D-Amino acid-based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy

Article abstract of DOI:10.1021/ml300288d

The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Because d-Amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of d-Amino acid analogues of our pan-PAD inhibitor Cl-Amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-Amidine and d-o-F-Amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-Amidine were moderately improved over those of l-Cl-Amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.

Full text of DOI:10.1021/ml300288d

Letter
pubs.acs.org/acsmedchemlett
D‑Amino Acid-Based Protein Arginine Deiminase Inhibitors:
Synthesis, Pharmacokinetics, and in Cellulo Efficacy
Kevin L. Bicker,† Lynne Anguish,‡ Alexander A. Chumanevich,§ Michael D. Cameron,† Xiangli Cui,§
Erin Witalison,§ Venkataraman Subramanian,† Xuesen Zhang,‡ Alena P. Chumanevich,§ Lorne J. Hofseth,§
Scott A. Coonrod,‡ and Paul R. Thompson*,†
†Department of Chemistry, The Scripps Research Institute, Scripps Florida, 120 Scripps Way, Jupiter, Florida 33458, United States
‡Baker Institute for Animal Health, College of Veterinary Medicine, Department of Molecular Biology and Genetics, and Proteomics
and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York 14853, United States
§Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina,
Columbia, South Carolina 29201, United States
S
* Supporting Information
ABSTRACT: The protein arginine deiminases (PADs) are
known to play a crucial role in the onset and progression of
multiple inflammatory diseases, including rheumatoid arthritis,
inflammatory bowel disease, and cancer. However, it is not
known how each of the five PAD isozymes contributes to
disease pathogenesis. As such, potent, selective, and bioavail-
able PAD inhibitors will be useful chemical probes to elucidate
the specific roles of each isozyme. Because D-amino amino
acids often possess enhanced in cellulo stability, and perhaps
unique selectivities, we synthesized a series of D-amino acid
analogues of our pan-PAD inhibitor Cl-amidine, hypothesizing
that this change would provide inhibitors with enhanced
pharmacokinetic properties. Herein, we demonstrate that D-Cl-amidine and D-o-F-amidine are potent and highly selective
inhibitors of PAD1. The pharmacokinetic properties of D-Cl-amidine were moderately improved over those of L-Cl-amidine, and
this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These
inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the
PAD isozymes.
KEYWORDS: D-amino acid, protein arginine deiminases, in cellulo efficacy, pharmacokinetic properties
generation PAD inhibitors, Cl-amidine has proven most
roteins undergo a host of post-translational modifications,
Pincluding acetylation, phosphorylation, methylation, ubiq- useful.13 This pan-PAD inhibitor reduces disease severity in
the collagen-induced arthritis (CIA) model of RA,16 as well as
in a mouse model of ulcerative colitis.17 Furthermore, Cl-
amidine was used to confirm that PAD4 plays an essential role
in neutrophil extracellular trap (NET) formation; Cl-amidine
prevents histone citrullination, chromatin decondensation, and
subsequent NET formation.18,19 Further testing and character-
ization of these inhibitors led to the identification of a PAD3
selective inhibitor, Cl-4-amidine.11 With the goal of producing
more potent inhibitors, second generation Cl-amidine ana-
logues were synthesized, using the principles of rational design
and structure−activity relationships. This work determined that
incorporation of an ortho-carboxylate on the benzoyl moiety of
Cl-amidine dramatically increased the potency (by up to 65-
fold) of these inhibitors.9
uitination, and citrullination.1 Catalyzed by the protein arginine
deiminase (PAD) family of enzymes, citrullination or
deimination is the hydrolysis of peptidyl-arginine to peptidyl-
citrulline. There are five PAD isozymes in humans and other
