Fragment-based Discovery of a Small-Molecule Protein Kinase C-iota Inhibitor Binding Post-kinase Domain Residues

Article abstract of DOI:10.1021/acsmedchemlett.8b00546

The atypical protein kinase C-iota (PKC-l) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-l with IC₅₀ = 424 μM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-l (IC₅₀= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-l.

Full text of DOI:10.1021/acsmedchemlett.8b00546

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Letter
Fragment-based Discovery of a Small-molecule Protein
Kinase C-iota Inhibitor Binding Post-kinase Domain Residues
Jacek Kwiatkowski, Nithya Baburajendran, Anders Poulsen, Boping Liu, Doris Hui
Ying Tee, Yun Xuan Wong, Zhi Ying Poh, Esther HQ Ong, Nurul Dinie, Joseph
Cherian, Anna Elisabet Jansson, Jeffrey Hill, Thomas H. Keller, and Alvin W Hung
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.8b00546 • Publication Date (Web): 15 Feb 2019
Downloaded from http://pubs.acs.org on February 17, 2019
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Fragment-based Discovery of a Small-molecule Protein
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Kinase C-iota Inhibitor Binding Post-kinase Domain Residues
Jacek Kwiatkowski,* Nithya Baburajendran, Anders Poulsen, Boping Liu, Doris Hui Ying
Tee, Yun Xuan Wong, Zhi Ying Poh, Esther HQ Ong, Nurul Dinie Binte Rahad, Joseph
Cherian, Anna Elisabet Jansson, Jeffrey Hill, Thomas H. Keller and Alvin W. Hung*
Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 11 Biopolis
Way, Helios #03-10/11, Singapore 138667 (Singapore)
KEYWORDS: Protein kinase C iota, fragment-based drug discovery, kinase inhibitor, C-terminal tail,
hepatocellular carcinoma, non-small cell lung cancer
Abstract: The atypical Protein Kinase C-iota (PKC-) enzyme is implicated in various cancers and has been put forward
as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine
fragment weakly inhibiting PKC- with IC50 = 424 M. Driven by structure-activity relationships and guided by docking
hypothesis, the weakly bound fragment was eventually optimized into potent inhibitor of PKC- IC50 = 270 nM.
Through the course of the optimization, an intermediate compound was crystallized with the protein and careful analysis
of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of
PKC-.
INTRODUCTION
Protein Kinase C (PKC) family consists of 11 isoenzymes that can be grouped into 3 subfamily types: the classical,
novel and atypical PKCs. The interest in PKC enzymes as targets for therapeutic intervention stems from the fact that
PKCs are important for critical cellular processes implicated in the promotion of tumor growth.1 The levels of most of
the PKC isoenzymes are altered in cancer cells and several PKCs are involved in regulation of apoptosis.2 However, the
complex network of processes dependent on PKCs is the cause of controversy whether PKC enzymes are viable drug
targets and consequently, out of the 11 isoforms, only PKC-epsilon and PKC-iota (PKC-) are described unambiguously
as bona fide oncogenes.3-9 Accordingly, isoform selective, high quality PKC inhibitors are needed to elucidate the
complex biology and explore the therapeutic potential of PKC modulation.
