Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation of IKKα

Article abstract of DOI:10.1021/acsmedchemlett.9b00613

The small molecule gibberellin JRA-003 was identified as an inhibitor of the NF-kB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKα (IκB kinase alpha) and IKKβ (IκB kinase beta). Analogs of JRA-003 were synthesized and NF-κB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 identified as the most potent synthetic gibberellin against cancer-derived cell lines, it displayed no cytotoxicity in cells derived from noncancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk, HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.

Full text of DOI:10.1021/acsmedchemlett.9b00613

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Letter
Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation of IKK#
James R. Annand, Andrew R. Henderson, Kyle S. Cole, Aaron J. Maurais, Jorge Becerra,
Yejun Liu, Eranthie Weerapana, Angela N. Koehler, Anna K Mapp, and Corinna S. Schindler
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00613 • Publication Date (Web): 21 May 2020
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Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation
of IKK
James R. Annand,1,2 Andrew R. Henderson,2 Kyle S. Cole,3 Aaron J. Maurais,3 Jorge Becerra,2 Yejun
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Liu,2 Eranthie Weerapana,3 Angela N. Koehler,4 Anna K. Mapp,1,2 and Corinna S. Schindler.*,1,2
1Department of Chemistry, Willard-Henry-Dow Laboratory, University of Michigan, 930 N University Ave., Ann Arbor, MI
48109
2Program in Chemical Biology, University of Michigan, 210 Washtenaw Ave, Ann Arbor, MI 48109
3Department of Chemistry, Merkert Center, Boston College, 2609 Beacon St., Chestnut Hill, MA 02467
4David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main St., Cambridge,
MA 02139
KEYWORDS: nuclear kappa-light-chain-enhancer of activated B cells (NF-κB), IκB kinase (IKK), gibberellins, inhibition of
nuclear translocation
ABSTRACT:
The small molecule gibberellin JRA-003 was identified as an
inhibitor of the NF-kB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and
significantly inhibits the nuclear translocation of pathway-activating kinases IKK (IB kinase alpha) and IKKβ (IB kinase beta). Analogs
of JRA-003 were synthesized and NF-B-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC
1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 identified as the most potent synthetic gibberellin against cancer-
derived cell lines, it displayed no cytotoxicity in cells derived from non-cancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk,
HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.
Chronic inflammation is known to affect all phases of carcino-
genesis and targeting inflammation in the tumor microenvironment
has been shown to significantly reduce the development, growth
and spread of malignancies.1,2 The NF-B (nuclear kappa-light-
chain-enhancer of activated B cells) signaling pathway is constitu-
tively active in the majority of cancers and considered a critical link
between chronic inflammation and tumorigenesis.3-5 Specifically,
NF-B dysregulation has been implicated in all stages of tumor-
igenesis including initiation,6,7 angiogenesis,8,9 metastasis,10,11 and
tumor survival.12-14 Though an important target for anti-cancer ther-
apy, the complex regulation of NF-B activation currently presents
significant challenges for the development of new therapeutics.
Consequently, selective inhibitors of the NF-B pathway hold great
potential to improve our current understanding of NF-B’s role in
carcinogenesis to ultimately design and advance new cancer thera-
peutics able to selectively target inflammatory pathways for the
prevention and treatment of malignancies.15,16
NF-B activity is tightly regulated in healthy cells. Transcrip-
tionally active subunits of NF-B are bound to inhibitory protein
subunits which are phosphorylated by activating kinases (IKK
and IKK) and subsequently proteolytically degraded by ubiquitin
dependent proteases before active NF-B can be translocated to the
nucleus (Fig. 1).17 While inhibition of the activating kinases was
shown to decrease NF-B signaling in cellular models, these strat-
egies were unsuccessful in the production of viable therapeu-
tics.18,19 Specifically, complete inhibition of canonical NF-B sig-
naling via IKK inhibition has been associated with systemic tox-
icity in vivo.20
Our laboratories have an active interest in identifying new strat-
egies for NF-B inhibition relying on small molecule inhibitors.