mammals (PAD 1−4 and 6), and all five PADs are calcium-
dependent enzymes.2,3 The PADs are distributed throughout
several tissue types, and while all of the isozymes are
cytoplasmic, only PAD2 and PAD4 are expressed in both the
cytoplasm and the nucleus,2−7 where they alter gene tran-
scription via their ability to citrullinate histones H3 and H4.8
The PADs are particularly intriguing given their role in a host
of autoimmune and inflammatory diseases, including rheuma-
toid arthritis (RA), ulcerative colitis, cancer, and Alzheimer's
disease, among others.2,3
We and others have developed a number of halo-
acetamidine-based PAD inhibitors;9−15 the halo-acetamidine
warhead covalently modifies the active site cysteine in the
PADs to irreversibly inhibit enzyme activity. Of the first
Received: September 15, 2012
Accepted: October 23, 2012
Published: October 26, 2012
© 2012 American Chemical Society
1081
dx.doi.org/10.1021/ml300288d | ACS Med. Chem. Lett. 2012, 3, 1081−1085

ACS Medicinal Chemistry Letters
Letter
Although these compounds are potent PAD inhibitors, their
L-amino acid-based structures leave them potentially susceptible
to proteolysis.20 For this reason, they have relatively short in
vivo half-lives and lose cellular potency quite rapidly. Given the
precedent of utilizing D-amino acids in peptide-based
compounds to improve stability,20−23 we hypothesized that
next generation Cl-amidine analogues incorporating D-amino
acids would produce PAD inhibitors with improved stability
and unique selectivities. To this end, a series of eight D-amino
acid-based Cl-amidine analogues were synthesized and tested
against PADs 1−4 to characterize their potency and selectivity.
The best inhibitors from this series are D-Cl-amidine and D-o-F-
amidine, as they exhibit enhanced selectivity for PAD1 over
PADs 2−4 and improved in cellulo efficacy in MDA-MB-231
cells and show a moderate increase in stability. Herein, we also
report for Cl-amidine, D-Cl-amidine, and D-o-F-amidine the
maximum tolerable dose (MTD) and the results of in cellulo
and pharmacokinetic studies.
this analysis, D-Cl-amidine and D-o-F-amidine are the most
potent and selective compounds from this initial series. For
example, these compounds preferentially inhibit PAD1, by
≥10-fold, with kinact/KI values of 13500 and 12100 M−1 min−1,
respectively (Figure 2). Given the altered stereochemistry and
preference for PAD1, we used dialysis experiments and
competitive activity-based protein profiling (ABBP) to confirm
that they irreversibly inhibit this isozyme (Figures S1 and S2 in
the Supporting Information, respectively). No enzymatic
activity was observed postdialysis, and the compounds
effectively competed labeling by rhodamine-conjugated Cl-
amidine (RCA), an active site-directed ABPP. It should also be
noted that D-Cl-amidine and D-o-F-amidine exhibited sigmoidal
inhibition curves when the inhibition experiments were
performed with PAD1 (Figure S3 in the Supporting
Information). Although this type of inhibition has not been
observed for any other previously reported PAD inhibitor, the
PADs are known to dimerize,24 and it is possible that these
compounds induce intrasubunit cooperativity, via a conforma-
tional change upon inhibition of one subunit of the dimer, that
makes the second subunit more susceptible to inhibition.
Further studies are required to confirm this hypothesis.
Given that F-amidine, Cl-amidine, o-F-amidine, and o-Cl-
amidine have been the most useful PAD inhibitors,9,13 we
initially synthesized their direct derivatives using D-ornithine, as
opposed to L-ornithine, as the starting material (Figure 1 and
Although D-Cl-amidine and D-o-F-amidine are generally less
potent than their L-counterparts, the D-series compounds
exhibit enhanced PAD1 selectivity versus their L-amino acid
counterparts (Figure 2). For example, L-Cl-amidine is only 3-
fold selective for PAD1 over PAD4, whereas D-Cl-amidine
shows roughly 10-fold selectivity between these two isozymes.