We were particularly interested in targeting atypical PKC-, due to its central role in the formation of an oncogenic
complex with epithelial cell transforming sequence 2 (ECT2) and partitioning defective 6 homolog (Par6). The assembly
of the three enzymes triggers the oncogenic Rac-Pak-Mek-Erk signalling cascade, which ultimately leads to tumor
growth and invasion, as demonstrated in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC).10,11
Correspondingly, high levels of ECT2 were correlated with poor prognosis for patients suffering from NSCLC and
HCC. More importantly, upregulation of PKC-, has been directly linked to poor cancer-specific survival in NSCLC. In
total, inhibition of PKC- would be an attractive approach for therapeutic intervention in HCC, through modulation of
ECT2 phosphorylation. To date, several atypical PKC inhibitors have been reported;12-15 herein we describe the
optimization of a fragment hit against PKC-
RESULTS
A fragment screen15 identified pyridine-amide fragment (compound 1, Table 1, IC50 = 424 M, LE = 0.29)16 as a hit
against PKC-. In the absence of an X-ray structure, we hypothesized the aminopyridine moiety in 1 to serve as a hinge
binder and begun a systematic structure-activity relationship-driven (SAR) exploration of the binding site by examining
substitutions of the phenylamine (synthesis described in Supporting Information). Interestingly, replacement of the
phenyl with pyridine resulted in a 20-fold increase in potency (compound 2, IC50 = 22 M), leading to a rapid
improvement in the overall efficiency of the molecule (LE = 0.40, LLE = 4.2). This simple substitution indicated the
possibility of a hydrogen bond (H-bond) interaction between the pyridine nitrogen and PKC-. Next, a close analogue
of compound 2 containing 2-aminopyrazine hinge binding motif in place of the 2-aminopyridine was found to be equally
active (compound 3). Based on the aminopyrazine scaffold, it was found that repositioning of the pyridine nitrogen (4)
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or replacing pyridine with phenol (5) did not result in a significant change in inhibitory activity as compared to
compound 3. On the other hand, compound 6 containing the more flexible pyridine-methylene amine was inactive.
Interestingly, replacement of the pyridine with a large bicyclic indole was found to be well tolerated in this position,
while 7-azaindole showed no inhibitory activity (compounds 7 and 8 respectively). Taken together, the equipotency of
phenol, indole (both H-bond donors) and pyridine (H-bond acceptor) could suggest water-mediated interaction, where
water could interact with either donor or acceptor.
9
Table 1. Fragment hit 1 and modifications of the amine moiety of the amide.a
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R
NH
X
O
H2N
N
IC50
[M]
IC50
[M]
Cmpd
1
R
X
LE
Cmpd
9
R
X
N
LE
N
CH 424
CH 22
0.29
19.6
24.0
0.36
Et
N
2
N
0.40
10
11
N
N
0.33
0.31
t-Bu
3
4
N
N
N
23
77
0.40
0.35
42.2
263
N
Me
N
5
HO
N
N
76
0.33
12
N
0.27
Me
N
6
7
N
N
> 384
34
-
13
14
N
N
> 384
> 384
-
-
Br
N
0.32
N
H
Br
8
N
> 384
-
N
H
N
a The two close analogues 2 and 3 showed improved potency and ligand efficiency. Subsequent modifications of compound
3 indicated little potential for improvement in IC50 by modifications of the peripheral pyridine.
Driven by the ability of the pocket to accommodate larger indole substituents, we next sought to explore potential
hydrophobic interactions through substitutions at the 2-position of the peripheral pyridine / phenyl. As shown in Table
1, none of the larger compounds containing ethyl (9), tert-butyl (10) or cyclopropyl (11) substituents brought any
significant improvement in potency. On the other hand, compounds bearing 2,6-dimethylpyridineamine (12) or
bromopyridine amines (13 & 14) were inactive. Overall, the initial SAR suggested the peripheral pyridine / phenyl
engaged in favorable interactions with protein; however due to apparent limited space and possible presence of water
molecule elaboration of the pyridine /phenyl did not transpire as an effective optimization strategy at this point.
Seeking further improvement, we next turned to molecular modelling to guide subsequent evolution of compound 2.
