Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00215

The protein arginine deiminases (PADs) catalyze the post-translational deimination of arginine side chains. Multiple PAD isozymes have been characterized, and abnormal PAD activity has been associated with several human disease states. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor and has been implicated in the neurodegenerative response to spinal cord injury. Here, we describe the design, synthesis, and evaluation of conformationally constrained versions of the potent and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series was also demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells.

Full text of DOI:10.1021/acsmedchemlett.6b00215

Letter
pubs.acs.org/acsmedchemlett
Cellular Activity of New Small Molecule Protein Arginine Deiminase 3
(PAD3) Inhibitors
Haya Jamali, Hasan A. Khan,† Caroline C. Tjin, and Jonathan A. Ellman*
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
S
* Supporting Information
ABSTRACT: The protein arginine deiminases (PADs) catalyze the post-translational
deimination of arginine side chains. Multiple PAD isozymes have been characterized, and
abnormal PAD activity has been associated with several human disease states. PAD3 has
been characterized as a modulator of cell growth via apoptosis inducing factor and has been
implicated in the neurodegenerative response to spinal cord injury. Here, we describe the
design, synthesis, and evaluation of conformationally constrained versions of the potent
and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series
was also demonstrated for the first time by rescue of thapsigargin-induced cell death in
PAD3-expressing HEK293T cells.
KEYWORDS: Protein arginine deiminases, small molecule inhibitor, apoptosis inducing factor, spinal cord injury
rotein arginine deminiases (PADs) catalyze the calcium-
Pdependent hydrolytic conversion of arginine residues to
citrulline side chains (Figure 1). Several PAD isozymes have
been identified and characterized.1−4 In particular, PADs 1, 2, 3,
and 4 have been shown to be catalytically active. PAD substrate
side chains contain potential hydrogen bond donors and are
also protonated at physiological pH, priming them for
interactions with negatively charged groups such as nucleic
acids.5,6 Due to the net loss of charge inherent in deimination
of arginine side chains, the post-translational modification
catalyzed by PADs may have dramatic effects on cell signaling.
Though the isozymes collectively possess a high degree of
sequence identity (50−55%),1,6 tissue-specific localization of
each isozyme in humans has been observed.3,7 Significantly,
abnormal activity of PADs has been demonstrated to play a role
in multiple human disease states.3,8
Figure 2. Previously described PAD inhibitors.
a
Scheme 1. Urea and Carbamate Inhibitor Synthesis
PAD3 in particular has been characterized as a modulator of
cell growth via AIF (apoptosis inducing factor) mediated
apoptosis. Citrullination by PAD3 of AIF in hNSCs is required
for its translocation to the nucleus to induce cell death,
identifying PAD3 as an upstream regulator of Ca2+ dependent
cell death.9 Notably, PAD3 activity has also been implicated in
the neurodegenerative response to spinal cord injury10 as well
as the citrullination of proteins during lactation.11
Cl-amidine 1, which irreversibly alkylates the active site
cysteine of PADs as confirmed by X-ray structure, was
developed by Thompson and co-workers and is the most
extensively evaluated small molecule PAD inhibitor in cells and
a
Reagents and conditions: (a) triphosgene, Et3N, CH2Cl2, 0 °C, 30
min followed by NH2(CH2)2NHBoc for 4 and HO(CH2)2NHBoc for
5, 0 °C, 1 h; (b) 20% CF3CO2H in CH2Cl2, rt, 1 h; (c) ethyl-2-
chloroacetimidate hydrochloride, Et3N, MeOH, rt, 1 h.
animal models12 and has furthered understanding of the role of
PADs in different diseases (Figure 2).13 However, Cl-amidine is
only moderately selective for PAD1, with significantly lower
potency against PAD2 and PAD3 isozymes.14 While Thompson
has subsequently developed significantly more potent cell
permeable analogs,14,15 these inhibitors uniformly show high
inhibitory activity against PAD1 and, depending on the
Received: May 22, 2016
Accepted: July 11, 2016
Figure 1. Conversion of arginine side chains by PADs.
© XXXX American Chemical Society
A
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a,b
Table 1. Inhibition of PAD3 by Second-Generation Benzamide Analogs
a
b
IC50 values are reported as mean SD and were run in duplicate. kinact/KI was determined using six concentrations of inhibitor at five time points.
Kobs = kinact/KI because [I] ≪ KI. The assays were run in quadruplicate. See Supporting Information for further assay details.
a
a
Scheme 2. Alkene Inhibitor Synthesis
Scheme 3. Alkyne Inhibitor Synthesis
a
a
Reagents and conditions: (a) 2 M LiOH, MeOH, rt, 1 h; (b) 3,
EDCI, Oxyma, Et3N, CH2Cl2, rt, 1 h; (c) 20% CF3CO2H in CH2Cl2,
rt, 1 h; (d) ethyl-2-chloroacetimidate hydrochloride, Et3N, MeOH, rt,
1 h.
Reagents and conditions: (a) 2 M LiOH, MeOH, rt, 1 h; (b) 3,
EDCI, Oxyma, Et3N, CH2Cl2, rt, 1 h; (c) 20% CF3CO2H in CH2Cl2,
rt, 1 h; (d) ethyl-2-chloroacetimidate hydrochloride, Et3N, MeOH, rt,
1 h.
structure, strong inhibition of PAD2 or PAD4. In all cases, low
inhibitory activity against PAD3 has been observed. A potent
and isozyme-selective inhibitor of PAD3 would be extremely
useful for deciphering the biological roles of this isozyme.
