Structure-guided design of α-amino acid-derived Pin1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.11.090

The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of α-benzimidazolyl-substituted amino acids.

Full text of DOI:10.1016/j.bmcl.2009.11.090

Bioorganic & Medicinal Chemistry Letters 20 (2010) 586–590
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-guided design of a-amino acid-derived Pin1 inhibitors
Andrew J. Potter, Stuart Ray, Louisa Gueritz, Claire L. Nunns, Christopher J. Bryant, Simon F. Scrace,
Natalia Matassova, Lisa Baker, Pawel Dokurno, David A. Robinson , Allan E. Surgenor, Ben Davis,
à
*
James B. Murray, Christine M. Richardson , Jonathan D. Moore
Vernalis (R&D) Ltd, Granta Park, Great Abington, Cambridge CB21 6GB, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics.
Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of
Received 21 October 2009
Revised 16 November 2009
Accepted 17 November 2009
Available online 22 November 2009
a
-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC50 <100 nM, but were
inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10–50-fold
loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked pro-
liferation of PC3 cells.
Keywords:
Pin1
Ó 2009 Elsevier Ltd. All rights reserved.
Cancer
Rotamase
Isomerase
Kinase signaling
Crystal structure
Small molecule inhibitor
NMR
Surface plasmon resonance
Pin1 provides the great majority of peptidyl prolyl cis/trans
isomerise (PPIase) activity capable of isomerising pSer/pThr-Pro
bonds.1 Isomerisation around such bonds can promote major con-
formational changes within proteins, enabling Pin1 to influence
dynamics and outcomes within pathways regulated by MAP kinases,
cyclin-dependent kinases and GSK-3b.2–6 Pin1 overexpression is
only weakly oncogenic in itself, but enhances transformation by
ErbB2 or activated Ras alleles.5 Remarkably, cells from Pin1 deficient
mice are resistant to transformation by Ras and ErbB2.7 As Pin1-defi-
cient mice are viable,8 there are considerable grounds for hope that
Pin1 inhibitors will have value for the therapy of cancer.
cently disclosed by Pfizer.16 The same report revealed that when
106 compounds were screened for activity against Pin1 no hits
were found whose binding to target could be verified by isothermal
titration calorimetry or NMR.16 This suggests high-throughput
screening is not an effective way of finding start points for drug
discovery programs targeting Pin1, but that alternative approaches
should be sought.
Vernalis have developed an NMR-based fragment screening
platform (SeeDs) capable of identifying compounds that compete
for binding to target with known competitor ligands.17,18 Two
competitor ligands had been identified whose binding to the Pin1
The irreversible inhibitor, Juglone (1), has been widely used to
probe the function of Pin1 inside cells, but is neither selective
nor a suitable start point for drug discovery.9–11 Several classes
of presumably reversible small molecule inhibitors have since been
reported.12,13Structural data confirming interactions of inhibitor
with Pin1, however, has been restricted to peptide antagonists,14,15
with the exception of a series of phenylalaninol phosphates, re-
active site had been verified by crystallography: the D-peptide
inhibitor 2, and the phenylalaninol phosphate 3.15,16 To identify
potential start points for drug discovery, a library of ꢀ1200 frag-
ments was screened by NMR for their ability to compete with
the binding of 2 and 3 to the PPIase domain of Pin1. This process
identified five competitively-binding compounds,19 of which the
indole 2-carboxylic acid 4 (IC50 16 lM) was the most potent inhib-
itor of Pin1’s PPIase activity.20
Pin1 crystals were readily obtained from an R14A (Arg14 mu-
tated to Ala) variant of full length Pin1,14 or a K77Q/K82Q mutated
variant of the isolated peptidyl-prolyl isomerase domain.16 Com-
pound 4 was soaked into crystals of Pin1R14A protein. This en-
abled the determination of a ligand-bound X-ray crystal structure
(Fig. 1), in which the indole moiety fits snugly into a hydrophobic
* Corresponding author. Tel.: +44 1223 895555.
E-mail address: j.moore@vernalis.com (J.D. Moore).
Present address: Division of Biological Chemistry & Drug Discovery, College of Life
Sciences, James Black Centre, University of Dundee, Dundee DD1 5EH, UK.
