligand (i.e. p5315-31Flu), k1 =
Kd, y = Lb* p5315-31Flu and x = [added titrant], G is an instrument
factor set to 1.
Single point fluorescence anisotropy screening for inhibition of
p53–hDM2 interaction
Expression and purification of 15N labelled hDM2 17-126 L33E.
E. coli BL21 (DE3) GOLD transformed with pET14b/hDM2 (17-
126) L33E were grown in minimal HCDM1 media (KH2PO4 10 g
l-1, K2HPO4 10 g l-1, Na2HPO4 7.2 g l-1, 15NH4Cl, 1.1 g l-1; with
added MgCl2 0.19 g l-1 and Glucose 4 g l-1 after autoclaving).
Protein was purified as described above.
A one point assay was carried out on 24 compounds at a fixed
concentration of inhibitor (10 mM), p53* (54.5 nM) and hDM2
(41.5 nM) in phosphate buffer. Each compound was assessed
in triplicate and left to equilibrate overnight in the dark. No
protein denaturation was observed over time, as evidenced by the
control/blank experiment lacking ligand in which p53* remains
fully bound to the protein. The anisotropy values were then
determined and used to calculate p53* fraction bound (Lb) as
above and then the percentage inhibition was calculated using
these values.
Fluorescence polarisation displacement assay
p5315-31 transactivation domain peptide (Ac-SQETFSDLWKLL-
PENNVC-NH2) (p53) and its fluorescein-labelled analogue (Ac-
SQETFSDLWKLLPENNVC(Flu)-NH2) (p5315-31Flu, p53*) were
purchased from Peptide Protein Research Ltd. Fluorescence
anisotropy assays were performed in 96 well plates. All experiments
were performed in 40 mM phosphate buffer at pH 7.42, containing
200 mM NaCl and 0.02 mg mL-1 bovine serum albumin (BSA).
A series of blank experiments were run to ensure that hDM2, p53
and BSA, did not contribute to the overall fluorescence of the well.
The serial dilutions of p53 and hDM2 in buffer (both with and
without BSA) correspondingly gave no significant fluorescence. In
all experiments the G factor, or ratio between the efficiency of the
S(ame) and P(erpendicular) channels, was set to 1.
Competition assay
Ligand (typically 2.5 nM–250 mM) was serially diluted into a
solution of p53* (54.5 nM) and hDM2 (41.5 nM) in phosphate
buffer containing BSA—the total volume of each well was
160 mL. The total fluorescence intensity and anisotropy were
both calculated. The anisotropy was then plotted against ligand
concentration in Origin 7 and fit to a dose response model (eqn
(5)) to extract an IC50 value.
(5)
Determination of the binding of p53* to hDM2
The procedure followed was described in our previous paper on
O-alkylated aromatic oligoamides.48 Briefly: hDM2 was serially
diluted (41.5 pM - 4.15 mM) into a solution of p53* (54.5 nM)
(40 mM Sodium phosphate buffer pH 7.54, 200 mM NaCl,
0.02 mg ml-1 of bovine serum albumin)—the total volume of
each well was 160 mL. Each experiment was run in triplicate
and the fluorescence polarisation measured using a Perkin Elmer
EnVisionTM 2103 MultiLabel plate reader, with excitation at
480 nm and emission at 535 nm (5 nM bandwidth) and the intensity
(eqn (1)) was calculated for each point. This was used to calculate
anisotropy (eqn (2)) and plotted to a sigmoidal fit in origin 7
to determine the minimum and maximum anisotropies (rmin and
HSQC perturbation shift analysis
All HSQC experiments were performed on a Varian Inova
spectrometer at 600 MHz in 60 mM sodium phosphate and 60 mM
sodium acetate buffer at pH 7.3 at a constant temperature of 25 ◦C.
There was 10% D2O present to allow for a lock signal. Shigemi
tubes were used and the volume of the solution was 300 mL.
A gradient filtered pulse sequence was used and the data was
phased using NMRpipe63 and visualised using NMRview 5.2264
The HSQC shift mapping was done on Accelerysis Weblab Viewer
by separating the difference in chemical shift to four categories
(Strong, Medium, Weak, and None) and colouring the residues
accordingly with darker colours representing the stronger shifts.
Grey was chosen for residues that were unassigned. The difference
in chemical shift was calculated (eqn (6)) where Ddoverall is the
overall change in chemical shift DdN is the change in the nitrogen
dimension and DdH is the change in the hydrogen dimension.
The change in hydrogen dimension is scaled by the ratio of the
magnetogyric radius of Nitrogen and Hydrogen to account for
the larger chemical shift range of Nitrogen.65 Each HSQC was
obtained from 300 mL of labelled hDM2 (0.125 mM). To obtain
the spectra with a mix of compounds a stock solution of either
the mimetic or p53 was added in DMSO, ensuring that the protein
would be saturated and the compound would still be soluble. In
r
max). Using eqn (3), the data for the anisotropy was converted to
fraction bound and multiplied by the p5315-31Flu concentration then
fitted in origin 7 (eqn (4)) to give the dissociation constant Kd =
74 4 nM.
