Development of bestatin-based activity-based probes for metallo-aminopeptidases

Article abstract of DOI:10.1016/j.bmcl.2008.09.021

A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be amenable to 'click' chemistry labeling for possible use in live cells. Finally, we demonstrate that the ABPs are able to label an aminopeptidase in a complex proteome. Thus, these bestatin-based probes should have wide utility to functionally profile aminopeptidases in many biological systems.

Full text of DOI:10.1016/j.bmcl.2008.09.021

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5932–5936
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of bestatin-based activity-based probes
for metallo-aminopeptidases
*
Michael B. Harbut, Geetha Velmourougane, Gilana Reiss, Rajesh Chandramohanadas, Doron C. Greenbaum
University of Pennsylvania, Department of Pharmacology, 433 S. University Avenue, 304G Lynch Laboratories, Philadelphia, PA 19104-6018, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2008
Revised 3 September 2008
Accepted 5 September 2008
Available online 10 September 2008
A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was
synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using
solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin
and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be ame-
nable to ‘click’ chemistry labeling for possible use in live cells. Finally, we demonstrate that the ABPs are
able to label an aminopeptidase in a complex proteome. Thus, these bestatin-based probes should have
wide utility to functionally profile aminopeptidases in many biological systems.
Keywords:
Aminopeptidase
Activity-based probe
Bestatin
Ó 2008 Elsevier Ltd. All rights reserved.
Solid-phase synthesis
Metallo-aminopeptidases (MAPs) are exopeptidases that cata-
lyze the hydrolysis and cleavage of a single N-terminal amino acid
from a peptide or protein substrate. The families of MAPs are a
large and diverse set of peptidases and comprise the M1, M17,
and M18 families, which in humans totals 16 potential enzymes
(www.merops.org). MAPs are widely distributed in organisms
from bacteria to humans and play essential roles in protein matu-
ration and regulation of the metabolism of bioactive peptides.1–3 In
addition, MAPs have been linked to several pathophysiological
states including cancer and hypertension.4,5
A major challenge in elucidating the function of peptidases
during normal or pathological processes lies in their complex
post-translational regulation. Petidase activity is usually tightly
controlled post-translationally, with mRNA levels frequently show-
ing little correlation to active protein levels. In addition, peptidases
can be localized to any part of a cell and are frequently found in
multiple locations with different functions. A targeted proteomics
approach whereby only active proteins are enriched by the use of
activity-based probes (ABPs) can address these complicating issues
and provide complementary data to more traditional genetic and
biochemical approaches. These ABPs typically possess three main
structural components: (i) a mechanism-based inhibitor scaffold
to covalently or non-covalently target catalytic residues or the
active site of peptidases, (ii) a linker, and (iii) a reporter tag, such
as a fluorophore or biotin, for the visualization and characterization
of labeling events, and eventual affinity purification of target pro-
teins. The mechanism-based inhibitor scaffold ensures that the
ABP binds to the peptidase in an activity-dependent manner. There-
fore, these ABPs can identify peptidases with differential levels of
activity during a biological process and potentially identify candi-
date enzymes that regulate the specific phenotype under study. In
addition, ABPs allow for screening of small molecule libraries and
identification of specific inhibitors that can be used to validate
the role of the target enzyme. To date, ABPs have been developed
for more than a dozen enzyme classes including: peptidases,6
kinases,7 phosphatases,8 glycosidases,9 and oxidoreductases.10
Although the MAP superfamily is quite large and divergent,
MAPs utilize a common catalytic mechanism by the coordination
of one or two Zn atoms in the active site to activate water for
nucleophilic attack of a peptide or protein substrate. To exploit this
mechanism several classic inhibitor scaffolds have been developed
to target the MAP family including, most prominently, phosphinic
acids,11 hydroxamic acids12,13 and the bestatin family.14,15 Both
2+
Zn
O
O
O
H O
S1
N
H
H
+
N
H
S1’
H
-
-
-
Glu
Glu
Glu
* Corresponding author. Tel.: +1 215 746 2992; fax: +1 215 746 6697.