Specifically, recent work by one of our groups showed that allogib-
beric acid (2) and gibberellic acid (3) selectively bind NF-B, spe-
cifically p50, and inhibit the NF-B pathway without inhibiting
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consistent with IKKα and IKKβ as specific targets of JRA-003. Ad-
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ditionally, proteome wide stable isotopic labeling with amino acids
in cell culture (SILAC) experiments were performed and only
eleven other proteins were found to be targets of JRA-003, none of
which are known to be modulators of the NF-κB pathway nor are
they known oncogenes (see Supporting Information, Fig. S2). Con-
sistent with these results, direct pulldown of IKKα from IL-1β stim-
ulated HEK 293T cells was observed upon treatment with JRA-031
but not inactive analog JRA-032 (Fig. 3B). Additionally, no loss of
binding was observed even with rigorous washing, suggesting that
JRA-031 is an irreversible covalent binder of IKK.
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In itia l M a s s S p e c P ro te o m ic s : N F k B
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Figure 1. The NF-B pathway is regulated by inhibitory com-
plexes that keep inactive NF-B localized in the cytoplasm. Upon
phosphorylation by IB kinases, and proteolytic degradation of in-
hibitory subunits, active NF-B is imported into the nucleus to bind
to DNA and promote gene expression.
a
b
K
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K
I
I
the activation and translocation of NF-B (Fig. 2).21 In related
studies aimed towards the synthesis of pharbinilic acid (JRA-008),
we identified JRA-003 as an active inhibitor of the NF-B path-
way.22 Herein we report the identification of IKKα and IKKβ as
protein targets of JRA-003. Additionally, we show that treatment
with JRA-003 significantly inhibits the nuclear translocation of
IKKα and IKKβ. We also report the synthesis and evaluation of
analogs of JRA-003 as inhibitors of the NF-κB pathway as well as
inhibitors of cancer cell viability. Specifically, our studies show
that JRA-003 is more than 500-fold selective towards inhibition of
lymphoma and breast cancer-derived cell lines than healthy fibro-
blast derived cell types.
Figure 3. Pulldown experiments using molecular probe JRA-
031 and inactive analog JRA-032. A. Pulldowns analyzed by mass
spectroscopy in TNFα stimulated HEK 293T cells. n = 3 (biological
replicates) B. Pulldowns analyzed by Western blotting in IL-1β
stimulated HeLa cells.
Next, we investigated the localization of NF-κB family mem-
bers. As IKK kinases are responsible for phosphorylating and de-
grading IκB, leading to the nuclear translocation of transcription-
ally active NF-kB subunits, RelA, RelB, and c-Rel, we anticipated
that IKK inhibitors would inhibit the nuclear translocation
Figure 2. Previously reported modulators of the NF-B pathway
related to the gibberellin family of natural products.23,24
JRA-003 is an effective inhibitor of canonical NF-κB signaling
upon pathway stimulation by either IL-1β (6.0 µM) or TNFα (2.6
µM) (see Supplemental Information, Fig. S1). To gain more direct
insight into the mechanism of action of JRA-003 pull down exper-
iments were performed with the alkyne-tagged analog of JRA-003
(JRA-031) (Fig. 3A). The experiments were conducted with pre-
treatment of cells with either JRA-003 or the negative control JRA-
002 followed by treatment with JRA-031; enrichment was im-
portant as many components of the NF-kB pathway are present in
low copy numbers.25 Under these conditions, it is expected that
upon pretreatment with JRA-003, but not with JRA-002 or DMSO,
specific targets of JRA-003 would be competed away from inter-
acting with the probe molecule, JRA-031. The results obtained are
Figure 4. Immunohistochemical staining and confocal micros-
copy of HeLa cells. Cells were pretreated with DMSO or JRA-003
before stimulation by IL-1β or treatment with vehicle. AF488
tagged antibodies were used to image NF-κB pathway members
and DAPI was employed as a nuclear stain.
