Development of activity-based probes for trypsin-family serine proteases

Article abstract of DOI:10.1016/j.bmcl.2006.03.012

A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased s

Full text of DOI:10.1016/j.bmcl.2006.03.012

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2882–2885
Development of activity-based probes for trypsin-family
serine proteases
Zhengying Pan,* Douglas A. Jeffery, Kareem Chehade, Jerlyn Beltman,à James M. Clark,
Paul Grothaus, Matthew Bogyo*,§ and Amos Baruch*
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 2 February 2006; revised 28 February 2006; accepted 2 March 2006
Available online 22 March 2006
Abstract—A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selec-
tively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This
increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS–PAGE methods.
We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell
implicated in allergy and autoimmune diseases.
Ó 2006 Elsevier Ltd. All rights reserved.
The primary goal of proteomics is to assign functions to
all proteins in a given cell, tissue or organism.1 The chal-
lenges implicit in this field have generated novel
approaches to functionally dissect the proteome. Chem-
ical proteomics or activity-based protein profiling
(ABPP) makes use of small molecule probes that form
covalent complexes with their targets using an activity-
dependent chemical reaction. These activity-based
probes (ABPs) can be used to profile enzymatic activities
in complex proteomes and provide information on pro-
tein targets at the functional, rather than the expression
level.2 Moreover, due to its use of small molecules, this
approach naturally focuses on ‘druggable’ enzymes that
show specific ligand binding.
expanded with the development of chemical probes that
target additional important enzyme families including
phosphatases5 and kinases.6 Many approaches have
focused on broad-spectrum probes that target multiple
related enzyme family members.7 Thus, there is a great
need to develop new chemical probes to selectively
target sub-classes of enzymes to study their roles in bio-
logical processes.
Serine hydrolases represent a large family of enzymes,
members of which participate in many crucial biologi-
cal processes. One of the most intriguing sub-classes of
this family is the trypsin-like serine proteases. This sub-
group is comprised of 65 enzymes in humans with un-
ique cellular and physiological regulatory roles in
health and disease.8 Several enzymes within this group,
such as thrombin, factor VIIa, factor Xa, and tryptase,
are being extensively pursued as drug targets in
cardiovascular and inflammatory indications. A fluor-
ophosphonate (FP) probe, similar to probe 1 (Bio-
FP), has been previously reported to target the serine
hydrolase family (Ref. 3). The broad reactivity of
probe 1 enables simultaneous labeling of a large num-
ber of enzymes including esterases, proteases, lipases,
and amidases. In many cases, this probe generates a
highly complex activity profile that prevents its usage
for studying low-abundance, specific enzymes using
simple analytical methods such as SDS–PAGE. To
A number of classes of ABPs have successfully been
designed and applied to serine hydrolases3 and cysteine
proteases.4 Recently the scope of this approach has
Keywords: Activity-based probes; Tryptase; Chemical proteomics.
*
Corresponding authors; e-mail addresses: zypan@yahoo.com;
zhengying.pan@celera.com; mbogyo@stanford.edu; amos.baruch@
celera.com
Present address: Novartis Institute of Biomedical Research, Boston,
MA 02139, USA.
à
Present address: Chiron Corporation, Emeryville, CA 94608, USA.
Present address: Department of Pathology, School of Medicine,
Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.
§
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.012

