Selective Bisubstrate Inhibitors with Sub-nanomolar Affinity for Protein Kinase Pim-1

Article abstract of DOI:10.1002/cmdc.201300042

Potent and selective: The unique nature of the ATP binding pocket structure of Pim family protein kinases (PKs) was used for the development of bisubstrate inhibitors and a fluorescent probe with sub-nanomolar affinity. Conjugates of arginine-rich peptides with two ATP mimetic scaffolds were synthesized and tested as inhibitors of Pim-1. Against a panel of 124 protein kinases, a novel ARC-PIM conjugate selectively inhibited PKs of the Pim family. Copyright

Full text of DOI:10.1002/cmdc.201300042

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DOI: 10.1002/cmdc.201300042
Selective Bisubstrate Inhibitors with Sub-nanomolar
Affinity for Protein Kinase Pim-1
Ramesh Ekambaram,[a] Erki Enkvist,[a] Angela Vaasa,[a] Marje Kasari,[a] Gerda Raidaru,[a]
Stefan Knapp,[b] and Asko Uri*[a]
The human provirus integration site for Moloney murine leuke-
mia virus (Pim) protein kinase (PK) family includes three consti-
tutively active serine/threonine kinases: Pim-1–3. At the amino
acid level, Pim-2 and Pim-3 show 61% and 71% identity to
Pim-1, respectively.[1] Pim kinases participate in several normal
biological processes, including cell survival, proliferation, differ-
entiation, and apoptosis.[2] Elevated expression levels of Pim-
1 and Pim-2 have been observed in hematologic malignancies
and prostate cancer, and increased Pim-3 expression has been
detected in some solid tumors.[2d] These findings suggest that
Pim kinases are the products of proto-oncogenes, which could
play an important role in the initiation and progression of
human cancer. The first small-molecule inhibitor of Pim-1 stud-
ied in the clinic was the imidazo[1,2-b]pyridazine SGI-1776,[2f]
used to treat refractory prostate cancer and relapsed/refractory
non-Hodgkin’s lymphoma; SGI-1776 was withdrawn from clini-
cal development because of dose-limiting toxicity through car-
diac QTc interval prolongation.[2e] Despite this, structurally di-
verse small-molecule inhibitors have continued to be devel-
oped for regulation of Pim kinase activity both as research
tools and drug candidates.[2e,3,4] More than 400 small-molecular
Pim-1 inhibitors with Ki (IC50) values <10 mm have been regis-
tered in the ChEMBL database.[3]
Several research groups,[8] including our own,[9] have previ-
ously used the bisubstrate approach for development of
highly potent inhibitors of various PKs. Bisubstrate inhibitors
incorporate two conjugated fragments, each targeted to the
binding site of a distinct substrate.[10] Chemically, the inhibitors
are conjugates of adenosine derivatives (or adenosine ana-
logues) and d-arginine-rich peptides (ARCs).[9a] Several ARCs
possess very high affinities for PKs—Kd values in the low pico-
molar range have been reported for protein kinase A (PKA)[11]
and Rho-associated protein kinase (ROCK)[12]—and high biolog-
ical and chemical stability.[9] ARCs labeled with fluorescent dyes
have revealed good cell-plasma-membrane-penetrative proper-
ties that have enabled their application for measurement of
kinase activity in living cells.[13] Although ARCs are generally
group-selective inhibitors that bind with high affinity to many
basophilic PKs (mostly belonging to the AGC group of PKs), in
the course of structure–activity relationship studies, structural
elements of ARCs were established[9c] that affected the kinase-
selectivity profile of the inhibitors.
In a recent paper,[14] we showed that, with a rational selec-
tion of the conjugate moieties targeted to distinct substrate
binding pockets of the PK, the bisubstrate approach can be
used for construction of fairly selective inhibitors for PKs. In
this way, an inhibitor with sub-nanomolar affinity and remark-
able selectivity was developed for casein kinase 2 (CK2), an
acidophilic PK that catalyzes phosphorylation of protein sub-
strates rich in acidic amino acid residues aspartic acid and glu-
tamic acid in close proximity to the phosphoryl acceptor site.