Figure 1. General model of interactions of bestatin in the active site of metallo-
E-mail address: dorong@upenn.edu (D.C. Greenbaum).
aminopeptidases.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.021

M. B. Harbut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5932–5936
5933
Fmoc SPPS
O
NHFmoc
O
NH
O
O
O
MH01
O
NH
H
N
H
N
H
N
a
b
O
c
O
O
S
N
H
N
H
BocHN
N
H
O
O
Fmoc
S
N
H
N
H
H
HN
Rink amide resin
OH
O
O
O
H
HN
H
H
NH
NH
O
O
Scheme 1. Synthesis of bestatin-based ABPs. Reagents and conditions: (a) i-20% Piperidine/DMF; ii-Fmoc-Lys(Biotin)OH, HBTU, HOBt, DIEA; (b) i-20% Piperidine/DMF;
ii-Fmoc-BpaOH, HBTU, HOBt, DIEA; iii-20% Piperidine/DMF; iv—Fmoc-NHPEGOH (20 atoms), HBTU, HOBt, DIEA; v-20% Piperidine/DMF; vi-Fmoc-LeuOH, HBTU, HOBt, DIEA;
vii-20% Piperidine/DMF; viii-N-Boc-(2S,3R)-3-amino-2-hydroxy-4-phenyl butyric acid, HATU, DIEA; (c) 95% TFA:2.5% TIS:2.5% H2O.
phosphinic acids and hydroxamic acids have the capacity to inhibit
metallo-endopeptidases and peptide deformylase while the besta-
tin scaffold appears specific for MAPs.
We have thus chosen to explore the bestatin scaffold to develop
ABPs to specifically target the MAP superfamily. Bestatin is a natu-
ral product of Actinomycetes that inhibits most MAP families,
including the M1, M17, and M18 families. Bestatin was originally
found to be a potent aminopeptidase B16 and leucine aminopepti-
dase inhibitor and has been crystallized with leucine aminopepti-
dase,17 leukotriene A4 hydrolase,18 and aminopeptidase N.19
Bestatin is thought to modulate many biological pathways, includ-
ing apoptosis.20,21 and inflammation.22 Therefore, MAP-specific
ABPs would be powerful tools to tease apart the functions of multi-
ple MAP pathways. In the present study, we set out to develop the
first MAP-specific ABP, exploiting bestatin for use as the inhibitor
scaffold.
Bestatin resembles a Phe-Leu dipeptide substrate. However, the
first residue contains a
a-hydroxy group that, along with the
neighboring carbonyl, co-ordinates the catalytic zinc atom result-
ing in a competitive active site-directed inhibitor (Fig. 1). In
MH01
O
NH2
O
O
O
H
N
H
N
H
N
O
O
S
N
H
N
H
H2N
N
H
O
O
H
OH
O
O
O
H
HN
NH
O
O
Bestatin Scaffold
Linker
Benzophenone
UV crosslinker
Biotin Affinity Tag
MH02
O
NH2
O
O
S
H
N
H
N
H
N
O
O
NH
HN
H2N
N
H
N
H
O
OH
O
O
O
O
Bestatin Scaffold
APN
Benzophenone
UV crosslinker
Linker
Biotin Affinity Tag
+ + + +
+ + +
APN
+ + + + + + + +
+ + + + + +
UV
UV
Bestatin
Preheat
+
μM probe
10
1
0.5 10
10
1
0.5 10
+
MH01
MH02
MH01
Figure 2. Anatomy of ABPs and labeling of aminopeptidase N. (a) Structure of ABPs MH01 and MH02. (b) Aminopeptidase N (0.13 U) was treated with 10, 1, or 0.5
either MH01 or MH02 for 1 h in 50 mM Tris–HCl, pH 7.8, 0.5 M ZnCl2 (buffer A). Certain reaction mixtures were UV crosslinked for 1 h on ice. Reactions were quenched with
SDS–PAGE buffer, and labeled protein was visualized via SDS–PAGE and western blotting for biotin. (c) Aminopeptidase N was treated with 100 M of the aminopeptidase
lM of
l
l
inhibitor bestatin or DMSO for 1 h in buffer A followed by labeling with MH01 for 1 h. Reactions were UV crosslinked (or not) for 1 h on ice, and labeled protein was visualized
as in Figure 2b.

