Molecular characterization of the boron adducts of the proteasome inhibitor bortezomib with epigallocatechin-3-gallate and related polyphenols

Article abstract of DOI:10.1039/c4ob02512a

The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the ability of Bortezomib (BZM) to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib's activity remain unclear. This paper describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of the boron adducts of EGCG and related polyphenols. The observed boron adducts included both boronate and borate derivatives, and their structural characteristics were correlated with cell-based evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent amide bond. The reported approach provides a useful method for determining the potential ability of polyphenols to form undesired adducts with boron-based drugs and interfere with their actions. This journal is

Full text of DOI:10.1039/c4ob02512a

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DOI: 10.1039/C4OB02512A
Organic &
Biomolecular Chemistry
Molecular characterization of the boron adducts
of the proteasome inhibitor Bortezomib with
epigallocatechin-3-gallate and related polyphenols
Cite this: DOI: 10.1039/x0xx00000x
Stephen J. Glynn,a,b Kevin J. Gaffney,a,b,c Marcos A. Sainz,a,b Stan G. Louie,c
Received
Accepted
and Nicos A. Petasis*a,c,d
DOI: 10.1039/x0xx00000x
The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the
ability of Bortezomib to induce apoptosis in cancer cells. This interaction was attributed to the
formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of
Bortezomib. However, the structural details of this boron adduct and the molecular factors that
contribute to its formation and its ability to inhibit Bortezomib’s activity remain unclear. This paper
describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of
the boron adducts of EGCG and related polyphenols. The observed boron adducts included both
boronate and borate derivatives, and their structural characteristics were correlated with cell-based
evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of
Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric
reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent
amide bond. The reported approach provides a useful method for determining the potential ability of
polyphenols to form undesired adducts with boron-based drugs and interfere with their actions.
www.rsc.org/
Introduction
The proteasome inhibitor Bortezomib (BZM), also known as
PS-341 and now marketed as Velcade™ (1, Fig. 1), is approved
for the treatment of multiple myeloma and mantle cell
lymphoma.1-6 A novel structural feature of BZM is its boronic
acid group, which forms reversibly a borate adduct with
threonine at the chymotrypsin-like active site of the 26S
proteasome, serving as a transition state mimic. Despite
extensive efforts in drug discovery aimed at the development of
boron compounds,7-13 BZM still remains the only approved
boron-containing drug. However, in recent years, a growing
number of boron-based therapeutics8-13 are being investigated
Fig. 1
Structures of boron-based proteasome inhibitors.
in clinical trials, including the new proteasome inhibitor
Delanzomib (CEP-18770)14-16 (2, Fig. 1).
The discovery1, 2 and clinical development4-6 of BZM as an
effective therapeutic agent demonstrated for the first time that
the selective inhibition of the 26S proteasome17 is a viable
approach for the treatment of cancer.3, 18-23 Cellular proteasome
is a key enzymatic complex in all cells that degrades and
recycles targeted and misfolded intracellular proteins. As a key
component of the ubiquitin proteasome pathway (UPP) and the
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VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4OB02512A
unfolded protein response (UPR), the proteasome plays a
critical role in cell division and cell survival. Selective and
temporary inhibition of the 26S proteasome by BZM or other
proteasome inhibitors results in a selective apoptotic effect in
cancer cells, which occurs via several mechanisms.24 By
blocking the degradation of IκB, proteasome inhibition partially
prevents the activation of the transcription factor NF-κB, and
results in cell cycle arrest and inhibition of cell growth.3, 18-23 It
also leads to the accumulation of ubiquitinated unfolded or
misfolded proteins resulting in the induction of endoplasmic
reticulum stress (ER stress).25,26 Based on its novel mechanisms
of action, BZM has been shown to be effective as a single agent
and in combination therapies.4, 6, 27
As part of our efforts towards the development of novel
proapoptotic agents for the treatment of various types of cancer,
including multiple myeloma,28 lung cancer,29 lymphoma,30
glioblastoma,31, 32 and breast cancer,33-35 we identified certain
molecules that selectively induce apoptosis in cancer cells by
increasing ER stress.31, 36-42 These ER-stress-aggravating agents
(ERSA) are novel anticancer therapeutics and are also effective
44
in certain combinations with other therapies.43,
In this
context, we have investigated the combination of such
molecules with BZM.31 We have shown that BZM promotes
enhanced killing of glioblastoma cells when used in
combination with the COX-2 inhibitor Celecoxib, as well as
2,5-dimethyl-celecoxib (DMC) a structural analog that does not
inhibit COX-2.31 This increased anticancer activity was
attributed to a modest increase in ER stress, evidenced by
elevated levels of key ER stress markers, such as the chaperone
protein GRP78 and the pro-apoptotic transcription factor
CHOP. Although cancer cells are able to survive under a hostile
microenvironment by adapting to chronic elevated ER stress
conditions, even a modest additional ER stress aggravation is
not tolerated, triggering cellular apoptosis.
