Optimizing the aryl-triazole of cjoc42 for enhanced gankyrin binding and anti-cancer activity

Article abstract of DOI:10.1016/j.bmcl.2020.127372

Gankyrin is an oncoprotein overexpressed in numerous cancer types and appears to play a key role in regulating cell proliferation, cell growth, and cell migration. These roles are largely due to gankyrin's protein-protein interaction with the 26S proteaso

Full text of DOI:10.1016/j.bmcl.2020.127372

Bioorganic&MedicinalChemistryLetters30(2020)127372
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimizing the aryl-triazole of cjoc42 for enhanced gankyrin binding and
anti-cancer activity
Dipti Kanabara,d, Pamela Farralesa,d, Abbas Kabira, Daniel Juanga, Manu Gnanmonyc,
Joseph Almasrib, Nicolas Torrentsa, Snehal Shuklaa, Vivek Guptaa, Vikas V. Dukhandea,
⁎
Amber D'Souzac, Aaron Mutha,
a Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY 11439, USA
b Department of Chemistry, College of Liberal Arts and Sciences, St. John’s University, Queens, NY 11439, USA
c Department of Pediatrics, Hematology and Oncology, University of Illinois College of Medicine, Chicago, IL 60612, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Gankyrin is an oncoprotein overexpressed in numerous cancer types and appears to play a key role in regulating
cell proliferation, cell growth, and cell migration. These roles are largely due to gankyrin’s protein-protein
interaction with the 26S proteasome. We previously published a study exploring the aryl sulfonate ester of
cjoc42 in an effort to enhance gankyrin binding and inhibit cancer cell proliferation. In order to further improve
the gankyrin binding ability of the cjoc42 scaffold, an extensive SAR for the aryl-triazole moiety of cjoc42 was
developed. Our cjoc42 derivatives exhibited enhanced gankyrin binding, as well as enhanced antiproliferative
activity against Hep3B, HepG2, A549, and MDA-MB-231 cancer cell lines.
Gankyrin
Liver cancer
Lung cancer
Breast cancer
Protein-protein interactions
Antiproliferation
Gankyrin is a key protein for regulating numerous oncogenic and
inflammatory pathways through its various protein-protein interactions
(PPIs).1–5 Additionally, gankyrin plays an integral role in signal trans-
duction and its overexpression is associated with poor prognosis as well
as aggressive proliferation in hepatocellular carcinoma, breast cancer
and lung cancer.1,6–13 Despite its prominent role in the onset and pro-
gression of these cancers, to date there is no small molecule inhibitor of
gankyrin which is a viable drug candidate. Therefore, there is a need to
develop novel inhibitors of gankyrin to explore their potential as an-
ticancer therapeutics.
role in the degradation of important tumor suppressor proteins
(TSPs).2,3,20 Gankyrin is also responsible for the degradation of other
TSPs, such as: CCAAT/enhancer binding protein α (C/EBPα), RNA CUG
triplet repeats binding protein 1 (CUGBP1), and hepatocyte nuclear
factor 4α (HNF4α).19,21–23 Reduced levels of TSPs due to gankyrin
overexpression results in the onset and progression of multiple cancer
types (e.g., lung, breast and liver). It has been shown that preventing
the degradation of TSPs by inhibiting specific PPIs results in decreased
cell growth and decreased cancer cell migration. Furthermore, de-
creased levels of gankyrin, and its related PPIs, has also been associated
with decreased cancer cell growth and migration, illustrating its po-
tential as a therapeutic target.5,16,24,25
A number of protein-protein interactions (PPIs) involving gankyrin
are responsible for the degradation of tumor suppressor proteins and
the subsequent development of certain cancers. For example, gankyrin
aids in the hyperphosphorylation of retinoblastoma protein (pRb) by
binding to cyclin-dependent kinase 4 (CDK4), resulting in the protea-
somal degradation of pRb. In a similar manner, gankyrin binds the
ubiquitin ligase MDM2, increasing its ubiquitination of p53, and
leading to the subsequent degradation of p53 by the proteasome.2,14–17
Additionally, gankyrin is one of a number of repeat-protein “chaper-
ones” required for the proper assembly of the proteasome.3,18,19 It binds
the S6 ATPase subunit of the 19S regulatory cap, which in turn en-
hances the targeting of p53, pRb, and other tumor suppressor proteins
to the proteasome for degradation, illustrating gankyrin’s prominent
The first small molecule inhibitor of the gankyrin-S6 ATPase inter-
action (cjoc42) was recently discovered in 2016.26 A subsequent study
demonstrated that cjoc42 can inhibit the proliferation of certain liver
cancer cell lines (huh6 and hepa1c1c7).21 Our previous work estab-
lishing an SAR for the aryl sulfonate ester of cjoc42 identified multiple
derivatives with enhanced gankyrin binding (higher ΔTm), as well as
improved anti-cancer activity against Hep3B and HepG2 cells as com-
pared to cjoc42 (Fig. 1, AFM-1-2).27 Therefore, we sought to explore
other structural features of cjoc42 to further shed light on the required
structural features for binding gankyrin while also seeking to identify
molecules with enhanced anti-cancer activity as compared to cjoc42.
