Identification of quinones as novel PIM1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.04.079

PIM1 is a proto-oncogene encoding the serine/threonine PIM1 kinase. PIM1 kinase plays important roles in regulating aspects of cell cycle progression, apoptosis resistance, and has been implicated in the development of such malignancies as prostate cancer and acute myeloid leukemia among others. Knockout of PIM1 kinase in mice has been shown to be non-lethal without any obvious phenotypic changes, making it an attractive therapeutic target. Our investigation of anthraquinones as kinase inhibitors revealed a series of quinone analogs showing high selectivity for inhibition of the PIM kinases. Molecular modeling studies were used to identify key interactions and binding poses of these compounds within the PIM1 binding pocket. Compounds 1, 4, 7 and 9 inhibited the growth of DU-145 prostate cancer cell lines with a potency of 8.21 μM, 4.06 μM, 3.21 μM and 2.02 μM.

Full text of DOI:10.1016/j.bmcl.2016.04.079

Accepted Manuscript
Identification of Quinones as Novel PIM1 Kinase Inhibitors
Richard L. Schroeder, Navneet Goyal, Melyssa Bratton, Ian Townley, Nancy
A. Pham, Phan Tram, Treasure Stone, Jasmine Geathers, Kathy Nguyen,
Jayalakshmi Sridhar
PII:
S0960-894X(16)30467-X
http://dx.doi.org/10.1016/j.bmcl.2016.04.079
BMCL 23841
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2016
26 April 2016
27 April 2016
Please cite this article as: Schroeder, R.L., Goyal, N., Bratton, M., Townley, I., Pham, N.A., Tram, P., Stone, T.,
Geathers, J., Nguyen, K., Sridhar, J., Identification of Quinones as Novel PIM1 Kinase Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.079
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Identification of Quinones as Novel PIM1 Kinase Inhibitors
Richard L. Schroedera, Navneet Goyala, Melyssa Brattonb, Ian Townleyb, Nancy A. Phama, Phan
Trama, Treasure Stonea, Jasmine Geathersa, Kathy Nguyena, and Jayalakshmi Sridhara,*
aDepartment of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA
bCell and Molecular Biology Core Facility, Xavier University of Louisiana, New Orleans, LA
70125 USA
*Corresponding Author:
1, Drexel Dr., New Orleans, LA 70125
E-mail: jsridhar@xula.edu

Abstract
PIM1 is a proto-oncogene encoding the serine/threonine PIM1 kinase. PIM1 kinase plays
important roles in regulating aspects of cell cycle progression, apoptosis resistance, and has been
implicated in the development of such malignancies as prostate cancer and acute myeloid
leukemia among others. Knockout of PIM1 kinase in mice has been shown to be non-lethal
without any obvious phenotypic changes, making it an attractive therapeutic target. Our
investigation of anthraquinones as kinase inhibitors revealed a series of quinone analogs showing
high selectivity for inhibition of the PIM kinases. Molecular modeling studies were used to
identify key interactions and binding poses of these compounds within the PIM1 binding pocket.
Compounds 1, 4, 7 and 9 inhibited the growth of DU-145 prostate cancer cell lines with a
potency of 8.21 M, 4.06 M, 3.21 M and 2.02 M.