Specifically, 2 was docked into published PKC- X-ray structure with the assumption that the 2-aminopyridine is
engaged in hydrogen-bonding interactions with PKC- hinge residues Glu333 and Val335. Furthermore, as guided by
the SAR in Table 1, the peripheral pyridine was oriented with the nitrogen pointing towards the wall of the binding site
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consisting of Ile332 and Lys283 (Figure 1).17 This docking conformation supported the formation of hydrogen-bonding
interactions between the peripheral pyridine nitrogen and a flexible lysine 283 as well as between the amide carbonyl
and Thr395. As shown in Figure 1b, expansion from the hinge-binding 2-amino-pyridine core could increase binding
energy through gaining hydrophobic interactions with isoleucine (Ile260) and a hydrogen-bond with aspartic acid
(Asp339). Notably, the model also suggested phenylalanine Phe552 as a possible partner for - interactions with
expanded ligands. It is noteworthy that since Phe552 is located in a flexible post-kinase loop (C-terminal tail), fulfilling
this interaction might not be straightforward. While Phe552 was shown to reside in the ATP site in both the apo and
ATP-bound structures of PKC- (PDB:3A8X and 3A8W), the residue is displaced by small molecule inhibitors, as
shown in X-ray structures of PKC--inhibitor complexes (e.g. PDB: 3ZH8 and 1ZRZ). Additionally, a varying degree
of structural rearrangement of the post-kinase domain dependent on the geometry of a ligand would likely affect the
clarity of SAR.
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Figure 1. Compound 2 (in green) modelled into X-ray structure of PKC-ι. a) Hydrogen bonds between 2 and protein
residues are shown as yellow dashed lines; b) a grid is superimposed on the binding site showing areas favorable for
binding ligand atoms with the following properties: hydrophobic (yellow), acceptor or negatively charged (red), donor
or positively charged (blue).
To enable the envisioned interactions in the inhibitor series, substitutions on the hinge-binding pyridine were next
attempted. Firstly, a bromine atom was introduced at the 5 position of the 2-aminopyridine moiety of compound 2 to
probe for space and as a handle for subsequent modifications (Table 2). Somewhat unexpectedly, the resulting
brominated compound 15 was inactive. On the other hand, a phenyl ring installed at the same position was well tolerated
(16). However, compared to compound 2, the addition of the phenyl ring did not bring about a significant improvement
in activity, which is also reflected in the decrease in ligand efficiency of compound 16 (LE = 0.28). The discrepancy
between the actual inhibitory results and the predicted improvement in potency from docking experiments could be
caused by the energy penalty paid for reorganization of the post-kinase domain of PKC-, offset by interactions of the
phenyl moiety in 16 with protein residues.
Table 2. Expansion from the hinge binding core.a
N
NH
X
R
O
H2N
N
IC50
IC50
Cmpd
15
X
R
LE
-
Cmpd
18
X
R
LE
-
[M]
[M]
O
NH2
Br
CH
> 384
CH
> 384
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NH
NH
N
N
16
17
CH
CH
32
0.28
0.35
19
20
CH
N
0.34
0.27
0.32
0.32
NH2
0.71
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a
The benzylamine improved potency by 45 folds indicating a strong interaction with protein, hypothetically the aspartic
acid (Asp339). This interaction seemed translatable to phenylpiperazine-containing compounds as well.
To further validate our docking model and capture additional enthalpic interactions through possible engagement of
aspartic acid (Asp-339), 3-methylamino- group was introduced into the phenyl ring of compound 16. This strategy was
successful and a 45-fold increase in potency was observed (17, IC50 = 0.71 M) as compared to compound 16. The
critical role of the base was further confirmed by converting the benzylamine to an amide (18). Expectedly, compound
18 was inactive. Next, seeking a less basic alternative of benzylamine, a piperazine group was tested as a replacement
of the methylamino- substituent. Gratifyingly, the resulting compound 19 was 2-fold more potent than its benzylamine-
containing counterpart, with IC50 reaching 0.34 M. The analogous compound containing 2-aminopyrazine as a hinge
binder was equally potent (20, IC50 = 0.27 M). At this stage, the three most potent compounds 17, 19 and 20 were also
tested against three other PKC isoenzymes representative of PKC sub-families: classical (PKC-), novel (PKC-) and
atypical (PKC- and , SI Table 1). In general compounds showed little selectivity within PKC family. However, SAR
across the four isoenzymes indicated opportunity to enhance selectivity towards atypical or classical PKCs (see SI for
details).