5934
M. B. Harbut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5932–5936
addition, the free amine of bestatin is co-ordinated by one or more
glutamate residues in the MAP active site.19 Bestatin family mem-
bers are slow- and tight-binding inhibitors15 with well-defined
interactions with the S1 and S10 active site pockets. Thus, bestatin
represents an ideal candidate for ABP development for MAPs due
to its family specificity, potency in the low nanomolar to micromo-
lar range, synthetic tractability and potential for expansion
through variation of the amino acid side chains in its core
structure.
To design an ABP family for MAPs, we chose to derivatize the
core bestatin inhibitor scaffold using a solid-phase synthetic strat-
egy (Scheme 1). Bestatin has a free carboxyl group available for
functionalization and previous X-ray crystal structures of MAP-
bestatin complexes indicated that extension at this carboxylate
was unlikely to perturb inhibitor binding.19 Thus, we attached
the inhibitor scaffold to solid-phase resin at the carboxyl end of
the molecule. Our first attempt to develop a MAP-directed ABP
probe involved the addition of a spacer, a UV crosslinker, and a
biotin affinity tag to the C-terminus of the core bestatin scaffold
(Scheme 1). We included a benzophenone UV crosslinker since this
is a non-covalent, reversible inhibitor scaffold. Synthesis was
accomplished on solid-phase using Rink amide resin and, to our
knowledge, represents the first reported solid-phase synthesis of
this class of molecules.23
the model aminopeptidase in an activity-dependent manner
(Fig. 2c). Our results show that, indeed, the bestatin-based probe
is an activity-dependent probe of MAPs. Firstly, competition with
the unbiotinylated parental compound, bestatin, blocked all label-
ing seen in lane 1, indicating that the probe was competitive and
therefore binding at the active site (Fig. 2c, lane 2). Preheating of
the sample, a process that denatures all protein targets, abrogated
labeling (Fig. 2c, lane 3), indicating that this labeling was depen-
dent on properly folded, active enzyme. Lastly, UV exposure was
necessary for labeling owing to the fact that bestatin is a non-cova-
lent, reversible inhibitor (Fig. 2c, lane 4).
While a biotin tag is ideal for affinity tagging purposes, its use is
not optimal for higher throughput activity-based profiling due to
the time and labor involved in producing western blots. Addition-
ally, many cells and tissues contain endogenously biotinylated
proteins that complicate analysis of biotinylated probe-based
western blots. To circumvent these shortcomings we synthesized
a fluorophore-tagged version of the bestatin-based probe, which
would allow for direct detection of labeled targets in a gel-based
read-out. For the fluorescent bestatin-based probe we added a
TAMRA group in addition to the biotin, creating a dual function
ABP, MH03, making it suitable for both affinity purification and
fluorescence applications (Fig. 3a).24 Labeling of purified porcine
aminopeptidase N with the dual label probe was performed under
the same conditions as with the original biotinylated MH01
(Fig. 3b). Labeled proteins were analyzed by SDS–PAGE coupled
with in-gel fluorescent scanning (Typhoon, GE). MH03 showed
similar activity-dependent labeling as MH01 (Fig. 3b) demonstrat-
ing the robustness of this ABP scaffold. Some residual labeling was
We initially explored the placement of the spacer and UV cross-
linker relative to the core bestatin scaffold (MH01 and MH02,
Fig. 2a) and found that there was little difference in labeling
efficiency of a model enzyme, purified porcine aminopeptidase N
(Sigma, Fig. 2b). We then assessed the ability of MH01 to label
MH03
O
O
NH2
O
O
O
H
N
H
N
O
S
HN
OH
O
N
H
N
H
N
H
H
HN
O
O
H
H2N
O
NH
O
O
NH
O
O
_
+
N
O
N
APN
80
70
60
50
40
30
20
10
0
BS
+
+
+
+
+
+ +
+
APN
UV
Bestatin
Preheat
+
100 nM 1 μM
10 μM 100 μM 1 mM
Bestatin
Figure 3. Aminopeptidase N ABP labeling by fluorophore-containing bestatin-based ABP. (a) Structure of MH03 probe. (b) Labeling of aminopeptidase N was performed as
described in Figure 2, but using MH03 and visualized using SDS–PAGE and in-gel fluorescent scanning. (c) Aminopeptidase N was pretreated with multiple concentrations of
bestatin (BS) for 1 h and then labeled with 10 lM fluorescent MH03. After in-gel fluorescent scanning labeling was quantified using ImageQuant software (GE).