Three isoforms (PIM-1, -2 and -3) of PIM kinases are encoded in the gene. The three
members of the PIM family are serine/threonine kinases that exhibit very high homology and
hence a certain amount of functional redundancy is found. These enzymes have little homology
with the other members of protein kinase family. The PIM1 gene was initially identified as a
proviral integration site in Moloney murine leukemia virus-induced mouse T-cell lymphomas1, 2.
PIM kinases are implicated in the development of solid tumors. DNA microarray analyses
showed the overexpression of PIM1 in human prostate cancer in relation to the grade of the
prostate cancer3. PIM kinases are critical mediators of hematopoeitic cell survival in both
immunology and oncology4, 5. PIM kinases potently cooperate with Myc, block apoptosis, and
induce oncogenic transformation6-8. Sustained PIM1 expression is induced by many cytokines,
mitogens and growth factors and PIM1 has been shown to be a major downstream target of the
STATs (signal transducer and activator of transcription)9, 10. PIM proteins role in human cancers
such as prostate, pancreatic, colon, chronic lymphocytic leukemia, non-Hodgkin’s lymphoma
and multiple myeloma is well established10-15. It has been found that the knockout of PIM1 in
mice is not lethal and its absence does not induce any immediately obvious phenotype16. This
makes PIM1 an attractive target for chemotherapy.
Many classes of ATP competitive small molecule PIM1 inhibitors have been recently
reported such as the pan-kinase inhibitor staurosporine and its related bisindolylmalemides17,
imidazo[1,2-b]pyridazines18, pyridones19, 20, flavonoids21, 22, benzoisoxazoles23, cinnamic acids24,
thiazolidinones25, etc. Most of these molecules inhibit a variety of protein kinases, leading to
questionable therapeutic value. Only SGI-1776 has been shown to fairly selective inhibitor of
PIM kinases and demonstrated to effectively induce apoptosis in lymphocytic leukemia cells26, 27
Given the prominent role of PIM kinases in numerous types of cancers, there is an urgent need to
find more lead compounds to be developed as PIM kinase inhibitors that can exhibit good kinase
selectivity profile.
.
Recently several reports have been published solving the crystal structure of PIM1
revealing several interesting features that is unique to PIM kinases24, 28-32. The PIM1 protein has
the typical secondary structural architecture with two domains connected by the hinge region
apart from a distinctive N-terminal peptide sequence. The PIM kinases contain the unique
consensus sequence ERPXPX in the hinge region. The hinge region is atypical due to presence
of a proline residue, Pro123, capable of making only a single hydrogen bond to the natural

substrate ATP. Of the five kinases that share such a characteristic, three of them belong to the
PIM kinase family. The P-loop of PIM1 is glycine rich containing residues GSGGFG. These
aspects could play an important role in determining the potency and specificity of inhibitors. Our
searches for lead molecules as kinase inhibitors lead us to quinolines and anthraquinones.
Emodin a natural anthraquinone isolated from Rheum emodi, is a Traditional Chinese Medicine.
Emodin is an established tyrosine kinase inhibitor33 and has been shown to inhibit several
kinases such as HER233, Pim134, Casein Kinase 235, DYRK1a36, SGK36, JAK237, etc.
Emodin was docked into the crystal structure of Pim1 kinase to understand the
interactions and the features of emodin responsible for these interactions (Figure 1). The
consensus docking postures revealed that the Emodin did not make any hydrogen bonds with the
hinge region residues. Emodin made two hydrogen bonds with the residues Lys67 and Glu89.
Additional -alkyl interaction of the emodin aromatic rings with the residue Ile185 was also
evidenced. Many groups have solved the crystal structure of Pim1 and a total of 53 crystal
structures were found deposited on the RCSB PDB website (http://www.rcsb.org)38. All the
ligands in the crystal structures were ATP-competitive or ATP-mimetic.A study of the
interactions of the numerous ligands to the Pim1 protein revealed that only on hydrogen bond
was made by the ligands with the hinge region to the residue Glu121. Other hydrogen bonds that
were made by the ligands involved the residues Gly45, Asp128, Glu171, Asn172, Asp186.
Additionally, hydrophobic interactions and-methyl interactions were found for these ligands
with the Pim1 residues Phe49, Ile185 and Leu174. These insights helped during the search for
Emodin analogs that could act as potential inhibitors of Pim1 kinase.
Glu
Lys
Emod
Phe
Ile1
Figure 1: (A) Binding mode of emodin to the ATP binding site of Pim1
kinase (PDB ID: 2O64.pdb). The protein residues are shown as stick
models and the emodin moecule is shown as yellow ball & stick model.

A 2D-pharmacophore involving the key functional groups of Emodin was designed and
Sybyl-UNITY search was performed of the PUBCHEM and ZINC databases. The hitlist
obtained was subjected to docking studies and based on the docking scores, a total of ~40
emodin analogues were chosen. The compounds 10, 12 and 14 were synthesized.
Figure 2: Schemes I and II outline the synthesis of compounds 12 and 14.
The precursor compound in the synthesis of compound 10, 2-formyl-1,4,5,8-
tetramethoxynaphthalene, was synthesized through known literature procedure39. Compound 11
was obtained through a double Friedel-Crafts acylation using 1,4-dimethoxybenzene and 2-
methylmaleic anhydride at high temperatures. Demethoxylation using HCl gave the desired
product 11 (55%). (Figure 2-Scheme I). Reported attempts using radical initiator catalysts in the
bromination of compound i resulted in no desired product formation40. Two 120W tungsten
lamps were used to initiate hemolytic bond cleavage of NBS by placing the lamps close to the
flask and allowing a vigorous reflux over 2h. Compound 12 was obtained after purification
(32%) (Figure 2-Scheme I). Formation of the trimethylsilyl diene 13 (74%) in the synthesis of 14
proceeded through an LDA enolate reduction41. (Figure 2- Scheme II). The Diels-Alder
cycloaddition between 13 and 1,4-quinone occurred slowly over 20h at 0oC. The corresponding