The increased selectivity for PAD1 is not restricted to PAD4, as
the inversion of stereochemistry also boosts selectivity against
PADs 2 and 3 to 50- and 200-fold, respectively. Similar
selectivity trends are observed with D-F-amidine, albeit with
reduced potency, consistent with the data for L-F-amidine; the
kinact/KI for PAD1 is only 1220 M−1 min−1. Interestingly, D-o-
Cl-amidine and D-o-F-amidine displayed the opposite potency
trends, with D-o-Cl-amidine showing limited inhibition toward
any of the PADs, whereas D-o-F-amidine was the second most
potent PAD1 inhibitor tested among the D-analogues. Even
though chloride is a better leaving group than fluoride, this
result is not unprecedented (e.g., Thr-Asp-F-amidine shows
higher potency and selectivity than Thr-Asp-Cl-amidine)10 and
likely reflects the steric constraints of the active site favoring the
smaller leaving group, as a result of the inversion of
stereochemistry. More significantly, D-o-F-amidine preferen-
tially inhibits PAD1 by 35-, 3900-, and 225-fold versus PADs 2,
3, and 4, respectively, making it the most selective PAD1
inhibitor ever discovered (Figure 2).
Figure 1. Structures of D-series compounds.
Scheme S1 in the Supporting Information). These compounds
are designated D-F-amidine, D-Cl-amidine, D-o-F-amidine, and
D-o-Cl-amidine; the “D” indicates the altered stereochemistry.
To evaluate their in vitro potency, kinact/KI values were
determined for each compound with all four active PAD
isozymes, that is, PADs 1−4 (Figure 2). Note that PAD6 was
not tested because it shows no activity in vitro. On the basis of
Previous studies demonstrated that the linker length between
the peptide backbone and the halo-acetamidine warhead lends
inhibitors unique selectivity.11 As such, the D-amino acid
versions of Cl2-amidine (two methylene linker) and Cl4-
amidine (four methylene linker) were also synthesized (Figure
1). However, these compounds were quite poor inhibitors
(kinact/KI < 150 M−1 min−1) for all of the PADs tested (Figure
2). This stresses the importance of linker length in the design of
PAD inhibitors, that is, the distance between the halo-
acetamidine warhead, which reacts with the active site cysteine,
and the peptide backbone is critical for potent inhibition. Given
that the carboxylate moiety of L-o-F-amidine and L-o-Cl-
amidine interacts favorably with Trp347 in PAD49 and that
inversion of the stereocenter for the D-amino acid inhibitors
may flip the orientation of the inhibitor in the enzyme active
site, we hypothesized that a negative charge on the C terminus
Figure 2. Inhibitor selectivity. kinact/KI values, determined with purified
enzyme, are displayed graphically and in table format (inset) for the D-
series compounds, as well as certain L-amino acid analogues, indicating
potency and selectivity for PADs 1−4.
1082
dx.doi.org/10.1021/ml300288d | ACS Med. Chem. Lett. 2012, 3, 1081−1085

ACS Medicinal Chemistry Letters
Letter
of the D-amino acid inhibitors may increase the potency of
these inhibitors. For this reason, D-Cl-amidine-OH and D-Cl-
amidine-Gly were synthesized (Figure 1). However, these
compounds were also poor PAD inhibitors (Figure 2; kinact/KI
< 230 M−1 min−1). Although this result could suggest that the
inhibitors do not bind to the enzyme in the opposite
orientation, relative to their L-amino acid counterparts,
structural studies will be necessary to confirm their true
binding modes. While D-Cl-amidine-OH turned out to be a
rather poor inhibitor for all of the PADs, it is interesting to note
that for PAD1 the amide isostere is 60-fold more potent. Thus,
if hydrolysis of the C-terminal amide occurs at an appreciable
amount during the initial breakdown of these compounds, the
hydrolysis of this bond in vivo would result in a significant loss
of potency, with consequent effects on in vivo efficacy. The fact
that L-Cl-amidine-OH is also a poor PAD inhibitor (Figure S4
in the Supporting Information) suggests that a similar
breakdown pathway would decrease the in vivo efficacy of Cl-
amidine. Given that core structure of L-Cl-amidine is similar to
benzoyl-arginine amide, a classical substrate for multiple
arginine directed proteases, we hypothesize that the C-terminal
amide may be proteolyzed in vivo.