Next, the pharmacokinetic profile of compound 19 was assessed through a series of in-vitro assays (Table 3). The
results confirmed the high solubility of the inhibitor at pH 7.4. Expectedly, the permeability as measured in a CACO-2
assay was found to be low. Additionally, 19 was stable in a metabolic assay with human liver microsomes, but
underwent fast degradation mediated by mouse liver microsomes. To evaluate the translation of biochemical activity
into antiproliferative effect against hepatocellular carcinoma cells, HUH-7 cells were treated with 19 and weak growth
inhibition was observed (GI50 = 11.3 M).
Table 3. Pharmacokinetic properties and cellular activity of compound 19.a
IC50 [M]
GI50 [M]
0.34
11.3
164
PKC-
Huh-7
Sol (pH 7.4) g/ml
CACO-2
(A-B [10-6cm/s])
0.28
MLM / HLM
71 / 5.1
(CL [µL/min/mg])
a PKC- inhibitor CRT006685412 was used as a positive control; GI50 against HUH-7 cells was 3.1 M
X-RAY CRYSTAL STRUCTURE AND BINDING MODE OF PKC- INHIBITORS
Concurrently to improving the potency of the scaffold, we attempted to elucidate the binding mode of the inhibitors
through X-ray crystallography. Our efforts were fruitful and compound 19 was successfully co-crystallized in complex
with PKC- (Figure 2, PDB: 6ILZ, SI). The analysis of the complex confirmed the key interactions between 19 and
PKC-: hydrogen-bond between 2-aminopyridine and the hinge residues of the protein - Val335 and Glu333 and
between the carbonyl oxygen of the amide and Thr395. The pyridine moiety of compound 19 was shown to engage in
lipophilic interactions with Val268, Thr395 and form a large number of van der Waals contacts with Asp396. While not
unambiguously shown in the X-ray, the arrangement of the protein residues around the peripheral pyridine of 19
suggested the existence of water-mediated hydrogen bond between the pyridine nitrogen, Lys283 and Asp396.
Molecular dynamics (MD) performed on the complex showed direct or water-mediated hydrogen bonds to Lys283
during 80% of the simulation time (see SI for details). Interestingly, analysis of the crystal structure revealed the phenyl-
piperazine substituent of compound 19 positioned in-between Ile260 and the sidechain of the post-kinase residue
Phe552. Furthermore, a clear  interaction of the phenyl group of 19 and Phe552, as well as lipophilic attraction to
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Ile260 were detected. The observed unusual alignment of the phenyl moiety of compound 19 on top of Phe552 enabled
additional van der Waals contacts with Tyr334. Furthermore, while not unequivocally shown in the X-ray structure of
the complex, the piperazine is likely to form hydrogen bond with Asp553 - another post-kinase domain residue
immediately following Phe552 and not with the expected Asp339 (see SI for additional comments). In contrast, many
kinase inhibitors have an aromatic ring in this region, which is sandwiched between the homologous residue of Ile260
and the protein backbone immediately following the kinase hinge region. Previously published PKC X-ray structures
with inhibitors have the post-kinase domain residue Phe552 displaced from the ATP-site and the inhibitors complexed
in these structures bind in the canonical binding mode (e.g. PDB: 3ZH8 and 1ZRZ). This binding conformation could
likely apply to compound 16, displacing the post-kinase loop and attracting Ile260 and explain no overall potency
improvement. However, the addition of a second strong interaction (H-bonding to Asp553, compounds 17, 19, 20) could
have resulted in the capture and rigidification of the post-kinase loop instead of displacing it.