⁎ Corresponding author.
d These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127372
Received 3 April 2020; Received in revised form 24 June 2020; Accepted 26 June 2020
Availableonline02July2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

D. Kanabar, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127372
Fig. 1. Molecular modeling of cjoc42 (1) and its derivative AFM-1-2 (2). (A) Cjoc42 (1) docked to its proposed gankyrin binding site (PDB: 1QYM). (B) AFM-1-2 (2)
docked to its proposed gankyrin binding site (PDB: 1QYM). (C) Structure of cjoc42 (1), AFM-1-2 (2) and targeted cjoc42 derivatives 11–15 (Ar = substituted aryl
group).
Cjoc42′s ability to bind gankyrin appears to be due to interactions
with 5 key amino acid residues (Y15, R41, W46, S49, W74, and K116)
as suggested by its proposed binding (Fig. 1A). From this, it appears
that the methyl 4-azidobenzoate group of cjoc42 is engaged in a π-π
interaction with W46 and a possible hydrogen bonding interaction with
R41.26 Utilizing this information, we sought to establish an SAR for this
aryl ring in an effort to improve gankyrin binding. Therefore, an ex-
tensive SAR was established by substituting a variety of different
functional groups (Fig. 1C, Ar substitutions) at the 2-, 3-, and 4-posi-
tions of the corresponding phenyl ring, as well utilize various hetero-
aromatic systems. The substitutions were made to optimize the π-π
interaction of the phenyl ring with the indole ring of W46, optimize a
possible hydrogen bonding interaction with R41, as well as probe for
potentially new interactions.
to generate azido intermediates 6a and 6c. These azido intermediates
then underwent a copper-catalyzed alkyne-azide cycloaddition (CuAAc)
with 4-pentyn-1-ol in the presence of sodium ascorbate and copper
sulfate to afford triazole intermediates 7–10 in modest to good yields
over 2 steps (41–76%). Triazole intermediates 7–10 then underwent a
nucleophilic substitution reaction with para-toluenesulfonyl chloride in
the presence of triethylamine and 4-dimethylaminopyridine to generate
desired cjoc42 derivatives 11–14 in modest to good yield (42–82%).
The relative ability of cjoc42 derivatives 11–15 to bind gankyrin
was determined via a protein thermal shift assay. A protein thermal
shift assay determines the temperature at which a protein denatures
(Tm). When a ligand effectively binds a protein, the Tm value shifts in
comparison to the protein without a bound ligand. This change in Tm
value is then represented as the ΔTm value or protein thermal shift,
where the larger the ΔTm, the more effectively the ligand binds the
protein of interest. Therefore, in order to evaluate the relative binding
affinities of our cjoc42 derivatives to gankyrin, the differences in Tm
values (ΔTm) were determined. This provided a method for ranking
cjoc42 derivatives 11–15 as well as compare them to cjoc42. The ΔTm
values obtained from thermal shift evaluation of cjoc42 derivatives
11–15 are shown in Fig. 2–5.