M. B. Harbut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5932–5936
5935
MH04
O
O
O
H
N
H
N
H
N
Fmoc SPPS
a
O
O
H
N
H
N
HN
OH
O
N
H
NHFmoc
O
NHAloc
HN
OH
O
N
H
O
O
H2
O
b
H2N
O
O
N
O
O
Rink resin
BocHN
O
O
HN
Scheme 2. Reagents and conditions: (a) i-20% Piperidine/DMF; ii-Fmoc-Lys(Aloc)OH, HBTU, HOBt, DIEA; iii -20% Piperidine/DMF; iv—Fmoc-BpaOH, HBTU, HOBt, DIEA;
v-20% Piperidine/DMF; vi—Fmoc-NHPEGOH (9 atoms), HBTU, HOBt, DIEA; vii-20% Piperidine/DMF; viii—Fmoc-LeuOH, HBTU, HOBt, DIEA; ix-20% Piperidine/DMF; x-N-Boc-
(2S,3R)-3-amino-2-hydroxy-4-phenyl butyric acid, HATU, DIEA; (b) i-Pd(PPh3)4, PhSiH3, DCM; ii-hexynoic acid, HBTU, HOBt, DIEA; iii-95% TFA:2.5% TIS:2.5% H2O.
observed (Fig. 3b, lane 2), which is normal in ABP labeling experi-
ments, although this may have been enhanced since bestatin is a
slow- and tight-binding inhibitor.
Next, we wanted to demonstrate that utility of the fluorescent
MH03 for relative quantification of enzyme labeling. To do this,
small alkyne tag to our MAP probe in order to utilize the popular
‘click’ bio-orthogonal chemistry, which employs a 1,3-dipolar
azide/alkyne cycloaddition.25,26 We thus replaced the biotin of
MH01 with a C-terminal alkyne resulting in a click probe, MH04
(Scheme 2 and Fig. 4).27 We observed facile ‘click’ addition of a
biotinylated azide following labeling of porcine aminopeptidase
N with MH04. Importantly the activity-dependent labeling of the
enzyme was not affected by this procedure, as illustrated by best-
atin preincubation, UV, and preheat controls.