of Rel.17 First, we analyzed the cellular compartmentalization of
NF-κB family members by immunohistochemical staining and
confocal microscopy in HeLa cells that were treated with either
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activity. We initially evaluated this compound library in Luciferase
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reporter gene assays in commercially available HEK 293T cell
lines in order to identify the structural features required to inhibit
the NF-κB pathway (Fig. 6). JRA-003 was found to be among the
most potent against the NF-κB pathway both upon pathway stimu-
lation by TNFα as well as IL-1β (Fig. 6, see Supplemental Infor-
mation, Fig. S3). Among the active analogs, the stereochemical
configuration of H-9, which is historically challenging to con-
trol,24,37-40 plays a significant role in the activity of NF-κB inhibit-
ing gibberellins (Fig. 6, JRA-022 vs. JRA-019). Additionally, only
gibberellins bearing electrophilic functionalities were found to be
active in the NF-κB pathway. Experiments to determine the contri-
bution of electrophile reactivity are ongoing (See Supplemental In-
formation, Fig. S4).
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Figure 5. Western blotting analysis in HEK 293T cellular cyto-
solic and nuclear fractions. Cells were pretreated with vehicle or
JRA-003 before stimulation by IL-1β treatment with vehicles. The
cells were then lysed, and the nuclei were separated for independent
analysis.
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In ensuing efforts, all gibberellin analogs were subjected to high
throughput evaluation (HTE) upon their ability to inhibit the
growth of 22 different cell lines derived from cancerous and non-
cancerous sources (Table 1 and Supporting Information Table S1).
Interestingly, the gibberellin analogs capable of modulating the
NF-κB pathway were also found to be active in the HTE Cell Titer
Glo® viability assay while non-NF-κB inhibiting gibberellins were
found to be inactive even at the highest concentrations tested. We
found that highly electrophilic gibberellins, namely JRA-019, JRA-
022, and JRA-026, were broadly cytotoxic with single digit mi-
cromolar EC50s across several cell lines and significant cytotoxicity
at the highest concentrations tested. In contrast, JRA-003 showed
unique potency, with EC50s <100 nM against breast cancer derived
cell lines (HS 578T, HCC1599), lymphoma derived cell lines (RC-
K8, Sud-HL4, CA 46) and a small cell lunch cancer derived cell
line (NCIH 446). Importantly, JRA-003 also displayed high selec-
tivity with a >10 µM EC50 against all non-cancer derived cell lines
as well as several non-inflammatory cancer cell types. Together,
this demonstrates that JRA-003 provides more than 500-fold selec-
tivity against inflammatory cancer in vitro.
DMSO or JRA-003 for four hours followed by one hour of path-
way stimulation with IL-1β or treatment with vehicle (Fig. 4, see
Supporting Information, Fig. S1). Additionally, we further corrob-
orated these data by Western blotting in HEK 293T cytosolic and
nuclear fractions in cells that had been similarly pre-treated with
DMSO or JRA-003 prior to stimulation with IL-1β or treatment
with vehicle (Fig. 5). Significant reduction of nuclear IKKα and
IKKβ was observed in these nuclear translocation assays. Nuclear
IK kinases are known to have a myriad of targets including co-tran-
scriptional mediators and histones. In particular, the nuclear activ-
ity of IKKα has been connected to an upregulation of NF-κB sig-
naling26 and has been linked to cell cycle regulation and survival in
colorectal,27,28 breast,29,30 pancreatic,31 gastric,32 osteo-sarcoma,33
and prostate cancers.34
These results prompted us to synthesize a small library of gib-
berellin and allogibberic acid analogs based on a synthetic strategy
we had previously developed towards the synthesis of pharbinilic
acid,24 with the goal of identifying structural features necessary for
Table 1. High throughput evaluation of synthetic gibberellin analogs.
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