Synthesis and Evaluation of Ginkgolic Acid Derivatives as SUMOylation Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.0c00353

SUMOylation has emerged as an important post-translational modification that has been shown to modulate protein activity associated with various signaling pathways, and consequently, it has emerged as an important therapeutic target. While several natural products have been shown to inhibit enzymes involved in the SUMOylation process, there has been little progress toward the development of more selective and potent SUMOylation inhibitors. Ginkgolic acid was one of the first natural products discovered to inhibit the SUMO E1 enzyme. Despite its use to mechanistically investigate the SUMOylation process, ginkgolic acid also modulates other pathways as well. In this Letter, preliminary structure-activity relationships for ginkgolic acid as a SUMOylation inhibitor are presented.

Full text of DOI:10.1021/acsmedchemlett.0c00353

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Letter
Synthesis and Evaluation of Ginkgolic Acid Derivatives as
SUMOylation Inhibitors
Christopher M. Brackett, Ana García-Casas, Sonia Castillo-Lluva, and Brian S. J. Blagg*
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ABSTRACT: SUMOylation has emerged as an important post-
translational modification that has been shown to modulate protein
activity associated with various signaling pathways, and con-
sequently, it has emerged as an important therapeutic target. While
several natural products have been shown to inhibit enzymes
involved in the SUMOylation process, there has been little
progress toward the development of more selective and potent
SUMOylation inhibitors. Ginkgolic acid was one of the first natural
products discovered to inhibit the SUMO E1 enzyme. Despite its
use to mechanistically investigate the SUMOylation process,
ginkgolic acid also modulates other pathways as well. In this Letter, preliminary structure−activity relationships for ginkgolic
acid as a SUMOylation inhibitor are presented.
KEYWORDS: SUMO, SAE inhibitors, cancer, autophagy, apoptosis
ost-translational modifications are critical to cellular
processes that regulate protein function and, to date,
ubiquitously expressed, SUMO4 mRNA is only present in the
kidneys, spleen, and lymph nodes.2 While there are significant
differences in their sequence homology, they all exhibit a
similar three-dimensional structure.
P
more than 450 unique modifications have been discovered.1
SUMOylation, the post-translational conjugation of the small
ubiquitin-like modifier (SUMO) to a protein, is an emerging
area of study due to the wide array of cellular processes that it
controls.2 Protein SUMOylation was first identified in 19963
and has been shown to be associated with DNA damage repair,
immunological response, protein stability, nuclear-cytosolic
transport, cell cycle progression, and apoptosis to name a few.4
Thus, it is not surprising that dysregulation of SUMOylation is
associated with many forms of cancer and neurodegenerative
disorders.5
The SUMOylation pathway is upregulated in several cancers
and has emerged as a potential target for the development of
small molecule inhibitors.6 In fact, several natural product
inhibitors of SUMOylation have been identified including
ginkgolic acid (1) and its structural analogue, anacardic acid
(2).9,10 While there are many structurally related ginkgolic
acids, the C15:1 derivative 1 and the fully saturated analogue 2
were the analogues first reported as SUMOylation inhibitors.
Ginkgolic acid exhibits anticancer activity and inhibits the
migration of several different cancer cell lines.11−13 RAC1 and
NEMO are proteins that control cellular migration, and
because SUMOylation modulates both of these proteins, it is
not surprising that SUMOylation inhibitors manifest anti-
metastatic activity.14,15 In fact, recent studies have demon-
strated that the antimigratory and anticancer activities
manifested by ginkgolic acid are linked to inhibition of
RAC1 and NEMO SUMOylation.16,17
The SUMOylation pathway is mechanistically very similar to
the process of ubiquitinylation. Initially, the E1 enzyme forms a
thioester bond with the C-terminus of the SUMO peptide.
Next, the activated SUMO is transferred to the E2 enzyme,
which is responsible for conjugation and transesterification,
resulting in a new thioester bond. The E3 ligases can then
associate with the loaded E2 enzyme, which catalyze the
transfer of SUMO to the ε-amino group on a specific lysine
within the substrate.6 This enzymatic pathway is also reversible
as sentrin specific proteases (SENPs) are responsible for
cleaving the bond between the substrate and SUMO (Figure
1).7
There are four SUMO isoforms: SUMO1−SUMO4.