Finally, to validate the utility of the probe as an ABP for MAPs in
the context of a complex proteome, we assessed labeling of an ami-
nopeptidase N homolog from the malarial parasite (PFA-M1) from
whole cell lysates. To facilitate the identification of PFA-M1 from
Plasmodium falciparum we utilized a parasite line that expresses
the endogenous PFA-M1 as a YFP C-terminal fusion.28 Homoge-
nized whole cell lysates from P. falciparum were labeled with
MH01. The resulting proteome was separated by SDS–PAGE and
western blot analysis was performed to first visualize biotinylated
proteins. A 150 kDa protein was labeled by MH01, which corre-
sponds to the approximate weight of the PFA-M1-YFP fusion pro-
tein (Fig. 5a). Preincubation of the protein lysate with
unbiotinylated bestatin (Fig. 5a, lane 2) resulted in loss of labeling
of the 150 kDa band illustrating that bestatin was competitive with
MH01 for this protein target. Additionally, the target was labeled in
porcine aminopeptidase
N was preincubated with increasing
concentrations of bestatin followed by the addition of a single con-
centration of the fluorescent MH03. The densities of each labeled
band, representing active enzyme, were quantified using a Typhoon
flatbed fluorescent scanner (GE). In-gel fluorescent scanning of the
labeled peptidase band showed a decrease in aminopeptidase N
labeling by MH03 relative to increasing concentration of bestatin
preincubation (insert in Fig. 3c). Percent competition values were
calculated by dividing the density of each of the bestatin preincu-
bated aminopeptidase N bands (lanes 2–5) by density of the
untreated band in lane 1 (Fig. 3c). We demonstrate a dose depen-
dent relationship that is amenable to quantification with a dynamic
range of several orders of magnitude, which will allow future
screening efforts of derivative libraries.
One of the technical challenges facing the development and use
of ABPs with biotin or fluorophore tags is limited or biased uptake
by live cells. In some cases, ABPs have been used to label enzymes
in living cells, but a more universal system for labeling would
employ a small, hydrophobic tag. We therefore chose to add a
MH04
O
O
H
N
H
N
H
N
N
Biotin -PEG -N3
N
O
N PEG-Biotin
HN
OH
O
N
H
O
O
CuSO 4
ligand
H2N
O
O
H2N
TCEP
5% t-butyl alcohol
APN
+
+
+
+
+
+
+
+
+
+
+
Biotin-N3
UV
+
+
+
Bestatin
Preheat
+
Figure 4. ‘Click’ chemistry-based ABP labeling of aminopeptidase N. (a) Structure of MH04 probe. (b) Aminopeptidase N labeling was performed as described in Figure 2c
using MH04. The ligation of the biotin-azide reporter tag was performed by adding 100 M of the biotin-azide tag, followed by 1 mM TCEP (tris(2-carboxyethyl) phosphine
hydrochloride), 100
M ligand (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) (17ꢀ stock in DMSO/tert-butanol 1:4), and 1 mM CuSO4. Reactions were allowed to
proceed for 1 h at room temperature, then quenched with equal volume of SDS–PAGE loading buffer. Labeled protein was visualized as in Figure 2b.
l
l

5936
M. B. Harbut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5932–5936
PfA-M1-YFP lysate
PfA-M1-YFP lysate
MH01
UV
Bestatin
Preheat
MH01
UV
Bestatin
Preheat
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
150kDa
Blot: Streptavidin-HRP
Blot: Anti-YFP
Figure 5. ABP labeling of the malarial M1 metallo-aminopeptidase in cell lysates. (a) P. falciparum cells (in buffer A) were freeze/thawed 3ꢀ on dry ice. Cell debris was
removed by centrifugation, and the lysate was retained for labeling. MH01 was incubated with parasite lysate for 1 h and then UV crosslinked for 1 h. In one reaction, 100
lM
unbiotinylated bestatin was preincubated with the lysate prior to probe addition. Labeled protein was visualized via a western blot for biotin. (b) The same blot was then
stripped and reprobed using anti-YFP.
13. Saghatelian, A.; Jessani, N.; Joseph, A.; Humphrey, M.; Cravatt, B. F. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 10000.
14. Wilkes, S. H.; Prescott, J. M. J. Biol. Chem. 1985, 260, 13154.
an activity-dependent manner, as preheating of the proteome prior
to labeling abrogated any labeling (Fig. 5a, lane 3). Finally, to iden-
tify this target the blot was stripped and reprobed using an anti-
YFP antibody (Fig. 5b). The anti-YFP revealed the presence of the
expected 150 kDa fusion protein in all lanes and this band overlaid
the exact position where the biotinylated protein appeared. It
should be noted that PFA-M1-YFP fusion appeared in all lanes with
the anti-YFP antibody, whereas the biotinylated species only ap-
peared when the active peptidase was labeled. This data thus con-
firmed the specific activity-based labeling of the PFA-M1
aminopeptidase by MH01 within a complex malarial parasite
proteome.