hydroxynaphthoquinone 14 (37%) was obtained through hydrolysis using HCl (Figure 2-
Scheme II). The synthesized compounds were characterized by 1H-NMR, 13C-NMR and Mass
spectroscopies. All compounds had characteristic peaks with appropriate spectroscopic values
(The experimental methods and spectroscopic charaterizations are detailed in Supporting
information). An initial high-throughput in-vitro screen at 10M concentration of all of the
compounds and emodin showed that eight compounds exhibited perceptible inhibition of Pim1
kinase (Figure 3).
Figure 3. Structures of Emodin and compounds 1-10, 12 and 14
investigated for inhibition of Pim1 kinase.

Since Emodin inhibited numerous kinases, the selectivity of the analogues chosen needed
to be evaluated first. At this time Chemical Computing group (CCG) introduced a new module in
7%
14%
14%
CDK
CK2
DAPK
PIM
36%
CAMK
CMGC
TK
29%
43%
SRC
50%
7%
Figure 4. The results of kinase search in MOE using the 2D structure of
compound 4. The pie charts represent the identity of kinase structure hits for each
family of kinases and the group for which these kinases belong.
MOE for kinase search. The kinase search module uses a database of kinases aligned globally.
The kinase detection algorithm employed in MOE is based on the alignments with the kinase
reference collections within the Hank’s regions. Additionally a database containing a broad
collection of chemical scaffolds or building blocks of available kinase inhibitors has been
compiled in MOE. This database contains structures belonging to 10 classes of inhibitors
(adenosines, staurosporines, oxoindoles, purines, isoquinolines, quinazlines, ureas, imidazoles,
aminopyrimidines, and anthraquinones) along with the SMARTS strings that can be used for
substructure search. The kinase search module had the option of ligand similarity search using
tanimoto distance cutoff property to identify kinases that could be the potential target of the
compound under study. Compound 4 was used as the study compound and an initial tanimoto
cutoff value of 0.80 was specified. The results displayed only those structures that belonged to
casein kinase II. This could have been the due of the presence of a crystal structure of casein
kinase II with Emodin as the bound ligand. The stringent tanimoto distance cutoff value was
relaxed to 0.60 for the next kinase search. The results (Figure 4) presented 5 protein kinases that
could be the potential target for inhibition- CK2 (43%), Pim kinases (29%), SRC kinase (14%),
CDK (7%) and DAPK (7%). These five kinases belonged to the three families CMGC (50%),
CAMK (36%) and TK (14%).

To understand the significance of the kinase selectivity search using MOE computing
program, compound 4 was evaluated against a panel of 100 kinases at KINOMEscan
(www.kinomescan.com). The compound(s) were screened at the 10 M concentration, and
results for primary screen binding interactions are reported as '% Ctrl' , where lower numbers
indicate stronger hits.
%Ctrl Calculation
The results presented in supporting information table 1 show that compound 4 that the
only protein kinases strongly inhibited are CSNK1D (30% control at 10M), Pim1 (34% control
at 10M) and Pim3 (36% control at 10M). The selectivity score S(35) was calculated as 0.022.
The KINOMEscan results were in excellent agreement with the kinase search performed in MOE
with casein kinase and Pim kinases being the main protein kinases to be targeted by compound 4.
The dose response curves were performed for the inhibition of Pim1 kinase by Emodin,
the eight compounds identified in the initial high-throughput screening (compounds 1-9) and
three synthesized compounds (compounds 10, 12 and 14) (Table 1). Emodin exhibited an IC50
value of 2.5 M in the in-vitro assay. Compounds 3, 6, 7 and 9 showed IC50 values of 7.4 M,
3.0 M, 3.6 M and 3.5 M which were equivalent to that of Emodin. Compound 2 and 4
presented IC50 values of 11.6 M and 19.2 M which were in the low micro molar range and
close to that of Emodin. Only compound 5 showed poor inhibition potency for Pim1 kinase with
an IC50 value of 57.1 M. Compounds 10, 12, and 14 were found to have IC50 values of 28 M,
27 M, and 28 M, respectively.