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In general, the binding mode derived from the X-ray crystal structure is in a good agreement with the computational
model developed in the early fragment-to-hit-to-lead stage of the project. While the empirical SAR remained our
ultimate guide in fragment optimization, the model aided the successful design of potent inhibitors with substituents
targeting specific protein residues. Importantly, the iterative process of monitoring the SAR and refinement of the
computational model was key to the acceleration of fragment optimization in the absence of an X-ray crystal structure
19
of a starting fragment hit.15
,18,
Figure 2. X-ray crystal structure of compound 19 in complex with PKC-. a) Hydrogen bonds are shown as yellow dashed
lines.  stacking interaction of the phenyl group of the inhibitor with a post-kinase domain residue Phe552 is shown as a
blue dashed line b) view of the binding site of PKC- in complex with 19 with surface applied; c) comparison of binding mode
of compound 19 (I) with two small molecule inhibitors RBT205 (II) and CRT0066854 (III). Post-kinase loop (colored blue
and indicated by an arrow) is shown to interact with compound 19, while being displaced in the other two instances (II, III,
flexible loop only partially resolved).
SUMMARY
Optimization of fragment hit (IC50 = 424 M) towards potent inhibition of PKC- resulted in the discovery of sub-
micromolar inhibitors of the target protein (IC50 = 340 - 270 nM). It is noteworthy that the advancement of the fragment
hit was driven exclusively by SAR and molecular modelling. The binding mode of a more potent and advanced
compound 19 was then elucidated through X-ray crystallographic analysis and revealed a unique binding conformation
involving the post-kinase residues Phe552 and Asp553 of PKC-. The PKC kinase family is distinct in having the post-
kinase domain that inserts side chains into the ATP-site and our X-ray crystal structure shows that this can be exploited
for inhibitor design.
ASSOCIATED CONTENT
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Supporting information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Provided in the SI are: selectivity data; biochemical and cellular assay procedures and notes; details on X-ray crystal structure;
description of molecular modelling and molecular dynamics; extended analysis of binding mode of compound 19; synthesis
and analytical data for new compounds.
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Author Information
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Corresponding author
*Email: j.kwiatkowski@uniquest.com.au; whung@eddc.a-star.edu.sg
Orcid
Jacek Kwiatkowski: 0000-0002-6900-8241; Anders Poulsen: 0000-0002-2790-9340; Joseph Cherian: 0000-
0003-2663-7192; Alvin W. Hung: 0000-0002-9985-6794
Present addresses
(J.K.) Queensland Emory Drug Discovery Initiative, UniQuest; Brisbane, Australia.
(A.E.J.) GE Healthcare, Uppsala, Sweden.
Acknowledgements
This work was in part supported by the Agency for Science, Technology and Research (A*STAR) Joint Council grant
1231B105. The analysis of PKC- – compound 19 complex was undertaken on the MX1 beamline at the Australian
Synchrotron, part of ANSTO.
ABBREVIATIONS
ECT2, epithelial cell transforming sequence 2; Par6, bind partitioning defective 6 homolog; HCC, hepatocellular carcinoma;
NSCLC, non-small cell lung cancer; HLM, human liver microsomes; MLM, mouse liver microsomes.
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17 Lys283 – is a crucial catalytic residue, conserved in all PKC isoenzymes
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19 Erlanson, D. A.; Davis, B. J.; Jahnke, W. Fragment-Based Drug Discovery: Advancing Fragments in the Absence of Crystal
Structures. Cell Chem. Biol. 2019, 26, 9-15.
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Fragment-based Discovery of a Small-molecule Protein
Kinase C-iota Inhibitor Binding Post-kinase Domain Residues
Jacek Kwiatkowski,* Nithya Baburajendran, Anders Poulsen, Boping Liu, Doris Hui Ying Tee, Yun
Xuan Wong, Zhi Ying Poh, Esther HQ Ong, Nurul Dinie Binte Rahad, Joseph Cherian, Anna
Elisabet Jansson, Jeffrey Hill, Thomas H. Keller and Alvin W. Hung*
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