à
Present address: Biofocus DPI, Chesterford Research Park, Saffron Walden, Essex
CB10 1XL, UK.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.090

A. J. Potter et al. / Bioorg. Med. Chem. Lett. 20 (2010) 586–590
587
(5) (IC50 630 lM) revealed the position of the methyl substituent
was dominant over the indole nitrogen in orientating the molecule
in the Pin1 active site.20 This result indicated that indole NH moi-
eties could hydrogen bond to residues on either side of the active
site pocket. To probe SAR in more detail, several benzimidazole
2-carboxylic acids were synthesized (Scheme 1).
Appropriately substituted o-phenylene-diamines were con-
densed (6) with 2,2,2-trichloroacetimidic acid methyl ester. Hydro-
lysis of the intermediate 2-trichloro-benzimidazoles (7) yielded the
corresponding benzimid-azole 2-carboxylic acids. Compound 8a
(IC50 63 lM), the benzimidazole analogue of 4, was less active than
its indole counterpart (see Table 1). Replacement of the 5-methyl
substituent of 8a with a nitrile (8b) increased potency ꢀ7-fold
(IC50 10 lM). The interaction between the carboxylic acid group
and Lys63 was essential for inhibition: a series of compounds where
the acid in 8a was replaced with amides (e.g., compound 9) were
inactive in the PPIase assay. This demonstrates the importance of
the bridging waters seen in the X-ray structure of 4 bound to Pin1.
The phenylalaninol phosphate 3 can be considered to make
three points of attachment with Pin1. The phenylalanine moiety
fills the hydrophobic pocket, the phosphate hydrogen bonds with
Ly63, Arg68 and Arg69, and the thiophene group binds to a
‘shelf-like’ flat hydrophobic surface located on the other side of
Cys113, away from the active site.16 The indole 2-carboxylic acid
fragment hit 4 mimicked the first two of these interactions, and
had the potential advantage of not having a phosphate group,
which might be expected to interfere with cell penetration. Conse-
quently, we were keen to explore means by which our ligands
could be elaborated to access this shelf. Unfortunately, 4 did not
present positions for elaboration that provided a suitable vector.
Analogue screening identified 3-(1H-benzo-imidazol-2-yl)-propi-
onic acid (10), which crystallized in the active site in the PPIase
K77Q/K82Q form of Pin1 so as to retain the interactions observed
for 4: the fused ring system binding to the hydrophobic pocket
and acid participating in a salt bridge with Lys63.20 Crucially, a vec-
tor towards the shelf is available by substitution off the methylene
Figure 1. Crystal structure of 4 bound to Pin1R14A (PDB ID: 3KCE). The indole
occupies the catalytic site of Pin1. H-bonding interactions (not shown) are observed
between the indole nitrogen and cysteine 113 and the indole carboxylic acid to
lysine 63. The position of water molecules is shown in red.
pocket in the Pin1 active site. The indole NH hydrogen bonds to the
side chain of Cys113, whilst the carboxylic acid substituent inter-
acts with Lys63 and a network of waters.
H
N
CONH2
CONH2
HN
O
O
O
OH
O
O
N
S
O
P
O
O
H
O
NH2
HN
carbon,
740 M) than 4, its ligand efficiency (LE = 0.29) was considered
acceptable for a start point, given the shallow highly-solvated
a
to the acid in 10.20 Whilst 10 was less active (IC50
HN
O
P
HN
O
l
CO2H
O
O
O
O
1
2
3
4
NH2
NH2
N
N
(a)
(b)
CCl3
CO2H
R
R
R
N
H
N
H
PPIase assay results for 4 (IC50 16 lM) implied a very high
ligand efficiency21 (LE = 0.48). To verify this value, binding affinity
was investigated by surface plasmon resonance spectroscopy
(SPR). At low ligand concentrations, less 4 bound to Pin1 than ex-
pected, but the bound quantity increased rapidly with concentra-
tion to super-stoichiometric levels.20 As binding of 4 to Pin1
could not be saturated, no KD for the interaction could be calcu-
lated. Two-dimensional NMR studies (HSQC), however, showed
that shifts in peak positions induced by 4 converged to a maximum
6
7
8
Scheme 1. Reagents and conditions: (a) 2,2,2-trichloroacetimidic acid methyl ester,
MeCO2H, 80%; (b) NaOH(aq); HCl(aq), 55–75%.