Table 1. Inhibition of
Pim1 kinase by emodin
and compounds 1 to 10,
12 and 14.
Pim1 is highly expressed in a significant fraction of human prostate cancer in which c-
MYC was also overexpressed. It was determined that synergistic interaction of Pim1 and c-MYC
was critically dependent on Pim1 kinase activity implying that inhibition of Pim1 kinase activity
should be effective in cancer treatment. To determine whether these compounds were effective
in-vivo, we tested the growth inhibitory properties of Emodin and compounds 1, 4, 7 and 8 on
the human prostate cancer cell line DU-145 that is known to express PIM-142. Figure 5 shows
Figure 5: Growth inhibition curve for DU-145 cells
treated with compounds 1, 4, 7, 9 and Emodin.

that compounds 1, 4, 7, 9 and Emodin inhibit the growth of DU-145 cells with an IC50 of 3.93
M, 8.21 M, 4.06 M, 3.21 M and 2.02 M. These data show that the Emodin and its analogs
show good promise in anticancer activities on prostate cancer cell lines.
Docking studies of Emodin and compounds 1-6 were performed on the known crystal
structure of Pim1 kinase protein (PDB ID: 2O64.pdb). The binding modes of compounds 1-6 to
Pim1 protein were similar to that of Emodin in that they occupied the ATP-binding pocket, but
did not show any hydrogen bonding interaction with the hinge region residues (Figure 5). One of
the carbonyl groups and its adjacent hydroxyl group of the compounds makes hydrogen bonds to
the side chains of two highly conserved residues, Lys67 and/or Glu89. The binding interactions
of the compounds with the best inhibition potency are shown in figure 5A for compound 3 and
figure 5B for compound 6. The binding postures show the planar portion of the molecules
aligned in the cavity in such a way that -alkyl group interactions could be maximized with the
numerous hydrophobic residues lining the top and bottom regions of the binding site. From a
study of all the available crystal structures and the co-crystallography study by the Bremer
group29, it is evident that all of the small molecule inhibitors bind in the ATP binding site of
Pim1, and most of them are localized near the hinge and the conserved Lys67/Glu89/Phe187
region. The fact that our compounds bind to Lys67 and/or Glu89, demonstrates that they satisfy
the requirements of hydrogen bonding to Lys67/Glu89 region as exemplified by the previous
studies Pim1. The docking scores of the compounds had a trend similar to that of their inhibition
potency with the more potent compounds having lower scores indicating more favorable poses
(supporting document, table 2).

Compound 3
Compound 6
A
B
Figure 6: Binding mode of compounds 3 (A) and 6 (B) to the
ATP-binding pocket of Pim1 kinase (PDB ID: 2O64.pdb). The
In conclusion, a new series of compounds have been identified as inhibitors of Pim1
kinase. They have excellent effect on the arrest of growth in the prostate cancer cells DU145.
The selectivity of the representative compound 4 for the Pim1, Pim2 and CSNK1D was
discovered by the KINOMEscan assay against a panel of 100 kinases. Docking studies have
revealed the binding orientations of these molecules in the ATP-binding cavity of the Pim1
protein. The insights obtained from the docking studies will help us to design and develop new
Pim1 inhibitors with improved potency while maintaining the selectivity for Pim kinases.
Acknowledgement
This research work was funded by the NIH-RCMI (2G12MD007595-06) from the National
Institute on Minority Health and Health Disparities and the startup grant by the Louisiana Cancer
Research Consortium. Additional support from the Department of Defense Award (W81XWH-
11-1-0105), the Louisiana Biomedical Research Network (LBRN- P20GM103424) is greatfully
acknowledged. We thank the NIH-BUILD Grant—TL4MD009607 and the Center for
Undergraduate Research at Xavier University of Louisiana for supporting the students who
worked on this project. The contents are solely the responsibility of the authors and do not
represent the official views of NIH.
Supplementary data
Supplementary data (synthetic protocols, compound characterization data, assay details, and
supplementary figures) associated with this article can be found in the online version.

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Graphical Abstract
Glu89
Lys67
Emodin
Phe49
Ile185