Having identified D-Cl-amidine and D-o-F-amidine as the
most promising PAD1 inhibitors in this series, we examined the
pharmacokinetics and pharmodynamics of these two com-
pounds. L-Cl-amidine was used as a reference. Initially, we
determined the MTD to examine the physiological effects of
these compounds on mice when given orally. The total body
weight changes, as well as observable changes in physiology of
mice that received varying doses of these compounds, given per
os, are shown in Tables S1−S3 in the Supporting Information.
Many of the mice in these studies exhibited signs of toxicity
(e.g., soft and bloody stool) at higher concentrations of
inhibitor, with the most significant observable toxicity being in
group 6 of L-Cl-amidine, where mice were notably shaking,
anxious, hunching, and breathing heavily from 1 to 4 h after
treatment. Thereafter, such signs and symptoms dissipated, and
these mice developed diarrhea or soft stool that lasted 2−3
days. Mice receiving 100 mg/kg (group 5) and 150 mg/kg
(group 6) L-Cl-amidine had significant weight loss 4 days (96
h) after consuming L-Cl-amidine (Figure 3). However, mice
given D-Cl-amidine or D-o-F-amidine maintained healthy weight
gains, similar to those mice given no compound at all (Figure
3). Given the definition of the MTD (i.e., the highest dose of a
compound that does not cause weight loss or significant sign of
toxicity), we conclude from this study that the MTD of L-Cl-
amidine in mice is 75 mg/kg and the MTD of the two D-series
compounds tested is greater than 150 mg/kg, the highest dose
tested. Although unclear why compounds synthesized from D-
amino acids have higher MTD values, it is possible that
metabolism of L-Cl-amidine generates a toxic metabolite that is
not produced as rapidly for the D-series compounds. We note
that 100 mg/kg of L-Cl-amidine has been given to mice daily by
ip for up to 56 days in our previous studies with no observable
signs of toxicity.16,17
The efficacy of the two most potent inhibitors, D-Cl-amidine
and D-o-F-amidine, were next tested in MDA-MB-231 cells, a
triple negative breast cancer cell line. L-Cl-amidine was again
used as the reference compound. MDA-MB-231 cells were
chosen for these studies because these cells overexpress PAD1
(Figure S5 in the Supporting Information). All compounds
were tested at varying concentrations (i.e., 100, 200, and 400
μM), and various measures of drug efficacy were evaluated (i.e.,
percent cell viability, cell number, and caspase 3 activity; Figure
4) 96 h after administration. Both L-Cl-amidine and D-Cl-
amidine were effective in significantly decreasing cell viability at
concentrations of 200 and 400 μM (P value <0.01; Table S2 in
the Supporting Information). Similarly, both compounds
significantly decreased cell number at a concentration of 400
μM (P value <0.05; Table S2 in the Supporting Information).
Unfortunately, the most selective compound in vitro, D-o-F-
amidine, had little efficacy in cellulo; we observed no effect on
cell viability and only a small decrease in cell number at a
concentration of 400 μM (P value <0.05; Table S2 in the
Supporting Information). The negatively charged carboxylate
likely limits cellular uptake.