In the present study, we have developed selective and
potent bisubstrate inhibitors for Pim kinases. Since 2005, nu-
merous crystal structures of ligand–Pim-1 complexes have
pointed to the uniqueness of hydrogen-bond networks and
hydrophobic interactions important for binding of the co-sub-
strate ATP (and its analogues) and inhibitors to Pim kinases.[15]
Most PKs form two hydrogen bonds with the adenine moiety
of ATP through residues in the hinge region, while Pim-
1 forms only one hydrogen bond, between the N6 atom of ad-
enine and the backbone carbonyl oxygen atom of the first
hinge residue (Glu121). Other kinases additionally form a hy-
drogen bond between the N1 atom of adenine and the back-
bone NH of the third hinge residue. The proline residue at po-
sition 123 in Pim-1 precludes the kinase from this capability.[3]
In the present study, this distinctness in the protein structure
was used for the construction of selective bisubstrate inhibi-
tors, ARC(PIM) compounds. We demonstrate that Pim selectivi-
ty and inhibitory potency of the ATP-competitive compounds
can be substantially improved by their conjugation with pep-
Most reported Pim inhibitors target the ATP binding site of
the enzymes. Some of these compounds have shown rather fa-
vorable Pim kinase inhibition profiles, at least in a small panel
of PKs.[5] Two recent reports have shown that peptides binding
to the substrate-protein binding domain of Pim-1 might also
possess nanomolar inhibitory potency and therefore be of in-
terest for biological research or even drug development ef-
forts.[6] The crystal structures have been solved of Pim-1 bound
to a high-affinity peptide substrate in complex with both an
ATP analogue (phosphoaminophosphonic acid-adenylate ester;
AMP-PNP) and a bisindolylmaleimide-based kinase inhibitor,[7]
that aids construction of protein-substrate-competitive inhibi-
tors.
[a] Dr. R. Ekambaram, Dr. E. Enkvist, Dr. A. Vaasa, Dr. M. Kasari, G. Raidaru,
Dr. A. Uri
Institute of Chemistry, University of Tartu
14A Ravila St., 50411 Tartu (Estonia)
E-mail: asko.uri@ut.ee
[b] Prof. S. Knapp
Nuffield Department of Clinical Medicine
Structural Genomics Consortium, University of Oxford
Roosevelt Drive, Oxford OX3 7DQ (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300042.
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tides targeted to the protein-substrate binding domain of the
kinase.
In the role of moieties targeted to the ATP binding site of
Pim-1, we used two compounds, 3-amino-5-bromobenzo[b]-
thiophene-2-carboxyic acid (ABBT) and a previously reported
ATP-competitive Pim kinase inhibitor, 3H-benzo-[4,5]thieno-
[3,2]-pyrimidin-4-one (BBTP).[5] Protected peptides were synthe-
sized on Rink-amide resin according to the conventional fluo-
renylmethoxycarbonyl (Fmoc) peptide synthesis methodolo-
gy.[9a] The heterocyclic fragment used for the synthesis of the
first series of ARC(PIM)s was prepared according to Scheme 1.
Scheme 2. Synthesis of the tricyclic ATP-competitive fragment BBTP and its
peptide conjugates. Reagents and conditions: a) ClCH2CN, 4n HCl in dioxane,
RT, 3 h; b) dioxane or DMF, D, 2 h; c) 1. NH or NH2–tether–peptide–resin,
DIPEA, DMF, 608C, 8 h, 2. TFA, RT, 2 h.
long lifetime photo-luminescence signal (t=20–270 ms) that is
proportional to the concentration of the ARC–Lum/PK complex
in solution.[16] The absence of the signal for the unbound
probe allows the use of ARC–Lum in great excess as compared
with the concentration of kinase. Consequently, the Cheng–
Prusoff relationship[17] can be used for simplified calculation of
Kd values from displacement curves.
Scheme 1. Synthesis of the bicyclic ATP-competitive fragment ABBT and its
peptide conjugates. Reagents and conditions: a) HSCH2COOCH3, KOH/DMF,
08C!RT, 3 h; b) 4m NaOH/EtOH, 608C, 3 h; c) 1. NH2–tether–peptide–resin,
HOBt, HBTU, DMF, RT, 1 h, 2. TFA, RT, 2 h.
The displacement experiments (Figure 2a and figures S2 and
S3 in the Supporting Information) were performed at relatively
high concentration of the ARC–Lum probe ARC-1139 (total
Fluorobenzonitrile 1 was treated with methyl-2-mer-
captoacetate. The resulting intermediate was sponta-
Table 1. Structures of ARC–Pim conjugates and their affinities toward Pim-1 and
PKAc.