Z. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2882–2885
2883
O
biotin. Following addition of an amino acid residue at
the P2 position, similar procedures produced biotinyla-
ted probes 4 and 5.
O
O
O
P
NH
H
O
F
O
O
HN
N
O
N
H
H
S
5
H
Figure 1. A general serine hydrolase probe 1.
Apparent inhibition constants [Ki(app)] were obtained for
these probes against four trypsin-like serine proteases
(Table 1). The addition of a P2 proline residue increased
the overall reactivity of probe 4 toward trypsin-like
serine proteases. Incorporation of an asparagine residue
at P2 yielded a 23-fold selectivity increase for probe 5
favoring tryptase over its closely related family member
trypsin.
circumvent this issue, we developed a series of selective
probes that specifically target the trypsin-like serine
proteases (see Fig. 1).
To achieve selectivity toward serine proteases, we fo-
cused our efforts on phosphonate-based probes. Ini-
tial attempts to use an a-amino fluorophosphonate
reactive group failed due to its short half-life in an
aqueous environment.9 The more stable reactive
group, diphenylphosphonate, has previously been
used to generate potent irreversible serine protease
inhibitors10. Here, we describe our efforts in develop-
ing diphenylphosphonate (DPP)-derived probes for
activity profiling of trypsin-like serine proteases11
(see Scheme 1).
To confirm that the potency and selectivity observed in
kinetic assays were reflected in enzyme labeling profiles,
probes 1, 4, and 5 were used for activity-based labeling
of a panel of recombinant enzymes from different serine
hydrolase families (Fig. 2). The extent of covalent label-
ing was determined by Western blotting with streptavi-
din detection of the biotin reporter group. Probe 1
labeled all recombinant enzymes tested including butyr-
ylcholine esterase (BCE), a hydrolase enzyme. In con-
trast, the lysine-DPP-based probes were completely
inactive against both chymotrypsin and BCE. The gen-
eral trypsin-family probe 4 efficiently labeled all trypsin-
Based on previous studies of substrate specificity,12
a
lysine residue was incorporated at the P1 site with either
a proline or asparagine at the P2 position to generate a
general trypsin-family protease probe 4 (Bio-PK-DPP)
and a b-tryptase-selective probe 5 (Bio-NK-DPP),
respectively. Lysine-based DPPs can be made through
the Oleksyszyn 3-component reaction using either con-
ventional heating or microwave-assisted agitation. Inter-
Table 1. Apparent inhibition constants [Ki(app) lM] of probes 1, 4, and
5 for trypsin-like serine proteases following 30 min of incubation13
Enzymes
Bio-FP 1
Bio-PK-DPP 4
Bio-NK-DPP 5
mediate
2
was obtained through three steps of
b-Tryptase
Trypsin
30
6.2
2.5
16.5
7.9
0.57
1.07
2.9
57.3
>100
4.03
protecting group manipulations. A biotinylated probe
3 (Bio-K-DPP) was made by coupling 2 with a commer-
cially available biotinylation reagent NHS-(PEG)4-
Thrombin
Plasmin
>100
O
O
P O
O
H
N
O
O
N
b
a
O
O
P(OPh)3
N
O
O
O
NH2
O
O
HN
NH
H
O
P O
O
H
H
H
N
O
3
O
N
S
O
O
O
O
NH2
c
O
H
O
H2N P O
O
N
P O
O
O
O
N
d; c
NH
H
O
O
O
HN
N
O
4
O
O
H
S
H
NH2
HN
O
e; c
O
H2N
2
O
O
NH
O
O
O
P O
O
H
H
O
O
N
HN
N
O
5
O
N
H
H
O
S
H
NH2
Scheme 1. Synthesis of ABPs with the diphenylphosphonate reactive group. Reagents and conditions: (a) HOAC as solvent, microwave, 150 °C,
5–10 min or oil bath, 70 °C, 1–3 h; (b) hydrazine; Boc2O; H2, Pd–C; (c) NHS-(PEG)4-biotin, triethylamine; then TFA; (d) Cbz-Pro-OH, EDCI,
HOBt; H2, Pd–C; (e) Cbz-Asn(Trt)-OH, EDCI, HOBt; H2, Pd–C.