Fig. 2
Structures of EGCG and other dietary polyphenols.
and a dimer of (-)-epicatechin (6, EC), was shown to block the
activity of NF-κB, by preventing its binding to DNA.56
The therapeutic effects of green tea have been primarily
attributed to its component catechins (Fig. 2), namely (-)-
epigallocatechin-3-gallate (3, EGCG), (-)-epicatechin gallate (4,
ECG) (-)-epigallocatechin (5, EGC), and (-)-epicatechin (6,
EC). These polyphenols were shown to interact with a number
of biological pathways including GRP78 a key chaperone
protein involved in ER stress.39 Interestingly, it was also
reported that EGCG and structural analogs inhibit the
proteasome, while their acetylated or methylated derivatives do
not show such activity.57-65
In order to examine the potential for synergistic effects of
BZM with green tea components, we have investigated the
combination of EGCG and related polyphenols with BZM for
targeting multiple myeloma and glioblastoma.39 Given the
presence of the boronic acid group in BZM, we considered the
possibility that a BZM+EGCG combination may result in the
formation of a boronate adduct. However, in the presence of
water and other hydroxylated molecules, these interactions
could be formed reversibly, allowing both BZM and EGCG to
exert their actions, despite the formation of this type of adduct.
Our studies39 revealed that EGCG and other polyphenols
instead of being synergistic, they effectively antagonized the
apoptotic effects of BZM and significantly reduced its ability to
induce cancer cell death in vitro and in vivo. The inactivation
of BZM by green tea extracts was demonstrated in a number of
experiments using the multiple myeloma cell line RPMI/8226
These interesting findings prompted us to investigate the
combination of BZM with (-)-epigallocatechin-3-gallate,
abbreviated as EGCG (3), the most bioactive polyphenol
component of green tea, a commonly used dietary supplement
(Fig. 2). Extracts from green tea have attracted great interest
from the scientific and alternative medicine communities,45 and
its chemopreventive46 and epigenetic47, 48 properties have been
shown in multiple animal models of cancer.49-51
53
Dietary polyphenols52,
(Fig. 2) are present in large
quantities in many common plant-based food components (e.g.
green tea, apples, grapes, wine, cocoa, etc.) and recent research
has pointed to their potential utility in the prevention of cancer,
cardiovascular diseases, osteoporosis, neurodegenerative
diseases and diabetes. For example, the bioactivity of (-)-
epicatechin (6, EC), and resveratrol (7, RSV),54 which are key
55
components of dark cocoa54,
and red wine54 respectively,
have received increased interest for their chemopreventive
properties. These polyphenols have shown several mechanisms
of action as anticancer agents, including regulation of oxidative
stress as well and inhibition of cell proliferation.55 Also,
procyanidin B2 (8), a common component of apples and grapes
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DOI: 10.1039/C4OB02512A
and U266, and the glioblastoma cell line LN229. These results
illustrated a complete abrogation of BZM-mediated antitumor
properties, with EGCG showing the most antagonistic effect. It
was shown that this result was duplicated in vivo by exhibiting
BZM’s inactivation by EGCG in nude mice models implanted
with multiple myeloma cells as well as in experiments using
bone marrow isolated from multiple myeloma patients.