I = (2PG) + S
(1)
(2)
(3)
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the case of the mimetic the final concentration was 250 mM and for
p53 it was 200 mM. In each case the amount of DMSO added was
less than 5% of the total volume of the protein. Fig. ESI 2a and b
show the HSQC spectra in the absence and presence of p53 and
mimetic respectively whilst Fig. ESI 3 illustrates the shift changes
by residue.† Large shifts were defined as greater than 0.3 ppm,
medium as between 0.3 to 0.09 and weak shifts those below 0.09.
15 L. T. Vassilev, B. T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic,
N. Kong, U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi and E. A. Liu,
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16 B. L. Grasberger, T. Lu, C. Schubert, D. J. Parks, T. E. Carver,
H. K. Koblish, M. D. Cummings, L. V. LaFrance, K. L. Milkiewicz,
R. R. Calvo, D. Maguire, J. Lattanze, C. F. Franks, S. Zhao, K.
Ramachandren, G. R. Bylebyl, M. Zhang, C. L. Manthey, E. C. Petrella,
M. W. Pantoliano, I. C. Deckman, J. C. Spurlino, A. C. Maroney, B. E.
Tomczuk, C. J. Molloy and R. F. Bone, J. Med. Chem., 2005, 48, 909–
912.
17 I. R. Hardcastle, S. U. Ahmed, H. Atkins, G. Farnie, B. T. Golding,
R. J. Griffin, S. Guyenne, C. Hutton, P. Ka¨llblad, S. J. Kemp, M. S.
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18 S. Shangary, D. Qin, D. McEachern, M. Liu, R. S. Miller, S. Qiu, Z.
Nikolovska-Coleska, K. Ding, G. Wang, J. Chen, D. Bernard, J. Zhang,
Y. Lu, Q. Gu, R. B. Shah, K. J. Pienta, X. Ling, S. Kang, M. Guo, Y.
Sun, D. Yang and S. Wang, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
3933–3938.
(6)
To compare the changes seen in the HSQC we also mapped the
shifts in a-carbons between the NMR structure of the apo protein
(PDBID: 1Z1M) with both the crystal structures of bound p53
(1YCR) and nutlin (1RV1). The structures were overlaid with the
least squares function in COOT66 and the distances between each
carbon were calculated (eqn (7))
19 E. A. Harker, D. S. Daniels, D. A. Guarracino and A. Schepartz, Bioorg.
Med. Chem., 2009, 17, 2038–2046.
20 O. M. Stephens, S. Kim, B. D. Welch, M. E. Hodsdon, M. S. Kay and
A. Schepartz, J. Am. Chem. Soc., 2005, 127, 13126–12127.
21 J. A. Kritzer, J. D. Lear, M. E. Hodsdon and A. Schepartz, J. Am.
Chem. Soc., 2004, 126, 9468–9469.
(7)
22 J. D. Sadowsky, W. D. Fairlie, E. B. Hadley, H.-S. Lee, N. Umezawa,
Z. Nikolovska-Coleska, S. Wang, D. C. S. Huang, Y. Tomita and S. H.
Gellman, J. Am. Chem. Soc., 2007, 129, 139–154.
23 W. S. Horne, M. D. Boersma, M. A. Windsor and S. H. Gellman,
Angew. Chem., Int. Ed., 2008, 47, 2853–2856.
The positional changes were separated into three groups (Large,
Medium, and Small) and mapped to the structure of hDM2 in a
similar manner as for the HSQC shifts.
24 E. F. Lee, J. D. Sadowsky, B. J. Smith, P. E. Czabotar, K. J. Peterson-
Kaufman, P. M. Colman, S. H. Gellman and W. D. Fairlie, Angew.
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Acknowledgements
25 T. Hara, S. R. Durell, M. C. Myers and D. H. Appella, J. Am. Chem.
Soc., 2006, 128, 1995–2004.
26 C. M. Goodman, S. Choi, S. Shandler and W. F. DeGrado, Nat. Chem.
Biol., 2007, 3, 252–262.
This work was supported by the Engineering and Physical Sciences
Research Council [EP/D077842/1 and EP/G022569/1] and The
European Research Council [240342]
27 R. Fasan, R. L. A. Dias, K. Moehle, O. Zerbe, D. Obrecht, P. R. E.
Mittl, M. G. Gru¨ttner and J. A. Robinson, ChemBioChem, 2006, 7,
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28 S. Kneissl, E. J. Loveridge, C. Williams, M. P. Crump and R. K.
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