To elucidate whether these inhibitors were inducing
apoptosis and/or inhibiting proliferation, caspase 3 and Ki-67
activity was monitored in MDA-MB-231 cells. Increased
caspase 3 activity is a hallmark of apoptosis,25 whereas Ki-67
is required for maintaining cellular proliferation, and a decrease
in Ki-67, only seen during G0 phase, is indicative of cell cycle
arrest.26 All of the compounds tested (i.e., L-Cl-amidine, D-Cl-
amidine, and D-o-F-amidine) had no significant effect on Ki-67
levels (Figure S6 in the Supporting Information). Only
increased amounts of tunicamycin, a positive control of cell
cycle arrest, resulted in a decrease in Ki-67 levels. However,
both L-Cl-amidine and D-Cl-amidine increased caspase 3
activity, similarly, in a dose-dependent manner, with a
significant increase in activity at a concentration of 400 μM
(Figure 4C; P value <0.01; Table S1 in the Supporting
Information). Following the trend seen in cell number and
viability studies, D-o-F-amidine treatment did not result in an
increase in caspase 3 activity.
Taken together, these data indicate that while D-o-F-amidine
is modestly potent and highly selective in vitro, it has poor
efficacy in cellulo. The lack of in cellulo efficacy with this
compound is likely due to poor cellular uptake, which we
hypothesize is caused by the presence of the negatively charged
carboxylate and lack of hydrophobic character. The data also
indicate that although L-Cl-amidine and D-Cl-amidine have
modestly different in vitro potencies toward PAD1 (kinact/Ki =
37000 and 13500 M−1 s−1, respectively), these compounds have
relatively equal in cellulo potencies. The fact that D-Cl-amidine
Figure 3. MTD studies. D-Series compounds are better tolerated in
vivo than L-Cl-amidine in MTD studies. Graph of percent change in
body weight of mice (n = 10) given varying doses of L-Cl-amidine, D-
Cl-amidine, and D-o-F-amidine. A significant decrease (*P value <0.05)
in weight is seen at higher doses of L-Cl-amidine but not with the D-
compounds tested.
1083
dx.doi.org/10.1021/ml300288d | ACS Med. Chem. Lett. 2012, 3, 1081−1085

ACS Medicinal Chemistry Letters
Letter
Figure 5. Stability and pharmacokinetic studies. (A) The stability of D-
Cl-amidine in mouse hepatic microsomes is similar to Cl-amidine. (B)
Mean plasma levels of Cl-amidine and D-Cl-amidine in mice over time,
administered by iv and ip injection.
within 2 h. However, D-Cl-amidine, administered by iv at a dose
of 2.5 mg/kg, was still detected after 2 h in serum at a
concentration of ∼21 nM and at 4 h at ∼10 nM. L-Cl-amidine,
administered by ip at a dose of 10 mg/kg, was completely
degraded within 4 h. Similar to the results for iv, D-Cl-amidine
was still observed in the blood serum at a concentration of ∼10
nM at 4 h when administered by ip at a dose of 10 mg/kg.
These data indicate that D-Cl-amidine is significantly more
stable within a mouse model as compared to L-Cl-amidine. This
increase in stability is possibly due to decreased proteolysis of
the inverted stereocenter.
Taken together, these data represent a new class of PAD
inhibitors with increased selectivity for PAD1 and increased in
vivo stability and tolerance. Although found in the skin where it
plays an undefined role in the cornification of the skin, little else
is known regarding the normal and pathological roles of
PAD1.2 Thus, these PAD1 selective inhibitors will be important
tool compounds that can be used to gain insight into the
function of this isozyme. Herein, we have shown that of the
eight D-series compounds synthesized, D-Cl-amidine and D-o-F-
amidine, are moderately potent and highly selective for PAD1
in vitro. MTD studies indicate that these compounds are
tolerated better in animal models than L-Cl-amidine, perhaps
due to their inherent proteolytic stability. Furthermore, we
show that D-Cl-amidine, although less potent in vitro, is equally
potent in cellulo as compared to L-Cl-amidine and shows better
pharmacokinetics. These properties make D-amino acid-based
PAD inhibitors a viable option for improving upon future
classes of compounds. Current efforts are focused on expanding
on these findings to increase the half-life, bioavailability, and
cell permeability of both L- and D-amino acid-based PAD
inhibitors.