Additional similar findings for BZM inactivation were also
reported by others,63, 66-70 while one study71 showed that under
certain conditions EGCG potentiates the effect of BZM.
We postulated39 that the inactivation of BZM was the
consequence of a covalent interaction to form a boronate adduct
between the polyphenols and BZM. Some preliminary evidence
for this transformation was obtained by 1H NMR and 13C NMR,
suggesting the formation of a new condensation product
between the two compounds, that prevents covalent binding of
the boronic acid group of BZM to the proteasome site.39
Despite these findings, the significant variability in the
inhibitory potency among the various polyphenols studied, did
not fully account for the strong ability of EGCG to inactivate
BZM. The condensation adducts of boronic acids with diols
have been extensively investigated,8, 72, 73 and are well known to
be formed reversibly. In principle, the boron adducts of
polyphenols,67,74,75 can also behave similarly, further
complicating the analysis of EGCG-mediated inactivation.
In order to gain additional insights regarding the effects of
dietary polyphenols on the efficacy of BZM and potentially the
growing number of other emerging boron-based drugs8-13 and
diol-based pro-drugs,74, 76 the work reported in this paper was
focused on a more detailed molecular characterization of the
postulated BZM/EGCG boron adduct. Towards this goal we
relied on a number of spectroscopic techniques and cell assays,
in an effort to identify the key factors involved in this process.
Results
In our previous report on the inactivation of BZM by EGCG,39
we provided direct evidence that the BZM/EGCG combination
generates a new molecular entity, evidenced by the appearance
of new peaks in H and 13C NMR. While these data confirmed
1
the formation of a new BZM/EGCG adduct, it was not possible
to fully characterize this species without additional studies.
EGCG contains three polyphenol rings (A,B,D), two of
which contain three adjacent hydroxyl groups, generating a
number of possibilities for forming adducts with the boronic
acid group of BZM. Moreover, the BZM/EGCG adduct could
involve the formation of a neutral boronate adduct or an anionic
borate group involving the participation of a third oxygen
substituent at the boron. Additionally, the conversion of boronic
acids to boronate or borate adducts with diols is a reversible
process, depending on the conditions and the relative stability
of the boron adducts. Thus, in order to fully compare the
relative stability and physiological relevance of adducts such as
the one formed from BZM and EGCG, it is important to
determine the equilibrium constant of this transformation.
Therefore, in order to elucidate the key structural features of
the BZM/EGCG adduct we relied on several NMR techniques,
modeling studies as well as cell-based assays.
Structure elucidation of the BZM/EGCG adduct by 1H NMR
1
The 400MHz H NMR spectra of BZM, EGCG, and the
1:2 BZM/EGCG combination were analyzed and all of the C-H
chemical shifts of EGCG and BZM were fully assigned (Fig.
3A,B). Interestingly, the 1H NMR spectrum of the BZM/EGCG
combination revealed several new peaks (Fig. 3C) that could be
attributed to two isomeric adducts 9 and 10 (Fig. 3D).
1
1
1
Fig. 3
A. 400MHz H NMR of EGCG in MeCN; B. 400MHz H NMR of Bortezomib (BZM) in MeCN; C. 400MHz H NMR of 1:2
1
mixture of EGCG:BZM in MeCN; D. Postulated structures of boronate adducts (9, 10) suggested by H NMR analysis. Arrows indicate
proton shifts corresponding to adducts of EGCG and BZM. Percentages indicate relative ratios of designated peaks.
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