Activity-based protein profiling reagents for protein arginine deiminase 4 (PAD4): Synthesis and in vitro evaluation of a fluorescently labeled probe

Article abstract of DOI:10.1021/ja0656907

Protein arginine deiminase 4 (PAD4), which catalyzes the post-translational conversion of peptidyl arginine to peptidyl citrulline, is widely regarded as one of the best new targets for the development of a novel rheumatoid arthritis therapeutic. In addition to its presumed role in this disease, PAD4 is also a calcium-dependent histone deiminase that acts as a transcriptional co-repressor. Herein we describe the design, synthesis, and in vitro evaluation of two fluorescently labeled activity-based protein profiling (ABPP) reagents that specifically and irreversibly modify the active, that is, calcium-bound, form PAD4 with equal affinity to previously described small molecule chemical probes of PAD4 function. These fluorescently tagged ABPPs will be useful for identifying the conditions under which this enzyme is activated in vivo and may prove to be useful RA diagnostics. Copyright

Full text of DOI:10.1021/ja0656907

Published on Web 10/25/2006
Activity-Based Protein Profiling Reagents for Protein Arginine Deiminase 4
(PAD4): Synthesis and in vitro Evaluation of a Fluorescently Labeled Probe
Yuan Luo, Bryan Knuckley, Monica Bhatia, Perry J. Pellechia, and Paul R. Thompson*
Department of Chemistry & Biochemistry, UniVersity of South Carolina, 631 Sumter Street, Columbia, South
Carolina 29208
Received August 11, 2006; E-mail: thompson@mail.chem.sc.edu
Protein arginine deiminase 4 (PAD4) has attracted significant
attention in the past several years because of its presumed role as
a causative factor in the pathophysiology of rheumatoid arthritis
(
RA), a disease affecting approximately 1% of the world’s
1,2
population. Additionally, this enzyme has attracted the attention
of molecular biologists interested in deciphering the factors
controlling eukaryotic gene transcription because this enzyme
downregulates the expression of genes under the control of the
estrogen receptor,3 presumably by catalyzing the post-translational
modification of arginine residues in histones H2A, H3, and H4 to
form peptidyl citrulline and ammonia.
-5
Because the activity of PAD4 appears to be upregulated in RA,
we and others suggested that this calcium-regulated enzyme (PAD4
is essentially inactive in the absence of millimolar amounts of
6
calcium) represents a valid therapeutic target for RA and initiated
efforts to develop PAD4 inhibitors.1
,6-9
Those efforts have led to
the successful identification of several bioavailable haloacetamidine-
based lead compounds, including F-amidine (1), Cl-amidine (2),
and 2-chloroacetamidine, that act as mechanism-based inactivators
7
-9
of PAD4 (Figure 1).
These compounds inactivate PAD4 by
covalent modification of Cys645,7 a key catalytic residue, through
one of two potential mechanisms (Figure 1)sCys645 facilitates the
deimination of arginine residues by forming a proposed thiouronium
intermediate that is ultimately hydrolyzed to form citrulline.
-9
-
1
-1 8
F-Amidine (kinact/K
I
of 3000 M min ) and Cl-amidine (kinact
/
Figure 1. Structures of inactivators and proposed mechanism of inactiva-
tion. (A) Structures of BAA, a PAD4 substrate, and the PAD4 inactivators
F- and Cl-amidine. (B) Proposed mechanisms of inactivation. (C) Fluores-
cently labeled PAD4 inactivators.
-1
-1 7
K
I
of 13 000 M min ) are the most potent PAD4 inhibitors to be
described to date and are significantly more potent than 2-chloroacet-
-1
-1 9
amidine (kinact/K ) 35 M min ). The increased potency is likely
I
due to the binding energy gained by the addition of the benzoylated
ornithine moiety that targets these inhibitors to the PAD4 active site.
In addition to their high potency and ability to irreversibly modify
PAD4, the fact that F- and Cl-amidine selectively modify the active,
that is, calcium-bound, form of PAD47 led us to consider that
these compounds could be adapted for use as activity-based protein
profiling reagents (ABPPs; reviewed in refs 10 and 11). Herein, we
describe the design, synthesis, and evaluation of two fluorescently
tagged PAD4 ABPPs, rhodamine-tagged F-amidine (RFA; 3) and
rhodamine-tagged Cl-amidine (RCA; 4) (Figure 1), that preferen-
tially label the calcium-bound, that is, active, form of the enzyme.
During the design of these fluorescently tagged ABPPs, we focused
on using the copper(I)-catalyzed azide alkyne [3 + 2] cycloaddition
reaction12 to generate a triazole linker because this chemistry affords
a level of versatility that is not provided by other linkers, that is, the
fluorescent tag can be added in a bio-orthogonal manner either be-
fore or after the inactivator has undergone reaction with the protein