Figure 4. Cellular efficacy studies. L- and D-Cl-amidine show equal
potency against PAD1 overexpressing MDA-MB-231 cells, in
decreasing cell viability (A) and cell count (B). (C) Both L- and D-
Cl-amidine increase caspase 3 activity, indicating that inhibition of
PAD1 leads to an increase in apoptosis (*P value <0.05; **P value
<0.01). No significant effects are observed with D-o-F-amidine.
is more selective than L-Cl-amidine suggests that this
compound will be useful for studying in vivo PAD1 activity
because its use will minimize off target effects.
Because the stability of several L-amino acid-based inhibitors
has been improved by synthesizing D-amino acid based mimics,
we next compared the stability and pharmacokinetic properties
of L- and D-Cl-amidine. We focused on D-Cl-amidine because it
is the most potent in cellulo PAD1 inhibitor from the D-series
of compounds. Initially, the stability of L- and D-Cl-amidine was
tested in a murine hepatic microsome stability assay. This assay
utilizes liver microsomes, which possess many of the enzymes
responsible for drug metabolism in vivo, and are a common
initial predictor of drug clearance properties.27 The results from
this assay indicate that both L- and D-Cl-amidine have similar
half-lives (37 and 33 min, respectively) (Figure 5A), indicating
that inversion of the stereocenter did not improve the stability
of Cl-amidine in this assay. The pharmacokinetic properties of
both L- and D-Cl-amidine were then examined in mice by both
intravenous (iv) and intraperitoneal (ip) injection methods
(Figure 5B). These data indicate that L-Cl-amidine, when
administered iv at a dose of 10 mg/kg, is completely degraded
1084
dx.doi.org/10.1021/ml300288d | ACS Med. Chem. Lett. 2012, 3, 1081−1085

ACS Medicinal Chemistry Letters
Letter
Specificity and Kinetic Studies of PADs 1, 3, and 4 Identify Potent
and Selective Inhibitors of Protein Arginine Deiminase 3. Biochemistry
2010, 49 (23), 4852−4863.
(12) Knuckley, B.; Causey, C. P.; Pellechia, P. J.; Cook, P. F.;
Thompson, P. R. Haloacetamidine-Based Inactivators of Protein
Arginine Deiminase 4 (PAD4): Evidence that General Acid Catalysis
Promotes Efficient Inactivation. ChemBioChem 2010, 11 (2), 161−
165.
(13) Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y.-H.; Stallcup,
M. R.; Sato, M.; Thompson, P. R. Inhibitors and Inactivators of
Protein Arginine Deiminase 4: Functional and Structural Character-
ization. Biochemistry 2006, 45 (39), 11727−11736.
(14) Stone, E. M.; Schaller, T. H.; Bianchi, H.; Person, M. D.; Fast,
W. Inactivation of Two Diverse Enzymes in the Amidinotransferase
Superfamily by 2-Chloroacetamidine: Dimethylargininase and Pepti-
dylarginine Deiminase. Biochemistry 2005, 44 (42), 13744−13752.
(15) Wang, Y.; Li, P.; Wang, S.; Hu, J.; Chen, X. A.; Wu, J.; Fisher,
M.; Oshaben, K.; Zhao, N.; Gu, Y.; Wang, D.; Chen, G. Anticancer
PAD inhibitors regulate the autophagy flux and the mammalian target
of rapamycin complex 1 activity. J. Biol. Chem. 2012, DOI: 10.1074/
jbc.M112.375725.
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, experimental details, and supplementary
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: pthompso@scripps.edu.
Funding
Financial support for this work was provided by NIH Grants
GM079357 (P.R.T.), CA151304 (P.R.T. and L.J.H.), and TSRI.
Notes
■
The authors declare no competing financial interest.