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Z. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2882–2885
enzymes were serine hydrolases that were not proteases.
In addition, several identified serine hydrolases were
abundant housekeeping enzymes such as acyl-transfer-
ase, lysophospholipase, and acyl-CoA thioester hydro-
lase. In sharp contrast, the trypsin-family-selective
probe 3 selectively labeled b-tryptase (Fig. 3, right pan-
el). Further analysis of the labeling with probe 1 re-
vealed that acyl-transferase and b-tryptase co-migrate
on the gel, thus making it difficult to distinguish activi-
ty-profiles for these two enzymes in HMC-1 cells. Since
no labeling of acyl-transferase was observed with probe
3, b-tryptase activity could be directly evaluated in the
mast cell proteome without a need for any further
purification steps. This exemplifies the utility of prote-
ase-selective probes for profiling trypsin-like proteases
within a complex proteome.
Plasmin
β-Tryptase
Chymotrypsin
BC Esterase
Trypsin
Thrombin
Figure 2. Labeling of recombinant enzymes with probes Bio-FP 1,
Bio-PK-DPP 4, and Bio-NK-DPP 5.
like enzymes tested. Interestingly, this probe was even
more potent in labeling thrombin than probe 1 even
though it has a much less reactive functional group.
Probe 5 labeled three of the four trypsin-like serine pro-
teases but not thrombin, with a clear preference for b-
tryptase. Together, these results suggest that the incor-
poration of a P2 element enhances both the selectivity
and potency of the probes for trypsin-like enzymes.
HMC-1 cells are immature cells that do not fully reflect
properties of mature mast cells. A prominent feature of
mast cell is the presence of granules that store pre-
formed mediators including proteases such as tryptases.
Upon activation, the content of the granules is released
into the extracellular milieu to promote local wound
healing and inflammatory responses. We characterized
serine hydrolase and protease activities in more physio-
logically relevant, mature mast cells that were derived
from CD34+ hematopoietic progenitors. These cells
were stimulated to degranulate via activation of the high
affinity receptor for IgE (FceR) (Fig. 4). Following acti-
vation, intact cells were incubated with probes for 1 h,
conditioned medium was collected, and labeled enzymes
were purified and identified. Probe 1 yielded a complex
labeling pattern regardless of the mast cell activation
state (Fig. 4, lanes 1 and 2). The tryptase-selective probe
5 predominantly labeled b-tryptase (Fig. 4, lanes 3 and
4). Notably, when the general trypsin-family probe 4
was used for labeling, both tryptase and cathepsin G
were labeled (Fig. 4, lanes 5 and 6). The observed reac-
tivity of probe 4 is consistent with the known substrate
specificity of these two enzymes, with tryptase exhibiting
trypsin-like activity and cathepsin G having both tryptic
and chymotryptic activities. Labeling with probes 4 and
5 showed clear up-regulation in the activity of these en-
zymes following IgE stimulation (Fig. 4, lane 3 vs lane 4
and lane 5 vs lane 6). This demonstrates that probes 4
Next we determined whether the probes can selectively
label trypsin-like serine proteases in a complex prote-
ome. Initially, the labeling pattern of the serine protease
probe 3 was compared to that of the general serine
hydrolase probe 1. Activity-based profiling was per-
formed using the human mast cell line HMC-1. Mast
cells are inflammatory cells that provide protection
against parasitic infections but are also known to play
a major role in allergic responses and autoimmune dis-
eases.14 Intact HMC-1 cells were incubated for 1 h with
probe 1 or probe 3. Cell lysates were then analyzed by
SDS–PAGE followed by Western blotting using strepta-
vidin-HRP detection reagent (Fig. 3). Multiple active
mast cell serine hydrolases were labeled by probe 1
(Fig. 3, left panel). Pretreatment with 4-(2-aminoeth-
yl)-benzenesulfonyl fluoride (AEBSF), a general serine
hydrolase inhibitor, blocked the labeling of several en-
zymes. Affinity purification of probe 1 modified enzymes
followed by tryptic digestion and analysis by mass spec-
trometry, resulted in identification of 15 membrane-
bound and secreted enzymes.15 The majority of these
Bio-FP Bio-NK Bio-PK
1
5
4
Bio-FP
Bio-K-DPP
1
3
IgE treatment
- + - + - +
kDa
- +
- +
AEBSF
prolyl endopeptidase
64
51
DPP7 +prolyl carboxypeptidase
Acyl-CoA thioester hydrolase
39
28
β Tryptase
Cathepsin G
β-tryptase+ acyl transferase
1
2
3
4
5
6
RA inducible serine carboxypeptidase
lysophospholipase
Figure 4. Selective activity-based labeling of mast cell trypsin-like
serine proteases by DPP-based ABPs. Mature human mast cells
derived from CD34+ hematopoietic stem cells were induced for
degranulation with IgE followed by anti-IgE treatment (lanes 2, 4,
and 6).
Figure 3. Selective labeling of b-tryptase in HMC-1 cell lysates by Bio-
K-DPP probe 3, with the absence (À) and presence (+) of AEBSF
pretreatment prior to probes addition.