In conclusion, we have developed a novel activity-based probe
class, with specificity for MAPs, based on the bestatin inhibitor
scaffold. The use of a biotin, fluorophore, or alkyne moiety did
not alter the activity-dependent labeling profile for the scaffold
and, thus, the suite of ABPs presented in this manuscript should
allow for a variety of labeling methodologies. We therefore believe
that this ABP may prove to be a valuable tool for future character-
ization of MAP activity in a wide variety of biological systems. We
are now currently pursuing the expansion and application of these
probes for use against the malarial parasite.
15. Rich, D. H.; Moon, B. J.; Harbeson, S. J. Med. Chem. 1984, 27, 417.
16. Suda, H.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. Arch. Biochem. Biophys. 1976,
177, 196.
17. Burley, S. K.; David, P. R.; Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
6916.
18.
Tsuge, H.; Ago, H.; Aoki, M.; Furuno, M.; Noma, M.; Miyano, M.; Minami, M.;
Izumi, T.; Shimizu, T. J. Mol. Biol. 1994, 238, 854.
19. Addlagatta, A.; Gay, L.; Matthews, B. W. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
13339.
20. Sekine, K.; Fujii, H.; Abe, F. Leukemia 1999, 13, 729.
21. Ezawa, K.; Minato, K.; Dobashi, K. Biomed. Pharmacother. 1996, 50, 283.
22. Penning, T. D. Curr. Pharm. Design 2001, 7, 163.
23. General method for solid-phase peptide synthesis of ABPs: Standard solid-
phase peptide synthesis was performed on Rink amide resin using HBTU/HOBt/
DIEA in an equimolar ratio in DMF for 30 min at RT. Coupling of the a-hydroxy-
b-amino acid required HATU for 1 h. Each amino acid was double coupled.
Fmoc protecting groups were removed with 20% piperidine/DMF for 30 min. To
cleave products from resin, a solution of 95% TFA:2.5% TIS:2.5% H2O was added
to the resin. After standing for 2 h, the cleavage mixture was collected, and the
resin was washed with fresh cleavage solution. The combined fractions were
evaporated to dryness and the product was purified by reverse phase-HPLC.
Fractions containing product were pooled and lyophilized. Reverse phase HPLC
was conducted on a C18 column using an Agilent 1200 HPLC. Purifications
were performed at room temperature and compounds were eluted with a
concentration gradient 0–70% of acetonitrile (0.1% Formic acid). LC/MS data
were acquired using LC/MSD SL system (Agilent). Solid-phase peptide
chemistry was conducted in polypropylene cartridges, with 2-way Nylon
stopcocks (Biotage, VA). The cartridges were connected to a 20 port vacuum
manifold (Biotage, VA) that was used to drain excess solvent and reagents from
the cartridge. MH01: C62H90N10O14S, predicted mass 1230.64, observed [M+H]
1231.4. MH02: C57H81N9O12S, predicted mass 1115.57, observed [M+H]
1116.3.
Acknowledgments
This work was supported by the University of Pennsylvania
Genome Frontiers Institute and the Ritter Foundation. We are
grateful to Dr. Michael Klemba (Virginia Tech) for the PFA-
M1-YFP parasite line.
24. The synthesis of the dual function ABP MH03 was accomplished using standard
solid-phase peptide synthesis as detailed above, However, the TAMRA was
added after product cleavage from resin due to the instability of TAMRA to TFA.
This was accomplished using a Boc protected lysine and addition of TAMRA
using HBTU/HOBt/DIEA in DMF after resin cleavage for 3 h. MH03:
C85H107N13O16S, predicted mass 1597.77, observed [M+2H] 799.8.
References and notes
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27. Synthesis of MH04 was accomplished as depicted in Scheme 2. The
deprotection of the Aloc group was conducted under
a positive flow of
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