Cjoc42 derivatives 11–14 were synthesized (Scheme 1) with the
goal of substituting the 4-methyl ester group of cjoc42 with different
functional groups as well as altering the position to the ortho, meta and
para positions on the phenyl ring linked to the triazole ring. The
synthesis of cjoc42 derivatives 11–14 (Scheme 1) commenced with
either the diazotization of a series of anilines followed by the addition
of sodium azide to generate a series of substituted azido intermediates,
or by the nucleophilic substitution of an aryl halide with sodium azide
Cjoc42 derivatives 11a-11l (Fig. 2) presented multiple derivatives
with increased ΔTm values as compared to cjoc42. Substitution of the
methyl ester of cjoc42 with a simple methyl group (11a) resulted in a 3-
fold increase in ΔTm. However, increasing the functional group size at
the 4-position with 4-Et (11b) and 4-iPr (11c) groups was met with
reduced ΔTm values as compared to their 4-Me counterpart (11a).
Furthermore, substitutions with other bulky groups such as 4-Ph (11d)
and 4-I (11j) demonstrated a complete loss in binding affinity. Inter-
estingly, large groups with increased electron withdrawing potential,
such as 4-CF3 (11e), 4-Cl (11h), and 4-Br (11i), exhibited significantly
increased ΔTm values as compared to cjoc42. However, smaller electron
withdrawing groups such as 4-F (11g) and 4-CN (11k) did not exhibit
Scheme 1. Reagents: (a) 4-pentyn-1-ol, sodium ascorbate, CuSO4,
THF/tBuOH/H2O; (b) p-toluenesulfonyl chloride, TEA, DMAP, CH2Cl2.
Ar = substituted aryl group.
2

D. Kanabar, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127372
Fig. 2. Evaluation of cjoc42 derivatives 11a-11l for relative gankyrin binding.
Compounds 11a-11l (300 µM) and gankyrin (20 µM) were incubated together
in a thermal shift assay and their respective ΔTm values were determined.
DMSO (5%) served as a negative control (vehicle), and cjoc42 (300 µM) served
Fig. 4. Evaluation of cjoc42 derivatives 13a-13h for relative gankyrin binding.
Compounds 13a-13h (300 µM) and gankyrin (20 µM) were incubated together
in a thermal shift assay and their respective ΔTm values were determined.
DMSO (5%) served as a negative control (vehicle), and cjoc42 (300 µM) served
as
a
positive control. All compounds were tested in triplicate.
as
a
positive control. All compounds were tested in triplicate.
**0.001 < p < 0.01, ***0.0001 < p < 0.001.
**0.001 < p < 0.01.
Fig. 3. Evaluation of cjoc42 derivatives 12a-12g for relative gankyrin binding.
Compounds 12a-12g (300 µM) and gankyrin (20 µM) were incubated together
in a thermal shift assay and their respective ΔTm values were determined.
DMSO (5%) served as a negative control (vehicle), and cjoc42 (300 µM) served
as
a
positive control. All compounds were tested in triplicate.
*0.01
<
p
<
0.05, **0.001
<
p
<
0.01, ***0.0001
< p < 0.001,
****0.00001 < p < 0.0001.
Fig. 5. Evaluation of cjoc42 derivatives 14a-14d for relative gankyrin binding.
Compounds 14a-14d (300 µM) and gankyrin (20 µM) were incubated together
in a thermal shift assay and their respective ΔTm values were determined.
DMSO (5%) served as a negative control (vehicle), and cjoc42 (300 µM) served
as a positive control. All compounds were tested in triplicate.
any significant enhancement in ΔTm. The electron donating and po-
tential hydrogen bond accepting 4-OMe group (11f) did exhibit a
substantial increase in ΔTm, suggesting that similar groups should also
be explored at this position in the future. The enhanced binding of 4-
OMe may be due to a gained hydrogen bonding interaction with D39
(Fig. 7) as well as an optimized π-π interaction with W46 (Fig. 7).
