The natural product hybrid of Syringolin A and Glidobactin A synergizes proteasome inhibition potency with subsite selectivity

Article abstract of DOI:10.1039/c0cc02238a

The preparation of a Syringolin A/Glidobactin A hybrid (SylA-GlbA) consisting of a SylA macrocycle connected to the GlbA side chain and its potent proteasome targeting of all three proteasomal subsites is reported. The influence of the syrbactin macrocycle moiety on subsite selectivity is demonstrated.

Full text of DOI:10.1039/c0cc02238a

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The natural product hybrid of Syringolin A and Glidobactin A synergizes
proteasome inhibition potency with subsite selectivitywz
a
b
a
c
d
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Jerome Clerc, Nan Li, Daniel Krahn, Michael Groll, Andre S. Bachmann,
Bogdan I. Florea,b Herman S. Overkleeft*b and Markus Kaiser*a
Received 30th June 2010, Accepted 17th August 2010
DOI: 10.1039/c0cc02238a
The preparation of a Syringolin A/Glidobactin A hybrid
(SylA–GlbA) consisting of a SylA macrocycle connected to
the GlbA side chain and its potent proteasome targeting of all
three proteasomal subsites is reported. The influence of the
syrbactin macrocycle moiety on subsite selectivity is demonstrated.
different subsite potencies could induce diverse pharmacological
effects and consequently demands for a deeper understanding
of the structural determinants governing subsite selectivity of
proteasome inhibitor classes.
We recently elucidated the syrbactin natural products
syringolin A (SylA), syringolin B (SylB) and glidobactin A
(GlbA) as mechanism-based irreversible proteasome inhibitors
(for the chemical structures of the syrbactins used during
this study, see ESIz).8 These natural products exert distinct
proteasomal subsite selectivities in biochemical activity assays
as well as in structural studies and selectively target the
proteasome in complex proteomes.9 SylA targets all three
proteolytic activities, albeit with different potencies. SylB
and GlbA on the other hand selectively inhibit b2 and b5 even
at high concentrations, inviting the hypothesis that the macro-
cyclic residue critically influences subsite selectivity while the
N-terminal lipophilic tail strongly enhances inhibition
potency. This hypothesis is supported by the finding that
model compound SylA–LIP, consisting of a SylA core structure
attached to a lipophilic side chain, inhibits all three proteolytic
sites, thereby displaying the most potent inhibition of all
syrbactins reported so far.10 While SylA, GlbA and SylA–LIP
showed promising anticancer activities in cell-based assays, the
order of activity was somewhat surprising, with GlbA being
more potent than SylA–LIP.11
Proteasomal protein degradation plays a critical role in
innumerous biological processes such as cell division, immune
responses, apoptosis and gene expression.1 Proteolysis takes
place in the 20S core particle (CP), a 670 kDa cylinder built up
from four stacked heptameric rings, in which only the b1, b2
and b5 subunits harbor distinct proteolytic activities with
different substrate specificities.2
Besides the constitutive 20S CP, alternative 20S proteasome
species are expressed in a tissue-specific manner. In the
immunoproteasome, the constitutive b1, b2 and b5 subunits
are replaced by the b1i, b2i and b5i subunits while the
thymoproteasome encompasses the proteolytically active b1i,
b2i and b5t subunits.3,4 These subunits display alternative
cleavage activities, thereby modulating the overall trimming
of proteins during degradation.
Active-site directed proteasome inhibitors are valuable
anticancer agents as well as versatile research tools.5
Consequently, various rationally-designed small molecules
and natural products have been explored in recent years.6
These inhibitors usually target the proteasomal proteolytic
subsites with different potencies. To date, the physiological
consequences of such subsite specific inhibition are not well
understood although distinct roles of individual proteasomal
subunits for certain biological processes have already been
observed.7 This pinpoints that proteasome inhibitors with
To gain further insights into syrbactins’ structure–activity
relationships, we decided to put to the test two additional
derivatives, being SylA–GlbA as a ‘true’ hybrid of Syringolin
A and Glidobactin A, together with sat-SylA as a negative
control. The synthesis of SylA–GlbA started with a Horner–
Wadsworth–Emmons (HWE) olefination of octanal (1) using
(E)-triethyl-4-phosphonocrotonate and LDA (Scheme 1). The
resulting ethyl ester 2 was hydrolyzed with LiOH to yield the
free acid 3. Peptide coupling of Boc–Thr(OtBu)–OH to the
previously reported SylA macrocycle 412 led to intermediate 5
which upon deprotection and PyBOP-mediated coupling of 3
yielded the desired SylA–GlbA hybrid (6). The synthesis of
sat-SylA is reported in the ESI.z
a Zentrum fur Medizinische Biotechnologie,
¨
Fakultat fur Biologie und Geographie, Universitat Duisburg-Essen,
¨
¨
¨
45117 Essen, Germany. E-mail: markus.kaiser@uni-due.de;
Fax: +49 201 1833135; Tel: +49 201 1832949
b Leiden Institute of Chemistry and Netherlands Proteomics Centre,
Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden,
The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl;
Fax: +31 71 5274307; Tel: +31 71 5274342
With these additional derivatives in hand, their inhibition
potency and subsite selectivity was established using competitive
activity-based protein profiling (ABPP) in cell lysates and
living cells in comparison with known syrbactins.13 To this
end, HEK cell lysates were preincubated with varying
concentrations of the syrbactins and profiled with the
proteasomal activity based probe (ABP) MV151 (Fig. 1, for
chemical structures of the ABPs, see ESIz).14 The results from
these experiments were generally in good agreement with the
results from the previous biochemical inhibition studies with
c Center for Integrated Protein Science at the Department Chemie,
Technische Universitat Munchen, Lichtenbergstr. 4, 85747 Garching,
Germany
¨
¨
d Cancer Research Center of Hawaii, University of Hawaii at Manao,
1236 Lauhala Street, Honolulu, Hawaii 96813, USA
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Experimental
details of synthesis of SylA–GlbA, synthesis of sat-SylA, chemical
structures of syrbactins and ABPs used during these studies, and
supplementary figures and experimental procedures for competitive
ABPP studies. See DOI: 10.1039/c0cc02238a
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 385–387 385