Z. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2882–2885
2885
8. Ossovskaya, V. S.; Bunnett, N. W. Physiol. Rev. 2004, 84,
579; Pawlinski, R.; Mackman, N. Crit. Care Med. 2004,
32, S293.
and 5 can be used to study the cellular b-tryptase’s
activity upon biological stimulation.
9. Internal data; see also Ni, L.-M.; Powers, J. C. Bioorg.
Med. Chem. 1998, 6, 1767.
10. Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E.
Chem. Rev. 2002, 102, 4639.
11. Hawthorne, S.; Hamilton, R.; Walker, B. J.; Walker, B.
Anal. Biochem. 2004, 326, 273.
In summary, we have demonstrated the design, synthesis,
and application of selective trypsin-family protease-selec-
tive probes 3, 4, and 5. These probes are capable of specif-
ically labeling trypsin-like serine proteases either in their
pure forms or as components of complex proteomes.
Thus, these probes represent powerful biochemical tools
for monitoring the activity of proteases in their natural
environment. Attachment of other reporter groups such
as fluorophores may also allow imaging of protease activ-
ity in situ as recently described for cysteine proteases.16
Finally, probes that target other sub-families of serine
proteases can be designed using a similar approach.
Increasing the number and type of chemical proteomics
probes targeting serine proteases will significantly en-
hance our ability to understand the activity, regulation,
and function of these important enzymes.
12. Harris, J. L.; Niles, A.; Burdick, K.; Maffitt, M.; Backes,
B. J.; Ellman, J. A.; Kuntz, I.; Haak-Frendscho, M.;
Craik, C. S. J. Biol. Chem. 2001, 276, 34941; Jackson, D.
S.; Fraser, S. A.; Ni, L. M.; Kam, C. M.; Winkler, U.;
Johnson, D. A.; Froelich, C. J.; Hudig, D.; Powers, J. C.
J. Med. Chem. 1998, 41, 2289.
13. Inhibition profiles for the serine proteases were generated
by incubating each enzyme in the presence of probes
(various concentrations) or 10% DMSO (vehicle control)
for 30 min in 96-well clear polystyrene plates at room
temperature prior to the addition of substrate. Enzyme
activity was measured by monitoring the hydrolysis of the
synthetic substrate tosyl-Gly-Pro-Lys-para-nitroanilide at
405 nM over 5 min using a UV/MAX kinetic plate reader
(Molecular Devices). The apparent inhibition constants
[Ki(app)] were calculated from the progress curves using the
software package Batch Ki (BioKin, Ltd). In each case, the
substrate concentration was at or below the Km for the
enzyme.
References and notes
1. Saghatelian, A.; Cravatt, B. Nat. Chem. Biol. 2005, 1, 130.
2. Jeffery, D. A.; Bogyo, M. Curr. Opin. Biotechnol. 2003, 14,
87; Adam, G. C.; Cravett, B. F.; Sorensen, E. J. Nat.
Biotechnol. 2002, 20, 805.
3. Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 14694.
14. Benoist, C.; Mathis, D. Nature 2002, 420, 875.
15. The following hydrolases were identified by LC/MS/MS
analysis (Q-Star) (serine protease in bold). Secreted
4. Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.;
Bogyo, M. Chem. Biol. 2000, 7, 569.
5. Kumar, S.; Zhou, B.; Liang, F.; Wang, W.-Q.; Huang, Z.;
Zhang, Z.-Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
7943.
6. Liu, Y.; Shreder, K. R.; Gai, W.; Corral, S.; Ferris, D. K.;
Rosenblum, J. S. Chem. Biol. 2005, 12, 99; Yee, M.-C.;
Fas, S. C.; Stohlmeyer, M. M.; Wandless, T. J.; Cimprich,
K. A. J. Biol. Chem. 2005, 280, 29053; Cohen, M. S.;
Zhang, C.; Shokat, K.; Taunton, J. Science 2005, 308,
1318.
7. Greenbaum, D. C.; Baruch, A.; Grainger, M.; Bozdech,
Z.; Medzihradszky, K. F.; Engel, J.; DeRisi, J.; Holder, A.
A.; Bogyo, M. Science 2002, 298, 2002; Jessani, N.; Liu,
Y.; Humphrey, M.; Cravatt, B. F. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 10335.
serine hydrolases: dipeptidyl peptidase
7 (54 kDa);
esterase 10 (37 kDa); tryptase b1 (34 kDa); lysophos-
pholipase 2 (24 kDa); platelet-activating factor acetyl-
hydrolase (26 kDa). Membrane serine hydrolases:
oxidized protein hydrolase (82 kDa); prolyl endopepti-
dase (81 kDa); angiotensinase C/propylcarboxypeptidase
(56 kDa); retinoid-inducible serine carboxypeptidase
(51 kDa); KIAA1363/carboxy esterase (48 kDa); perox-
isomal acyl-coenzyme A thioesterhydrolase 2a (46 kDa);
aldolase A (39 kDa); protein FLJ11342/lipase (32 kDa);
similar to hypothetical protein 4833421E05Rik/lipase
(28kDa); OVCA2/tumor suppressor in ovarian cancer 2
(25 kDa).
16. Joyce, J. A.; Baruch, A.; Chehade, K.; Meyer-Morse, N.;
Giraudo, E.; Tsai, F. Y.; Greenbaum, D. C.; Hager, J. H.;
Bogyo, M.; Hanahan, D. Cancer Cell 2004, 5, 443.