SUMO2, and SUMO3 share 97% sequence homology and
are often referred to as SUMO2/3, as they have not yet been
functionally distinguished.8 Their sequence is only 50% similar
to that of SUMO1, whereas SUMO4 is the least similar and
the least studied of the isoforms. While SUMO 1−3 are
Despite the increased attention that SUMOylation has
received in recent years and ginkgolic acid’s widespread use as
Received: June 23, 2020
Accepted: September 24, 2020
Published: September 24, 2020
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© XXXX American Chemical Society
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Figure 1. SUMOylation catalytic cycle: (1) maturation, (2) activation, (3) conjugation, (4) ligation, and (5) hydrolysis.
a SUMOylation inhibitor, no assessment of its structure−
activity relationships nor its ability to inhibit SUMOylation has
occurred. The original study demonstrated ginkgolic acid as an
inhibitor of the E1 enzyme and showed that methylation of the
acid was not tolerated, however, acetylation of the hydroxyl
moiety did not affect inhibitory activity. Furthermore, salicylic
acid, which is identical to ginkgolic acid but lacks the
hydrocarbon tail, was completely inactive.10 In addition,
ginkgolic acid inhibits other biological processes as well,
which complicates its use as an inhibitor of SUMOylation.18
Therefore, efforts to develop more potent and selective
inhibitors are highly desired. Herein, we report preliminary
structure−activity relationships between ginkgolic acid/ana-
cardic acid and the inhibition of SUMOylation.
Initial studies aimed to determine the significance and
optimal position of the alkyl chain and its effect on
SUMOylation. Four 2-hydroxybenzoic acid scaffolds were
chosen to generate all possible regioisomers as presented in
Figure 2, including the 2,6-substitution pattern present in the
natural product.
Synthesis of these ginkgolic acid derivatives commenced
with commercially available dihydroxybenzoic acids, which
were condensed with acetone to provide the corresponding
acetonides, 3a−d, in modest yields. The free phenols were
Figure 2. (A) Ginkgolic acid (1) and anacardic acid (2). (B)
Compound numbering scheme. The first letter represents position of
the alkyl tail, and the second letter represents alkyl tail length.
Example compound 8bc shown.
subsequently converted to the corresponding trifluormethyl-
sulfonates, 4a−d, via reaction with trifluoromethanesulfonic
anhydride. Initially, the triflates were coupled with different
primary alkenes via a Heck reaction. The 11-, 13-, and 15-
carbon chains were chosen to mimic the natural products,
which have alkyl chains ranging from 13−17 carbons. After the
Heck coupling, the alkenes were reduced or left unsaturated
prior to base-mediated hydrolysis of the acetonide (Scheme 1).
The initial library of compounds was screened for its ability
to inhibit RanGAP1 SUMOylation in vitro at 50 μM. All
compounds were soluble in the reaction media at this
concentration. RanGAP1 is a GTPase activating protein that
is involved in the transportation of proteins to and from the
nucleus. RanGAP1 associates with the nuclear core complex
and initiates transport only after SUMOylation has occurred.19
Furthermore, it does not require an E3-ligase for SUMOyla-
tion, which makes the use of this recombinant system useful
for the evaluation of SUMOylation inhibitors. The presence of
SUMOylated RanGAP1 was probed via Western blot analysis
(Figure 3). The results obtained from this in vitro assay are
summarized in Table 1. The data from the initial screen
suggest that any alteration of the substitution pattern about the
central aromatic ring completely ablates SUMOylation
inhibition. All 2,6 disubstituted analogues (6da−6dc and
8da−8dc) were able to successfully inhibit RanGAP1
SUMOylation at 50 μM, suggesting that unsaturation did
not exhibit a negative effect on activity. To determine whether
there was an optimal alkyl tail length, the compounds
manifesting inhibitory activity at 50 μM were screened at 5
B
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a
Scheme 1. Synthesis of Ginkgolic/Anacardic Acid Analogues
a
(a) SOCl2, DMAP, acetone, 1,2-DME, 24 h (77%); (b) trifluoroacetic anhydride, trifluoroacetic acid, acetone, 24 h (27−53%); (c) Tf2O,
pyridine, CH2Cl2, 3 h (35−89%); (d) alkene, Pd(dppf)Cl2, K2CO3, DMF, 75 °C, 12 h (38−86%); (e) KOH, DMSO, 80 °C, 2 h (42-94%); (f) H2,
EtOAc, Pd/C, 16 h (64−98%).
Figure 3. Western blot results from initial library screen. All compounds were tested at 50 μM.
μM, which revealed the unsaturated compounds, 6db and 6dc,
to be most active (Supporting Information (SI), Figure S1).
The length of the alkyl chain for ginkgolic acids range from
13 to 17 carbons, but the initial study identified ginkgolic acid
as a SUMOylation inhibitor that contained a C15 side chain.