When our focus moved to the 3-position (Fig. 3), the gankyrin
binding affinity relative to cjoc42 was increased for all derivatives
(12a-12g). At this position, small electron withdrawing groups dis-
played the greatest increases in ΔTm, with 3-F (12c) and 3-CN (12h)
exhibiting 5-fold and 6-fold increases in ΔTm, respectively, as compared
to cjoc42. This enhanced binding may be due to a gained hydrogen
bonding interaction with R41 (Fig. 7) as well as an optimized π-π in-
teraction with W46. These 2 compounds also exhibit the highest ΔTm
values for gankyrin to date.
resulted in 2 derivatives (13d and 13e) with improved gankyrin
binding relative to cjoc42. While 2-OMe (13d) is electron donating and
2-CF3 (13e) is electron withdrawing, they resulted in similar ΔTm va-
lues. This suggests that sterics may be a significant factor in binding.
This was further supported by 2-Me (13a) and 2-Ph (13c) exhibiting
similar gankyrin binding to cjoc42, while 2-Et (13b) demonstrated a
substantial decrease in relative binding affinity. It was also found that
other electron withdrawing groups such as 2-F (13f), 2-CN (13g), and
2-CO2Me (13h) exhibited decreased gankyrin binding affinities as
compared to cjoc42. The enhanced binding affinity of 2-CF3 (13e) may
be due to a gained hydrophobic interaction of the trifluoromethyl group
Substitutions at the 2-position (Fig. 4, compounds 13a-13h) only
3

D. Kanabar, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127372
Scheme 2. Reagents: (a) Biphenyl-4-sulfonyl
chloride or 4-iodobenzenesulfonyl chloride, DMAP,
TEA, CH2Cl2.
with I79 (Fig. 7) as well as an optimized π-π interaction with W46
(Fig. 7).
suggests a change in binding mode causing each aryl substitution to no
longer optimally interact with gankyrin. Future derivatives will utilize
these findings to help guide our design.
We next turned our attention to substituting the phenyl ring at-
tached to the triazole with various heteroaromatic systems. Compounds
14a-d demonstrated modest but not significant improvements in gan-
kyrin binding as compared to cjoc42. Substituting the phenyl ring of
cjoc42 with the 2- and 4-pyridine rings (14a and 14c, respectively)
exhibited similar gankyrin binding affinities, whereas 3-pyridine (14b)
displayed a slightly better binding affinity than cjoc42, 14a, and 14c.
Substituting the phenyl ring with a thiazole (14d) ring also resulted in a
modest but not significant improvement in gankyrin binding affinity as
compared to cjoc42. Since compounds 14a-d did not show a significant
improvement in gankyrin binding affinity as compared to cjoc42, they
were not further evaluated in cell-based assays.
The 5 most potent binders of gankyrin (11a, 11e, 11f, 12c, and 12g)
had their anti-proliferative activity assessed in four gankyrin-over-
expressing cancer cell lines (Table 1, HepG2, Hep3B, A549, and MDA-
MB-231).10,28–30 Cjoc42 did not exhibit any ability to inhibit pro-
liferation in these four cell lines when evaluated up to 100 µM. How-
ever, all 5 cjoc42 derivatives (11a, 11e, 11f, 12c, and 12g) demon-
strated an ability to inhibit cell proliferation at 100 µM, resulting in IC50
values less than 100 µM. Specifically, 11a exhibited the greatest anti-
proliferative effect in MDA-MB-231 cells with an IC50 value of 36.6 µM.
Compound 11e demonstrated the greatest ability to inhibit prolifera-
tion in both HepG2 and Hep3B cells with IC50 values of 42.6 µM and
42.7 µM, respectively. Compound 11f then displayed the best ability to
inhibit A549 cell proliferation resulting in an IC50 value of 74.6 µM.
While compounds 12c and 12 g also exhibited an ability to inhibit
Hep3B and HepG2 cells, neither proved more potent than compound
11e. Interestingly, compounds 11a, 11e, 11f, 12c, and 12 g did de-
monstrate similar anti-proliferative activity to cjoc42 in certain cell
lines despite their enhanced gankyrin binding ability. Specifically, each
of these compounds exhibited an IC50 value greater than 100 µM in at
least one of the cell lines they were evaluated in (Table 1). Although the
reason for this is currently unknown, it could be due to poor membrane
permeability and/or a lack of metabolic stability (i.e., sulfonate ester)
and will be the focus of future studies.