Table 1
Biological data for indole/benzimidazole Pin1 inhibitors
value, suggesting a KD of ꢀ200
l
M.20 Adjusting buffer and salt con-
R2
X
centrations in the PPIase assay so as to mimic conditions in the SPR
or NMR experiments made no difference to the IC50 obtained. The
high affinity of the interaction of 4 with Pin1 that would be in-
ferred from the PPIase assay results was therefore not confirmed
by biophysical techniques, but 4 nevertheless remained an inter-
esting start point for drug discovery. Possible explanations for
these contradictory results are discussed in the Supplementary
data.20
R1
R3
N
H
R1
R2
R3
X
Pin1 IC50
(l
M)
a
4
5
CO2H
CO2H
CO2H
CO2H
CONHEt
(CH2)2CO2H
(CH2)2CO2H
Me
H
Me
CN
Me
H
H
Me
H
H
H
C
C
16
630
63
8a
8b
9
10
11
N
N
N
N
C
10
SAR was investigated amongst several commercially available
indole-2-carboxylates. Given the disagreement between the PPIase
assay and other techniques, only tentative conclusions regarding
ligand activity can made from PPIase assay data alone. Neverthe-
less, the crystal structure of the 6-methyl substituted compound
>2000
740
450
H
H
H
a
All IC50 values are the mean of at least two determinations and are rounded to
two significant figures where appropriate.

588
A. J. Potter et al. / Bioorg. Med. Chem. Lett. 20 (2010) 586–590
Table 2
Pin1 active site. Compound 11, the indole analogue of 10, had sim-
ilar activity (IC50 440 M).
Biological data for benzimidazole propionate Pin1 inhibitors
l
N
Study of the ligand bound crystal structure of 10 suggested a
viable way to access the hydrophobic shelf was via elaboration of
the 2-position. (R)-Amino acid derivatives were of particular inter-
est and the required amides were prepared by the route shown in
Scheme 2. The commercially available aspartic acid derivative (12)
was esterified with potassium carbonate and methyl iodide and
the resulting diester (13) then hydrogenated using palladium on
carbon, yielding the mono acid (14) in good overall yield. Benz-
imidazole formation was achieved through coupling of o-phenyl-
enediamine to the mixed anhydride of the mono acid followed
by cyclization under acidic conditions to yield 15. The BOC-pro-
tected benzimidazole (16) was then de-protected using TFA and
coupled to the desired carboxylic acids using carbodiimide/trieth-
ylamine. Finally, esters (17) were saponified to yield the corre-
sponding acids (18–20) on work up. Analysis of these final
compounds showed that some degree of racemization had taken
place along the synthetic route. Chiral HPLC showed that the
R-enantiomers were approximately 75% ee.20
O
O
R2
N
H
HN
R1
O
R1
Et
R2
H
Pin1 IC50
78
(lM)
LE21
0.28
18a
18b
H
N
H
6.3
0.24
18c
18d
H
H
7.5
0.26
0.30
S
0.83
O
19a
H
H
6.6
0.29
O
The propionyl amide 18a (78
tency over the parent 10, but the indolyl-propionyl amide 18b
(6 M) was more active still (Table 2). X-ray crystal structures of
lM) had ꢀ10-fold improved po-
19b
17b
19c
19d
19e
20
0.13
92
0.29
0.16
0.28
0.28
0.32
0.29
O
O
O
O
O
O
N
l
CH3
H
these amides were not obtained, but the structure obtained when
the phenylpropionyl amide (18c) was soaked into crystals of Pin1-
R14A/Q131A (a Pin1 variant bearing a mutation facilitating crystal-
lisation with a more open binding site) revealed that the phenyl
moiety was positioned on the shelf as expected.20 The flexibility
inherent in the side chains of compounds 18b and 18c suggested
there would be an entropic penalty to their binding to Pin1, so
other, more rigid examples were also prepared. This tactic led to
rapid success: the benzothiophene substituted derivative (18d)
0.32