ABBREVIATIONS
PAD, protein arginine deiminase; MTD, maximum tolerable
dose; iv, intravenous; ip, intraperitoneal
■
(16) Willis, V. C.; Gizinski, A. M.; Banda, N. K.; Causey, C. P.;
Knuckley, B.; Cordova, K. N.; Luo, Y.; Levitt, B.; Glogowska, M.;
Chandra, P.; Kulik, L.; Robinson, W. H.; Arend, W. P.; Thompson, P.
R.; Holers, V. M. N-α-Benzoyl-N5-(2-Chloro-1-Iminoethyl)-l-Orni-
thine Amide, a Protein Arginine Deiminase Inhibitor, Reduces the
Severity of Murine Collagen-Induced Arthritis. J. Immunol. 2011, 186
(7), 4396−4404.
(17) Chumanevich, A. A.; Causey, C. P.; Knuckley, B. A.; Jones, J. E.;
Poudyal, D.; Chumanevich, A. P.; Davis, T.; Matesic, L. E.; Thompson,
P. R.; Hofseth, L. J. Suppression of colitis in mice by Cl-amidine: A
novel peptidylarginine deiminase inhibitor. Am. J. Physiol. Gastrointest.
Liver Physiol. 2011, 300 (6), G929−G938.
(18) Wang, Y.; Li, M.; Stadler, S.; Correll, S.; Li, P.; Wang, D.;
Hayama, R.; Leonelli, L.; Han, H.; Grigoryev, S. A.; Allis, C. D.;
Coonrod, S. A. Histone hypercitrullination mediates chromatin
decondensation and neutrophil extracellular trap formation. J. Cell
Biol. 2009, 184 (2), 205−213.
(19) Hemmers, S.; Teijaro, J. R.; Arandjelovic, S.; Mowen, K. A.
PAD4-mediated neutrophil extracellular trap formation is not required
for immunity against influenza infection. PLoS One 2011, 6 (7),
e22043.
REFERENCES
■
(1) Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J. Protein
Posttranslational Modifications: The Chemistry of Proteome
Diversifications. Angew. Chem., Int. Ed. 2005, 44 (45), 7342−7372.
(2) Jones, J. E.; Causey, C. P.; Knuckley, B.; Slack-Noyes, J. L.;
Thompson, P. R. Protein arginine deiminase 4 (PAD4): Current
understanding and future therapeutic potential. Curr. Opin. Drug
Discovery Dev. 2009, 12 (5), 616−627.
(3) Vossenaar, E. R.; Zendman, A. J. W.; van Venrooij, W. J.; Pruijn,
G. J. M. PAD, a growing family of citrullinating enzymes: genes,
features and involvement in disease. BioEssays 2003, 25 (11), 1106−
1118.
(4) Zhang, X.; Bolt, M.; Guertin, M. J.; Chen, W.; Zhang, S.;
Cherrington, B. D.; Slade, D. J.; Dreyton, C. J.; Subramanian, V.;
Bicker, K. L.; Thompson, P. R.; Mancini, M. A.; Lis, J. T.; Coonrod, S.
A. Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26
citrullination facilitates estrogen receptor alpha target gene activation.
Proc. Natl. Acad. Sci. U.S.A. 2012, DOI: 10.1073/pnas.1203280109.
(5) Cherrington, B. D.; Zhang, X.; McElwee, J. L.; Morency, E.;
Anguish, L. J.; Coonrod, S. A. Potential Role for PAD2 in Gene
Regulation in Breast Cancer Cells. PLoS One 2012, 7 (7), e41242.
(6) Nakashima, K.; Hagiwara, T.; Ishigami, A.; Nagata, S.; Asaga, H.;
Kuramoto, M.; Senshu, T.; Yamada, M. Molecular characterization of
peptidylarginine deiminase in HL-60 cells induced by retinoic acid and
1alpha,25-dihydroxyvitamin D(3). J. Biol. Chem. 1999, 274 (39),
27786−27792.