However, the data presented herein demonstrate that an alkyl
chain of 11 carbons is also an effective inhibitor. To determine
whether compounds with shorter alkyl chains could also inhibit
SUMO-E1, 6- and 8-carbon analogues were synthesized as
shown in Scheme 1. As other substitution patterns had proven
deleterious toward SUMOylation inhibition, only derivatives
based on the 2,6-substitution pattern were pursued. Com-
pounds 6dd, 6de, 8dd, and 8de, shown in Figure 4, were
screened at 50 μM and found to be completely inactive at the
inhibition of SUMOylation. Furthermore, the results clearly
show that a long alkyl chain is required for ginkgolic acid
derivatives to inhibit SUMO-E1.
The highly metastatic prostate cancer cell line, PC3, as well as
the triple negative breast cancer cell line, MDA-MB-231, were
treated with compounds and overall SUMOylation levels were
probed via Western blot analysis. Consistent with the in vitro
results, global SUMOylation levels were significantly dimin-
ished at 10 and 20 μM in PC3 cells after 24 h treatment.
Consistent with the preliminary studies, treatment with
compound 8ab did not affect SUMOylation at either
concentration tested (Figure 5A). Similar results were obtained
in MDA-MB-231 (SI, Figure S2). Additionally, the ability of
the compounds to inhibit SUMOylation was independent of
the SUMO isoform (SI, Figure S3).
The original study demonstrated ginkgolic acid to manifest
activity against SUMOylation and identified SUMO E1 as the
target.10 While the mechanism of action was not investigated,
it is believed that due to structural similarities between the lead
compounds and ginkgolic acid, 6db, 6dc, 8db, and 8dc are
likely manifesting a similar mechanism of inhibition.
Compounds 6db, 6dc, 8db, and 8dc were chosen for further
evaluation, whereas 8ab was used as an inactive control,
compound 8db is fully saturated ginkgolic acid derivative,
anacardic acid, 2, from Figure 1. After establishing the ability of
these compounds to inhibit RanGAP1 SUMOylation in vitro,
the cellular activity of the lead compounds was investigated.
Prior studies demonstrated that the inhibition of SUMOy-
lation via silencing or small molecule inhibition induces cell
death via an autophagy-mediated apoptosis mechanism.17
Increased cleavage of Poly(ADP-ribose) polymerase (PARP) is
a classic hallmark of apoptosis,20 and indeed, treatment with
C
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Table 1. Results of Initial Library Screen
saturated
entry
unsaturated
entry
substitution pattern
tail length
active at 50 μM
tail length
active at 50 μM
2,3
2,4
2,5
2,6
pentadecyl (C15H31)
8aa
8ba
8ca
8da
no
no
no
yes
pentadecenyl (C15H29)
6aa
6ba
6ca
no
no
no
yes
6da (2)
2,3
2,4
2,5
2,6
tridecyl (C13H27)
undecyl (C11H23)
8ab
8bb
8cb
8db
no
no
no
yes
tridecenyl (C13H25)
undecenyl (C11H21)
6ab
6bb
6cb
6db
no
no
no
yes
2,3
2,4
2,5
2,6
8ac
8bc
8cc
8dc
no
no
no
yes
6ac
6bc
6cc
6dc
no
no
no
yes
either 10 or 20 μM of compounds 6db, 6dc, 8db, or 8dc led to
increased levels of cleaved PARP (Figure 5B). Treatment of
PC3 cells with 10 μM 8ab did not induce levels of cleaved
PARP or LC3II, however, treatment with 20 μM 8ab did lead
to increased levels of both species, albeit to a lesser extent than
the active compounds, indicating that at the higher
concentration, 8ab may induce apoptosis, albeit an alternative
mechanism of action not related with SUMO inhibition.
Consistent with previous studies,17 the active compounds also
increased levels of LC3II, which is the phosphatidylethanol-
amine form of LC3 that is associated with the autophagosome
(Figure 5C). Taken together, these results provide evidence
that SUMOylation inhibition induces cell death via autophagy.
Cell viability was measured directly against both cell lines
and at concentrations wherein SUMOylation was suppressed.
Clearly, inhibition of cell growth was also affected by the
administration of these compounds. Time course studies were
also performed and demonstrated that compounds 6db, 6dc,
8db, and 8dc induced cell death after 24 h treatment, which is
consistent with the immunoblot data linking inhibition of
SUMOylation to cell viability. (Figure 6).
Figure 4. C8 and C6 ginkgolic acid derivatives.