0.18
0.025
0.26
CN
H
CH2N
H
N
H
inhibited Pin1 with an IC50 <1 lM in the PPIase assay and could
also be soaked into crystals of Pin1 R14A/Q131A.20
SPR indicated the over-binding phenomenon observed with the
earlier fragment was no longer apparent. Binding of 18d to Pin1
was shown to saturate, with one ligand molecule bound per recep-
tor. The KD for the interaction was estimated by SPR to be
this moiety would provide a good basis for this exercise. The crys-
tal structure of 19a in Pin1R14A20 suggested that further elabora-
tion off the 5-position of the furan would be beneficial, and this
resulted in the identification of a superior compound with sub-
200 nM potency (19b). The substantial potency gain is considered
to be associated with good van der Waals contacts between the
ligand side chain and shelf, combined with liberation of water mol-
ecules otherwise constrained against the hydrophobic ligand and
protein surfaces, giving rise to a substantial entropic gain on their
displacement back into solvent. The negatively charged carboxyl-
ate remains a requirement for activity as 17b, the methyl ester of
19b, loses activity by >100-fold. Several 5-phenyl-substituted fur-
an compounds were synthesized and assayed for their ability to in-
hibit Pin1. These compounds differed only in terms of 4-position
substitution off the phenyl ring. The most potent compound, the
amino-methyl substituted phenyl derivative 19e inhibited Pin1
ꢀ10
l
M.20 The structure of 18d bound to Pin1 suggested that there
was an opportunity to improve potency by increasing the area of
shelf surface that they covered. The effectiveness of other ring sys-
tems linked through the amide was therefore explored. The
2-methylfuran-3-carbonyl amide 19a was prepared to establish if
BnO
BnO
HO
CO2H
Boc
CO2Me
Boc
CO2Me
Boc
(a)
(b)
O
HN
O
HN
O
HN
12
13
14
MeO2C
O
NHBoc
N
with an IC50 of ꢀ20 nM. SPR confirmed a sub-lM KD (530 nM) for
(c)
(d)
(e), (f)
the interaction between 19e and Pin1 and a 1:1 stoichiometry.20
Neither 19b nor its close relatives could be crystallized in Pin1,
but modelling was consistent with a crystal structure subsequently
derived for 20, a 5-phenyl-2-methyl pyrazole substituted com-
pound (IC50 260 nM). In the crystal structure of 20 in Pin1R14A
(Fig. 2) the benzimidazole binds as previously observed, though
the carboxylic acid group is laterally displaced relative to 10. On
the shelf, the phenyl and pyrazole rings of 20 make good contact
with the surface, though the nitrogen of the pyrazole ring does
not seem to make direct polar interactions with either protein res-
idues or ordered waters.
N
H
CO2Me
Boc
NH
NH2
HN
15
16
N
N
(g)
N
H
CO2Me
O
N
H
CO2H
O
HN
HN
R
R
17
18-20
Compounds 19b and 20 were unable to inhibit the PPIase activ-
ity of FKBP12 or cyclophilin A, suggesting that they specifically
antagonised Pin1. Furthermore, the (S)-enantiomers of these two
Scheme 2. Reagents and conditions: (a) Na2CO3, MeI, DMF, 90%; (b) H2, Pd/C,
MeOH, 100%; (c) IBCF, NEt3, phenylenediamine; (d) AcOH, 65 °C, 80% over 2 steps;
(e) CF3CO2H; (f) RCO2H, EDAC, NEt3, DMF, 50–60%; (g) LiOH, THF, H2O, 90–95%.

A. J. Potter et al. / Bioorg. Med. Chem. Lett. 20 (2010) 586–590
589
Figure 3. Crystal structure of 23b bound to Pin1R1 (PDB ID: 3KAI). Replacement of
the benzimidazole with naphthyl group does not alter binding mode.
Figure 2. Crystal structures of 20 bound to Pin1R14A (PDB ID: 3KAH). The phenyl
pyrazole group makes Van der Walls interactions with the hydrophobic shelf.
Incorporation of the benzimidazole achieves a second H-bonding interaction to
serine 154.