(7) Cherrington, B. D.; Morency, E.; Struble, A. M.; Coonrod, S. A.;
Wakshlag, J. J. Potential Role for Peptidylarginine Deiminase 2
(PAD2) in Citrullination of Canine Mammary Epithelial Cell
Histones. PLoS ONE 2010, 5 (7), e11768.
(8) Thompson, P. R.; Fast, W. Histone Citrullination by Protein
Arginine Deiminase: Is Arginine Methylation a Green Light or a
Roadblock? ACS Chem. Biol. 2006, 1 (7), 433−441.
(9) Causey, C. P.; Jones, J. E.; Slack, J. L.; Kamei, D.; Jones, L. E.;
Subramanian, V.; Knuckley, B.; Ebrahimi, P.; Chumanevich, A. A.;
Luo, Y.; Hashimoto, H.; Sato, M.; Hofseth, L. J.; Thompson, P. R. The
Development of N-α-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoeth-
yl)-l-ornithine Amide (o-F-amidine) and N-α-(2-Carboxyl)benzoyl-
N5-(2-chloro-1-iminoethyl)-l-ornithine Amide (o-Cl-amidine) As
Second Generation Protein Arginine Deiminase (PAD) Inhibitors. J.
Med. Chem. 2011, 54 (19), 6919−6935.
(10) Jones, J. E.; Slack, J. L.; Fang, P.; Zhang, X.; Subramanian, V.;
Causey, C. P.; Coonrod, S. A.; Guo, M.; Thompson, P. R. Synthesis
and Screening of a Haloacetamidine Containing Library To Identify
PAD4 Selective Inhibitors. ACS Chem. Biol. 2011, 7 (1), 160−165.
(11) Knuckley, B.; Causey, C. P.; Jones, J. E.; Bhatia, M.; Dreyton, C.
J.; Osborne, T. C.; Takahara, H.; Thompson, P. R. Substrate
(20) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J.
M.; Moos, W. H. Comparison of the proteolytic susceptibilities of
homologous L-amino acid, D-amino acid, and N-substituted glycine
peptide and peptoid oligomers. Drug Dev. Res. 1995, 35 (1), 20−32.
́
(21) Tugyi, R.; Uray, K.; Ivan, D.; Fellinger, E.; Perkins, A.; Hudecz,
F. Partial d-amino acid substitution: Improved enzymatic stability and
preserved Ab recognition of a MUC2 epitope peptide. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102 (2), 413−418.
(22) Werle, M.; Bernkop-Schnurch, A. Strategies to improve plasma
̈
half life time of peptide and protein drugs. Amino Acids 2006, 30 (4),
351−367.
(23) Adessi, C.; Soto, C. Converting a Peptide into a Drug: Strategies
to Improve Stability and Bioavailability. Curr. Med. Chem. 2002, 9 (9),
963−978.
(24) Liu, Y.-L.; Chiang, Y.-H.; Liu, G.-Y.; Hung, H.-C. Functional
Role of Dimerization of Human Peptidylarginine Deiminase 4
(PAD4). PLoS ONE 2011, 6 (6), e21314.
(25) Alnemri, E. S.; Livingston, D. J.; Nicholson, D. W.; Salvesen, G.;
Thornberry, N. A.; Wong, W. W.; Yuan, J. Human ICE/CED-3
Protease Nomenclature. Cell 1996, 87 (2), 171.
(26) Scholzen, T.; Gerdes, J. The Ki-67 protein: From the known
and the unknown. J. Cell. Physiol. 2000, 182 (3), 311−322.
(27) Baranczewski, P.; Stanczak, A.; Sundberg, K.; Svensson, R.;
Wallin, A.; Jansson, J.; Garberg, P.; Postlind, H. Introduction to in vitro
estimation of metabolic stability and drug interactions of new chemical
entities in drug discovery and development. Pharmacol. Rep. 2006, 58
(3), 453−472.
1085
dx.doi.org/10.1021/ml300288d | ACS Med. Chem. Lett. 2012, 3, 1081−1085