of interest. Note that, while we only report our efforts to develop flu-
orescently tagged ABPPs in this communication, it will be trivial to
adapt our methodologies to perform the couplings post-inactivation.
The synthesis of RFA and RCA, which is described in detail in
the Supporting Information, utilized a solid-phase synthetic meth-
,8
Figure 2. Representative IC50 data for 4 determined with and without
preincubation with calcium.
odology that involved the on-resin coupling of an ethyl haloace-
timidate hydrochloride to N-R-4-azidobenzoyl ornithine (Scheme
S1). Subsequently, this compound was cleaved from the resin and
13
coupled to a previously described rhodamine-alkyne construct
via the copper(I)-catalyzed azide alkyne [3 + 2] cycloaddition
reaction and then purified by reverse phase HPLC.
To evaluate the inhibitory properties of RFA and RCA, IC ’s were
50
8
determined using previously established methods. Briefly, the com-
pounds were preincubated with PAD4 in the absence or presence
of saturating amounts of calcium (10 mM final) for 15 min prior
to assaying (Figure 2). The IC ’s of RFA and RCA are 23.7 (
50
4.1 and 7.4 ( 0.8 µM, respectively, when preincubated with cal-
cium, remarkably consistent with those obtained for F-amidine (21.6
14468
9
J. AM. CHEM. SOC. 2006, 128, 14468-14469
10.1021/ja0656907 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. In vitro labeling of PAD4 with RFA and RCA. The ratio of
inactivator to protein is indicated at the top of the gel. The coomassie stained
gels are shown to confirm equal loading of PAD4 in each lane. The numbers
refer to the ratio [RXA]/[PAD4].
(
2.1 µM) and Cl-amidine (5.9 ( 0.3 µM),7,8 thereby indicating
that the reporter tag does not influence the interaction between these
compounds and PAD4. Also noteworthy is the fact that the IC50’s
are significantly higher for both RFA (>76 µM) and RCA (>50
µM) when calcium is omitted from the preincubation step. This
result is significant because it is consistent with the modification
of an active-site residue;7,8 upon calcium binding, active-site
residues are moved into positions that are competent for catalysis.14
Figure 4. In vitro labeling of E. coli cell lysates with RFA and RCA. The
lysates were prepared from bacteria overexpressing either wild-type or the
C645S mutant. The coomassie stained gels (bottom) are shown to confirm
equal loading of protein in each lane.
to this enzyme during its in vivo activation, potentially offering an
explanation for why nonphysiological concentrations of calcium
are required for in vitro enzyme activity. Furthermore, these probes
may prove to be useful RA diagnostics and will undoubtedly aid
the identification of nonspecific targets of these compounds whose
identities will aid the successful design and synthesis of PAD4-
specific inhibitors. Finally, the fact that RFA is highly selective
for the active form of PAD4 suggests that fluoroacetamidine-
containing compounds will have better pharmacological charac-
teristics than chloroacetamidine-containing compounds because they
are likely to be more selective for PAD enzymes and would
therefore be expected to have fewer off-target effects.
Having established that RFA and RCA inhibit PAD4 with
comparable potency to their parent compounds, we then evaluated
their ability to act as ABPPs by incubating them with purified
recombinant PAD4 in the absence and presence of calcium. The
reaction components were then separated on a 12% SDS-PAGE
gel and fluorescently labeled proteins visualized (Figure 3). The
results of these studies clearly demonstrate that RFA and RCA
preferentially modify the active form of the enzyme, that is, calcium-
bound, PAD4; although at higher concentrations of RCA, PAD4
is modified in the absence of calcium by this compound. In contrast,
a C645S mutant, which lacks the active-site nucleophile and is
essentially inactive, was not modified by RFA and only minimally
modified by RCA (Figure S1), consistent with crystallographic and
mass spectrometry experiments demonstrating that this is the only
Acknowledgment. This work was supported by start up funds
provided to PRT from the USC Research Foundation.
Supporting Information Available: Scheme S1, Figure S1,
materials, methods, and spectral characterizations. This material is
available free of charge via the Internet at http://pubs.acs.org.
residue modified in PAD4 by F-amidine.7,9 MALDI-MS experi-
ments on full-length PAD4 treated with RFA and RCA showed
respective mass shifts of 937 and 956 Da relative to control samples,
within instrument error of the expected 940 Da mass shift, which
is consistent with the preferential modification of a single site on
the enzyme. Limit of detection assays demonstrated that g125 ng
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