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Fig. 1 Competitive ABPP of HEK cell lysates using 1 mM MV151 as
ABP and varying concentrations of syrbactins. MV151-targeted
proteasomal subsites (for assignment, see ref. 14) are marked by
arrows on the left side of the gel. 20 mg protein/lane was loaded.
M = 25 kDa marker lane.
Scheme
1 Structure and synthesis of the SylA–GlbA hybrid.
inhibit this subsite. These findings support our hypothesis that
the type of macrocycle moiety critically influences b1 subsite
selectivity.
(A) Chemical structures of SylA and GlbA and their mergence to
the hybrid compound SylA–GlbA. (B) Chemical synthesis of
the SylA–GlbA hybrid. (a) (E)-triethyl-4-phosphono crotonate
(1.5 eq.), LDA (2 eq.), THF, ꢀ78 1C – rt, 2.5 h, 67%. (b) LiOH
(3 eq.), MeOH/H2O (1 : 1), 50 1C, 1 h, 82%. (c) Boc–Thr(OtBu)–OH
(1.2 eq.), PyBOP (1.5 eq.), HOAt (1.5 eq.), DIPEA (2 eq.), DMF,
0 1C ꢀ rt, 1 h, 62%. (d) (i) 20% TFA in DCM, rt, 30 min; (ii) 3
(1.2 eq.), PyBOP (1.5 eq.), HOAt (1.5 eq.), DIPEA (4 eq.), rt,
o/n, 25%.
Encouraged by these results, we continued our investigations
by competitive ABPP of thymus lysates which besides the
constitutive 20S CP also contain the immuno- and thymo-
proteasome (Fig. 2).15 For the immunoproteasome, a similar
trend for subunit selectivity and inhibition potency was
observed with all syrbactins. While the negative control
sat-SylA was completely inactive, the other derivatives
preferentially bound to the b5i subunit, followed by the b2i
subunit. Direct comparison of syrbactins revealed GlbA and
SylA–GlbA as the most potent compounds, followed by
SylA–LIP and SylA and lastly SylB. Interestingly, SylA–GlbA
also bound to the b1i subunit while GlbA showed (if any) only
very weak inhibition. Thus, the immunoproteasome displays a
similar inhibition pattern as the constitutive proteasome.
Moreover, despite b5t visualization by the MVB003 probe is
generally only weak15 (b5t being expressed in parts of the
thymus only,4 whereas we applied extracts from the organ as a
whole), we observed that GlbA concentrations of 10 mM
completely abolished b5t labelling, while no such activity
could be seen for any SylA-derived syrbactin. This different
activity profile is in accordance with literature predictions4,15
that b5t displays a rather unique cleavage specificity, preferring
hydrophilic residues such as the hydroxylysine and threonine
moieties of GlbA in their active site pockets. Finally, as GlbA
shows promising anti-cancer activities in cell-based and mouse
purified 20S CP, with only slight deviations in the activity
pattern. All syrbactins except the non-reactive negative control
sat-SylA bind the b2 and b5 subsites, although with different
affinities. Furthermore, all syrbactins inhibited the b5 subunit
more potently than b2. A direct comparison of the different
syrbactins revealed GlbA as the most potent inhibitor,
followed by the only slightly less potent SylA–GlbA hybrid.
Surprisingly and in contrast to what was found in the
biochemical assay, SylA–LIP showed less affinity than GlbA,
and we argue this discrepancy is the result of unspecific
binding of the highly hydrophobic SylA–LIP to other proteins
in the cell lysate. This finding might explain the unexpected
activity pattern of GlbA and SylA–LIP in the previous cancer
cell assays.11 SylA and in particular SylB appeared much
less active than the syrbactins with the N-terminal lipophilic
chain. Importantly, while the syrbactins with
a SylA
macrocycle and in particular SylA–GlbA also bind, at high
inhibitor concentrations, to the b1 subunits, GlbA did not
c
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Financial support from The Netherlands Organization for
Scientific Research (NWO) and the Netherlands Genomics
Initiative (NGI) to H.S.O. and the Deutsche Forschungsge-
meinschaft (DFG grant KA2894/1-1) to M.K. is greatly
acknowledged.
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c
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Chem. Commun., 2011, 47, 385–387 387