neously cyclized to benzothiophene moiety 2 under
basic conditions. Hydrolysis of the methyl ester gave
3-amino-5-bromobenzo[b]thiophene-2-carboxylic acid
(3) that was coupled to the N terminus of the pep-
tide on solid phase using N,N,N’,N’-tetramethyl-O-(1H-
Compd
Structure
Kd [nm][a]
Pim-1
PKAc
3
6
7
8
ABBT
>100000
0.5ꢀ0.2
1.8ꢀ0.6
52ꢀ8
ND
25ꢀ8
benzotriazol-1-yl)uronium
hexafluorophosphate
ABBT-Ahx(d-Arg)6-(d-Lys)-NH2
ABBT-Ahx-(d-Arg)-Ahx-(d-Arg)6-(d-Lys)-NH2
BBTP-Piperidine
BBTP-Ahx-(d-Arg)-Ahx-(d-Arg)6-(d-Lys)-NH2
BBTP-Hyp-Ahx-(d-Arg)6-(d-Lys)-NH2
BBTP-Hyp-Ahx-(d-Arg)2-NH2
BBTP-Hyp-Aox-(d-Arg)2-NH2
BBTP-Hyp-Ahx-(d-Arg)-Ahx-(d-Arg)6-(d-Lys)-NH2 <0.2
BBTP-Hyp-Ahx-(d-Arg)4-NH2
BBTP-Hyp-(d-Arg)8-NH2
BBTP-Hyp-Ahx-(d-Arg)9-NH2
BBTP-Hyp-Ahx-(d-Arg)6-(d-Lys-PF-647)-NH2
90ꢀ25
(HBTU)/hydroxybenzotriazole (HOBt) activation.
>100000
36ꢀ9
Subsequently, the reaction of compound 2 with
chloroacetonitrile (4n HCl in dioxane) led quantita-
tively to intermediate 4, which underwent cyclization
under reflux in DMF or dioxane to produce com-
pound 5 (Scheme 2). The chloromethyl group of the
pyrimidinone ring of 5 readily reacted with nucleo-
philic amines (piperidine, hydroxyproline), or the
N terminus of an arginine-rich peptide (Scheme 2).
In the second set of peptide conjugates (Table 1),
the chloromethyl group of the pyrimidinone ring of 5
was reacted with nucleophilic amino groups of the
tether in the presence of N,N-diisopropylethylamine
(DIPEA) in DMF at 608C. Finally, the conjugates were
cleaved and their protecting groups were removed
by treatment of the resin with the mixture of tri-
fluoroacetic acid (TFA)/H2O/triisopropylsilane (90:5:5,
9
<0.2
10
11
12
13
14
15
16
17
<0.2
1700ꢀ300
>100000
>100000
510ꢀ50
30000ꢀ5000
3300ꢀ700
790ꢀ120
ND
180ꢀ50
250ꢀ70
10ꢀ2
<0.2
<0.2
0.6ꢀ0.2
(d-Arg)9-NH2
Staurosporine
SGI-1776
290ꢀ110
5.2ꢀ1.8
4.8ꢀ1.6
35000ꢀ7000
3.5ꢀ1.3
220ꢀ60
[a] Kd values were determined using an ARC–Lum binding/displacement assay (de-
tailed description provided in the Supporting Information); data represent the mean
of 3–5 independent experiments; ND: not determined.
concentration 100 nm) compared with that of Pim-1 (2 nm).
v/v/v). The compounds obtained were purified by reversed-
phase HPLC on a C18 column.
This was needed for shifting the displacement IC50 curves to
higher concentration values for characterization of tight-bind-
ing inhibitors.[16] As ARC(PIM) inhibitors have relatively low af-
finity toward PKAc, ARC-1188 at a concentration of 5 nm was
used for displacement experiments with PKAc (2 nm). The ob-
tained IC50 values were used to calculate dissociation constants
(Kd). ARC–Lum probes possess Kd values of 8 nm against the
For the biochemical characterization of ARC(PIM) com-
pounds, their affinity toward Pim-1 and PKA catalytic subunit
(PKAc) was measured in a binding/displacement assay with
ARC–Lum probes and time-gated luminescence intensity read-
out (Figure 1).[16] Binding of PKs to ARC–Lum probes induces
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The inhibitory potency of some ARC(PIM) compounds was
also determined using the Omnia kinetic assay (Figure 2b).[18]
The calculated inhibition constant (Ki) and Kd values were simi-
lar for both the reference compound SGI-1776 (Kd =4.8ꢀ
1.6 nm, Ki =4.5ꢀ1.5 nm) and the newly synthesized ARC(PIM)
compounds 7 (Kd =1.8ꢀ0.6 nm, Ki =1.4ꢀ0.6 nm), 10 (Kd <
0.2 nm, Ki <0.5 nm), and 11 (Kd =180ꢀ50 nm, Ki =145ꢀ
45 nm).
A bicyclic ABBT moiety alone (3) showed no binding to Pim-
1 even at 100 mm concentration of the kinase, whereas upon
its conjugation with arginine-rich peptides, the obtained com-
pounds (conjugates 6 and 7) exhibited low to sub-nanomolar
affinity toward Pim-1, and over 50-fold selectivity over PKAc.