The IC50 values manifested by the five compounds were
determined against both cancer cell lines. Previous studies have
reported that MYC dependent cell lines such as MDA-MB-231
are more susceptible to SUMOylation inhibition.21,22 Con-
sistent with these reports, our compounds were generally more
effective against MDA-MB-231 cells as compared to the PC3
cell line (SI, Figure S4). Compound 6db was the most active
against both cell lines and manifested IC50 values of 10.77 and
8.2 μM against PC3 and MDA-MB-231, respectively. Against
PC3 cells, both unsaturated compounds were the most active,
however, against MDA-MB-231 cells, the alkyl chain length
appeared important for both C13 derivatives, 6db and 8db,
which were most active. In addition, the IC50 values manifested
by the active compounds correspond to the concentrations at
which cleaved PARP and LC3II were induced. Compound 8ab
exhibited the weakest antiproliferative activity with IC50 values
Figure 5. (A) Effects of lead compounds on global SUMOylation
with SUMO1, apoptosis, and autophagy related factors on PC3 cells
after 24 h treatment. (B) Densitometric quantification of the cleaved
PARP data from A using ImageJ. (C) Densitometric quantification of
the LC3II data from A using ImageJ. All experiments were performed
at least three times, and the data are presented as the mean SEM.
D
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00353.
■
sı
Synthetic experimental details, characterization of
compounds, and biological data (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Brian S. J. Blagg − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556, United
States; orcid.org/0000-0002-6200-3480; Email: bblagg@
nd.edu
Authors
Christopher M. Brackett − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
́
́
Ana García-Casas − Departamento de Bioquimica y Biologia
́
́
Molecular, Facultad de Ciencias Quimicas y Biologicas,
Universidad Complutense, Madrid 28040, Espana; Instituto de
Investigaciones Sanitarias San Carlos (IdISSC), Madrid 28040,
Espana; orcid.org/0000-0002-1186-8054
Sonia Castillo-Lluva − Departamento de Bioquimica y Biologia
Molecular, Facultad de Ciencias Quimicas y Biologicas,
Universidad Complutense, Madrid 28040, Espana; Instituto de
Investigaciones Sanitarias San Carlos (IdISSC), Madrid 28040,
Espan
̃
Figure 6. (A) The 48 h viability studies of four active and one inactive
compound at 10 or 20 μM against PC3 cells. (B) The 48 h viability
studies of four active and one inactive compound at 10 or 20 μM
against MDA-MB-231 cells. (C) Time-dependent viability studies
with two concentrations against PC3 cells (*P ≤ 0.05 significant
versus nontreated cells). All experiments were performed at least three
times and the data are presented as the mean SEM.
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́
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́
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a
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00353
of 14.8 and 12.8 against PC3 and MDA-MB-231 cells,
respectively. The IC50 value manifested by 8ab against PC3
cells is lower than the concentration at which 8ab induced
cleaved PARP and LC3II, suggesting that 8ab affects cell
viability by an alternative mechanism of inhibition.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
SUMOylation is an important post-translational modifica-
tion owing to the wide range of pathways that it modulates. In
several cancers, the E1, E2, or SENPs have been altered to
produce a dysregulated SUMOylation pathway and has now
emerged as a novel target for the development of new cancer
treatments. The MYC oncogenic pathway plays a key role in
many cancers, and its activity relies upon the SUMOylation
pathway to function properly, making the development of
SUMOylation inhibitors even more important. In fact,
numerous natural products have been discovered that inhibit
SUMOylation, but most affect other enzymatic processes as
well. For example, ginkgolic acid was the first natural product
SUMOylation inhibitor discovered but also exhibits anti-
depressant, antifungal, and antimicrobial activities. Despite
these concerns, ginkgolic acid continues to be used as a
selective SUMOylation inhibitor. This study represents a first
step toward the establishment of structure−activity relation-
ships for ginkgolic acid as an inhibitor of the SUMOylation
pathway. While ginkgolic acid and some of the inhibitors in
this study contain unsaturation, the location of unsaturation
did not affect inhibitory activity. This data highlights the nature
of the alkyl chain, which is less important than the sterics, as
compounds with shorter alkyl chain did not exhibit any activity
in the initial in vitro assay. Future studies will further explore
this trend and optimize the selectivity of these compounds
toward SUMOylation inhibition.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
B.S.J.B. is grateful to the NIH for financial support
(CA213566). S.C.L.L. is the holder of a Ramon y Cajal
■
́
research contract from the Spanish Ministry of Economy and
Competitiveness (MINECO), and the work was partially
supported by a MINECO/FEDER research grant (RTI2018-
094130-B-100). A.G.C. is a recipient of a FPU grant from the
Spanish Ministry of Science, Innovation and University.
ABBREVIATIONS
SUMO, small ubiquitin-like modifier; SENP, sentrin-specific
protease
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