Table 3
Biological data for naphthyl-substituted amino acids
compounds (determined by chiral HPLC to be 100% ee) were inac-
tive in the Pin1 PPIase assay (IC50 >10 l
M).19 The more potent
CO2H
benzimidazole-based Pin1 inhibitors were assayed for their ability
to inhibit growth of PC3 prostate cancer cells under serum-free
conditions where Pin1 plays an important role in survival and pro-
liferation.22 Unfortunately, none of the compounds were active in
cell-based assays, which is consistent with their high polar surface
area (PSA) and, where tested, extremely low permeability in CaCo-
2 assays (data not shown). To generate Pin1 inhibitors with re-
duced PSA, the commercially available napthyl alanine derivative
(21) was coupled to the desired carboxylic acids using carbodiim-
ide/triethylamine and esters (22) were saponified to yield the cor-
responding acids. This resulted in 23a and 23b, the napthyl
substituted analogues of the benzimidazoles 19b and 20, respec-
tively (Scheme 3).
O
HN
R
R
Pin1 IC50
2.6
(
lM)
PC3 GI50
12.5
(lM)
O
23a
23b
MeO
N
N
3.9
4.7
For 23a in particular, good evidence was obtained that its mode
of action on cells was, at least in part, via Pin1 inhibition (Fig. 4). In
serum starved PC3 cells, 23a phenocopied transfection of siRNAs
Compound 23b, the naphthyl-substituted version of 20, was a
less efficient inhibitor of Pin1 (IC50 3.9 lM; LE 0.23) but crystal-
versus Pin1 in suppressing cyclin D1 expression (EC50 ꢀ 10
lM)
lized in Pin1R14A with a similar binding mode (Fig. 3). Although
the acid group is slightly less well-positioned to interact with
Lys63, the pyrazolophenyl side chain retains good stacking against
the shelf-like hydrophobic surface. The naphthyl ring of 23b occu-
pies a similar position to the benzimidazole ring of 20, but pro-
trudes beyond it, packing against the side chains of Phe134,
His157, Met130 and Gln131. The similar binding modes of 20
and 23b imply the >10-fold loss in potency is likely due to loss
of H-bond interactions formed by the benzimidazole 20 with
Cys113 and Ser154. Encouragingly, 23b, along with the naphthyl-
but leaving cyclin E1 levels unaffected. 23a also prevented the
substituted phenyl furan 23a (IC50 2.6 lM, LE 0.22) was able to in-
hibit the growth of PC3 cells under serum-free conditions (Table 3).
CO2Me
CO2Me
Boc
(a), (b)
HN
O
HN
R
21
22
CO2H
(c)
Figure 4. Compound 23a modulates biomarkers dependent upon Pin1 function. (A)
Transfection of siRNA versus Pin1 reduces expression of cyclin D1, but cyclin E1
levels are unaffected. (B) 6 h exposure to 23a reduces cyclin D1 levels, but cyclin E
levels are unaffected. (C) 20 min stimulation with insulin increases phosphorylation
of p70S6 kinase on Thr389; this response is blocked by 60 min pre-treatment with
23a. All data shown is from cells cultured in serum-free conditions for at least 24 h
before cells were harvested.
HN
O
R
23
Scheme 3. Reagents and conditions: (a) CF3CO2H, (b) RCO2H, EDAC, NEt3, 70–75%;
(c) LiOH, THF, H2O, 100%.

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A. J. Potter et al. / Bioorg. Med. Chem. Lett. 20 (2010) 586–590
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insulin23 (EC50 ꢀ 30
lM). Further characterization of 23a will be
reported elsewhere.
In conclusion, this study reports the identification of a fragment
hit (4) in an NMR-based screen, and then the elaboration of a re-
lated fragment (10) to compounds with nM potency against Pin1
using a structure-based approach. Cell active compounds (23a
and 23b) inhibiting Pin1 in the low
lM range were obtained fol-
lowing a further scaffold switch. These results suggest identifica-
tion of reversible Pin1 inhibitors with properties consistent with
their use as drugs may be possible and that structure-guided meth-
ods constitute an attractive route towards this goal.
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Acknowledgements
We thank Günter Fischer (MPI,Halle, Germany) and Martin
Drysdale for useful discussions, Douglas Williamson and Nicolas
Foloppe for critical reading of the manuscript and David Bell
(Analytical Research and Services, PA, USA) for ee analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.090.
19. Data not shown.
20. See Supplementary data.
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