Piperidine-modified tricycle BBTP 8 revealed moderate affinity
toward Pim-1 (Kd =52ꢀ8 nm) and very low affinity toward
PKAc (Kd >100 mm). In line with the previously mentioned ten-
dency, conjugation of BBTP with a peptide (compound 9) led
to a 50-fold improvement in affinity of the compound toward
Pim-1. However, an even greater increase in affinity toward
PKAc was determined for this conjugate. Conjugate 9 incorpo-
rating the BBTP moiety was more Pim-1-selective than conju-
gates 6 and 7 with the ABBT fragment.
Figure 1. Binding assay used for determination of affinity of inhibitors: dis-
placement of an ARC–Lum probe from its complex with a protein kinase
(PK) by competitive inhibitors.[16]
It has been shown previously that the attachment of a hy-
droxypyrrolidinyl group to the pyrimidinone ring leads to in-
creased Pim selectivity of inhibitors.[5] Therefore, an l-hydroxy-
proline spacer was introduced into the conjugate to increase
its Pim-1/PKAc selectivity. The resulting compound (10) re-
vealed very high affinity toward Pim-1 (Kd <0.2 nm), together
with almost a 50-fold decreased affinity toward PKAc (Kd =
1700ꢀ300 nm).
Thereafter, the effect of the tether length and number of ar-
ginine residues in the peptide moiety on the affinity of conju-
gates was tested. The affinity of conjugates containing either
a 6-aminohexanoic acid or an 8-amino octanoic acid linker was
compared. To keep the affinity of the compounds in an easily
measurable region, only two arginine residues were incorporat-
ed into the conjugates. The two conjugates possessed coincid-
ing affinity toward Pim-1 (Kd =180ꢀ50 nm and 250ꢀ70 nm
for 11 and 12, respectively). Both conjugates had even lower
affinity than fragment 8. The increase in the number of argi-
nine residues in the peptide moiety from two (11) to four (14)
enhanced the affinity of the conjugate toward Pim-1 by 18-
fold. Conjugate 10 incorporating six arginine residues and
a lysine residue had an even higher affinity (>50-fold increase
over 11). Conjugates with six (9, 10, 13), eight and nine (15
and 16) arginine residues had sub-nanomolar affinities that
could not be differentiated with the analytical assay used in
the present study. These findings are in agreement with the
supposed spatial positioning of BBTP-type ATP-competitive in-
hibitors and a substrate peptide, Pimtide (KRRRHPS) in the
active site of Pim-1. Superpositioning of the corresponding
crystal structures (PDB codes 3JY0 and 2BIL, respectively)
points to the distance between these compounds of ~8 ꢁ (fig-
ure S5 in the Supporting Information) that could be retained
by the application of a single 6-aminohexanoic acid linker. Ac-
cording to this model for ARCs, the optimal positioning of argi-
nine residues requires the application of a longer linker for
Figure 2. Characterization of Pim-1 inhibitors 7, 10, 11, and SGI-1776 by
a) displacement of ARC-1139 (100 nm total concentration) from the complex
with Pim-1 (2 nm) and b) inhibition of Pim-1.
ARC-1139–Pim-1 complex and 10 nm against the ARC-1188–
PKAc complex, as determined by titration of the probes with
PKs (figure S1 in the Supporting Information). In spite of using
the high-affinity probes at high concentration, displacement
IC50 values of several ARC(PIM) compounds were still in the
low nanomolar range (close to the kinase concentration in the
binding assay), which made the calculation of reliable Kd
values for these compounds impossible.
The affinities of two previously characterized commercially
available Pim inhibitors staurosporine and SGI-1776 were also
determined. A generic PK inhibitor, staurosporine, bound to
Pim-1 and PKAc with similar affinity (Kd =5.2ꢀ1.8 and 3.5ꢀ
1.3 nm, respectively), whereas a selective Pim inhibitor, SGI-
1776, displayed Kd values of 4.8ꢀ1.6 nm and 220ꢀ60 nm for
Pim-1 and PKAc, respectively (literature Pim-1 IC50 =7 nm[2f]).