As we previously reported, substituting the p-tosyl group of cjoc42
with a biphenyl-4-sulfonate ester group or 4-iodobenzene sulfonate
ester group resulted in a ~2-fold improvement in gankyrin binding
(Fig. 1, AFM-1-2 and JA-1-38, respectively). Armed with this knowl-
edge, we sought to combine these features with our most potent aryl-
triazole substitutions. Therefore, we synthesized cjoc42 derivatives
15a-d to combine the best features of the R1 and R2 substitutions to
cjoc42 (Scheme 2). The synthesis relied on constructing key triazole
intermediates 7–9 as previously described. Intermediates 7–9 then
underwent
a nucleophilic substitution with biphenyl-4-sulfonyl
chloride or 4-iodobenzenesulfonyl chloride to afford compounds 15a-d
in good yield (57–71%).
Cjoc42 derivatives 15a-d presented only 2 derivatives, 15a and
15d, which exhibited improved gankyrin binding affinity as compared
to cjoc42 (Fig. 6). However, all four derivatives demonstrated de-
creased ΔTm values as compared to AFM-1-2, JA-1-38, 11f, and 12e
In summary, a novel series of cjoc42 derivatives with a modified Ar
group (Fig. 1) were designed, synthesized, and evaluated for their
ability to bind gankyrin and inhibit liver, lung and breast cancer cell
proliferation. Modifications to the aryl triazole moiety resulted in 3 new
derivatives of cjoc42 (11f, 12g, and 13e) with superior anti-cancer
activity in Hep3B, HepG2, A549, and MDA-MB-231 as compared to
cjoc42. In terms of gankyrin binding, an extensive SAR (Fig. 8) revealed
that electron donating substituents were favored at the R = 4-position
(11e), while at the R = 3-position the electron withdrawing CN group
was favored (12g), and at the R = 2-position the electron withdrawing
CF3 group was preferred (13e). These second generation cjoc42 deri-
vatives also exhibited enhanced gankyrin binding compared to AFM-1-
2 (Fig. 1), our most potent first generation cjoc42 derivative.27 How-
ever, AFM-1-2 demonstrated similar or superior anti-proliferative ac-
tivity against Hep3B and HepG2 cells than our most potent gankyrin-
binding second generation cjoc42 derivatives 11a, 11e, 11f, 12c, and
12g. In addition, second generation cjoc42 derivatives 11f, 12g, and
13e all exhibited improved anti-proliferative activity against Hep3B,
HepG2, A549 and MDA-MB-231 cells compared to cjoc42. These find-
ings mark a significant advance over cjoc42 in terms of both gankyrin
binding as well as anti-cancer activity in liver, lung, and breast cancers.
(ΔTm
< 1.0). Although disappointing, this loss in binding affinity
Declaration of Competing Interest
Fig. 6. Evaluation of cjoc42 derivatives 15a-15d for relative gankyrin binding.
Compounds 15a-15d (300 µM) and gankyrin (20 µM) were incubated together
in a thermal shift assay and their respective ΔTm values were determined.
DMSO (5%) served as a negative control (vehicle), and cjoc42 (300 µM) served
as a positive control. All compounds were tested in triplicate.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4

D. Kanabar, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127372
Fig. 7. Predicted binding of cjoc42 derivatives 11f (A), 12g (B), and 13e (C) to gankyrin based on in silico docking studies (PDB: 1QYM).
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cjoc42
AFM-1–2
11a
> 100
> 100
> 100
NT
> 100
NT
26.0 ( 3.5)
> 100
46.2 ( 6.1)
> 100
79.9 ( 4.7) 36.6 ( 5.5)
11e
42.7 ( 5.8)
> 100
42.6 ( 6.5)
> 100
> 100
> 100
11f
74.6 ( 4.4) 72.8 ( 7.1)
12c
82.1 ( 16.0)
44.9 ( 8.5)
61.8 ( 12.1)
> 100
> 100
> 100
> 100
> 100
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12g
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Fig. 8. Summary of aryl-triazole SAR.
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The authors thank the College of Pharmacy and Health Sciences, the
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