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conjugates with two arginine residues. Therefore ARCs with
two arginine residues did not possess increased affinity com-
pared with BBTP alone, but compounds with four or more argi-
nine residues revealed much stronger binding ability.
to the bisubstrate character of these inhibitors. The dual bind-
ing mode of generic ARC-type inhibitors with PKAc was firmly
established through X-ray analysis of ARC/PKAc co-crystals and
via displacement of ARC-Fluo probes with single substrate-
competitive inhibitors.[9c,19] From the other side, the established
sub-micromolar affinity of a widely used transport peptide (d-
Arg)9-NH2 toward Pim-1 points to the possibility that the effect
of the peptide on the properties of the cargo is not limited to
increase of its cell uptake efficiency, but rather that protein
phosphorylation balances could also be regulated by inhibition
of Pim and other PKs by the transporter.[20]
Introduction of an additional aminocaproic acid (Ahx) linker
unit and a d-arginine spacer into the tether between sub-
strate-competitive moieties caused only moderate changes in
binding affinities, and the effect was opposite for conjugates
incorporating either an ABBT (6 and 7) or a BBTP (11 and 13)
fragment. All tricycle-containing conjugates with a hydroxypro-
line spacer had remarkable Pim-1-selectivity over PKA. For
PKAc, an increase in the number of arginine residues also led
to higher affinity of ARC(PIM) compounds, whereas in the case
of conjugates incorporating a hydroxyproline spacer (10–16),
even compounds with eight arginine residues possessed only
sub-micromolar affinity toward PKAc.
To evaluate the PK selectivity of compound 10, its was
tested in a panel with 124 PKs (carried out by the Division of
Signal Transduction Therapy, University of Dundee, UK). Residu-
al activities of PKs were determined at a compound concentra-
tion of 50 nm (table S2 in the Supporting Information). The
testing revealed the highest inhibitory potency of 10 toward
the Pim family (17%, 40%, 31% for Pim-1, Pim-2 and Pim-3, re-
spectively) and very weak or no inhibition against all other kin-
ases tested.
According to the results of structure–affinity studies with ge-
neric ARC compounds and structural analysis of ARC/PKAc co-
crystals,[19c] ARCs can be tagged with voluminous groups at the
C terminus of the peptide moiety without diminishing their af-
finity toward PKs. Here, a high-affinity inhibitor (10) was la-
beled with the fluorescent dye PromoFluor-647 via the side
chain amino group of the lysine residue. The obtained fluores-
cent probe (17) was titrated with Pim-1 to determine the affini-
ty of the probe (figure S4 in the Supporting Information). The
calculated Kd value of 0.6ꢀ0.2 nm for 17 indicated that the
dye did not disturb binding of the inhibitor to the kinase and
pointed to the applicability of the probe for screening of Pim
inhibitors in biochemical and cellular assays.
In summary, the bisubstrate approach was used to develop
inhibitors with sub-nanomolar affinity for Pim-1. The bisub-
strate character—simultaneous association with binding sites
of distinct substrates—of novel inhibitors was shown by dis-
placement of the dye-labeled conjugate from the complex
with Pim-1 by both an ATP-competitive inhibitor SGI-1776 and
a protein-substrate-competitive inhibitor (d-Arg)9-NH2. Thus, bi-
substrate inhibitors possessing sub-nanomolar affinity can be
constructed for pharmacologically important protein kinases.
In a binding assay with fluorescence anisotropy (polariza-
tion) readout, the bisubstrate character[10] (simultaneous associ-
ation with both substrate binding sites of Pim-1) of the labeled
ARC(PIM) inhibitors was demonstrated by displacement of
probe 17 from its complex with Pim-1 by both the ATP-com-
petitive inhibitor SGI-1776 and protein-substrate-competitive
peptide (d-arginine)9-NH2 (Figure 3).
Experimental Section
Experimental details for the synthesis and characterization of new
compounds (UV, NMR, HRMS and selectivity data), and details of
the biochemical assays used in this study are available in the Sup-
porting Information.
The substantially higher Pim-1 affinity of ARC(PIM) com-
pounds (e.g., Kd <0.2 nm for 10) if compared with the affinity
of their structural constituents, ATP mimics 8 (Kd =52ꢀ8 nm)
and the peptide (d-Arg)9-NH2 (Kd =290ꢀ110 nm), also points
Acknowledgements
This work was supported by grants from the Estonian Science
Foundation (8230, 8419, ERMOS30) and the Estonian Ministry of
Education and Sciences (SF0180121s08). S.K. is funded by the
Structural Genomics Consortium,
a registered charity (no.
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canada Foundation for Innovation, Genome
Canada, GlaxoSmithKline, Pfizer, Eli Lilly, Takeda, AbbVie, the No-
vartis Research Foundation, the Ontario Ministry of Research and
Innovation, and the Wellcome Trust.
Keywords: bisubstrate inhibitors · fluorescent probes · Pim-1 ·
protein kinases
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Received: January 29, 2013
Revised: April 5, 2013
Published online on April 24, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 909 – 913 913