We have recently reported on the use of a fragment-based
substrate screening approach for the discovery of potent PAD3-
selective inhibitors, the best of which are >10-fold selective for
PAD3 over the other isozymes.16 These low molecular weight
and nonpeptidic inhibitors represent the only potent, PAD3-
selective inhibitors described in the literature. Herein, we report
on the further optimization of inhibitor 2 (Figure 2) to provide
more potent inhibitors where the amide has been replaced by a
heterocyclic functionality. Moreover, we have established that
these inhibitors are active in cell culture by their protection of
thapsigargin-induced cell death of HEK293T cells expressing
PAD3.
Inhibitor 2, which was one of the most potent and selective
PAD3 inhibitors that we had previously identified, was an
appealing starting point for optimization. The flexible alkyl
chain connecting the chloroacetamidine mechanism-based
pharmacophore to the remainder of the inhibitor structure
provides a key region for optimization with conformational
constraints potentially benefiting inhibitor potency and/or
selectivity. These types of conformational constraints have
B
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ACS Medicinal Chemistry Letters
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a
Scheme 4. Synthesis of Imidazole Inhibitor
a
Reagents and conditions: (a) CuBr, EtOAc/CHCl3, 65 0 °C, 5 h; (b)
(i) hexamine, CH2Cl2, rt, 1 h; (ii) MeOH, HCl, rt, 1 h; (c) Boc-
GABA-OH, TBTU, Et3N, CH2Cl2, rt, 1 h; (d) NH4OAc, AcOH, 140
°C, 2 h; (e) 20% CF3CO2H in CH2Cl2, rt, 1 h; (f) ethyl-2-
chloroacetimidate hydrochloride, Et3N, MeOH, rt, 1 h.
Figure 3. Rescue of thapsigargin-induced cell death in PAD3-
expressing HEK293T cells by inhibitors (a) 1, (b) 2, and (c) 18.
Log % cell survival is reported as an average of four values SD.
a
Scheme 5. Synthesis of Chloroimidazole Inhibitor
and carbamate substitution had orthogonal effects on PAD1
and 4 inhibition. The carbamate in 6 resulted in a considerably
weaker PAD1 inhibitor with little effect on PAD4, while the
urea in 7 showed little effect on PAD1 inhibition but gave 3-
fold weaker PAD4 inhibition.
Conformational constraints were also introduced in the
center of the hydrocarbon chain by incorporation of an E
alkene (Scheme 2). Saponification of the literature compound
N-Boc γ-amino ester 818 was followed by coupling the resulting
acid with 3-phenylbenzylamine to provide amide 9. Removal of
the Boc group with trifluoroacetic acid followed by coupling
with ethyl-2-chloroacetimidate then gave alkene inhibitor 10.
This conformationally constrained inhibitor showed compara-
ble potency against PAD3 to parent inhibitor 2 though it did
provide reduced selectivity over PAD2 (Table 1). More
rigorous determinatination of kinact/KI for alkene inhibitor 10
confirmed that its potency was comparable to that of 2.
An alkyne conformational constraint was also introduced
(Scheme 3). As opposed to the alkene constraint in inhibitor
10, the alkyne results in a much more significant displacement
of the chloroacetamidine mechanism-based pharmacophore
from the remainder of the inhibitor structure (Scheme 3). The
N,N-bis-Boc γ-amino ester 11, which was prepared according to
literature procedures,19 was treated with excess NaOH to
remove one of the Boc groups along with concomitant
saponification of the ester. Coupling with 3-phenylbenzylamine
then provided amide 12. Removal of the Boc group and
coupling with ethyl-2-chloroacetimidate then gave alkyne
inhibitor 13.
Given the greater perturbation of the structure relative to the
alkene isostere 10, it is perhaps not surprising that inhibitor 13
incorporating the alkyne resulted in >10-fold reduction in
PAD3 inhibition as well as weaker inhibition of all of the other
PAD isozymes (Table 1).
Next, we explored heterocycle replacements of the amide
present in inhibitor 2. Imidazoles are well-known isosteres for
amide bonds.20,21 Therefore, the introduction of this
functionality in place of the amide backbone was first explored
(Scheme 4). Bromination of 3-phenyl acetophenone 1422 was
followed by amine displacement to provide aminoketone 15,
which was then coupled with N-Boc γ-aminobutyric acid to give
16. Heating 16 at elevated temperatures with NH4OAc then
provided imidazole 17, which was converted to the imidazole
a
Reagents and conditions: (a) NCS, CH3CN, rt, 12 h; (b) 20%
CF3CO2H in CH2Cl2, rt, 1 h; (c) ethyl-2-chloroacetimidate hydro-
chloride, Et3N, MeOH, rt, 1 h.
a
Scheme 6. Synthesis of Triazole Inhibitor
a
Reagents and conditions: (a) 1-azido-3-phenylbenzene, Na ascorbate,
CuSO4, H2O/EtOH/PhMe, rt, 12 h; (b) ethyl-2-chloroacetimidate
hydrochloride, Et3N, MeOH, rt, 1 h.
contributed to greatly enhanced selectivity in histone deactylase
(HDAC) inhibitors,17 but they have not previously been
explored for PAD inhibitors. Replacement of the amide in 2
with heterocycle isosteres is also of value because it would
eliminate susceptibility to hydrolases.
Replacement of the amide in 2 with carbamate and urea
functionality reduces rotational flexibility while also modulating
hydrogen bonding properties (Scheme 1). These modifications
could readily be introduced using amine salt 3 as a common
starting material, which was treated with triphosgene followed
by coupling with either N-Boc ethylene diamine or N-Boc
ethanolamine to give 4 and 5, respectively. Cleavage of the Boc
group followed by reaction with ethyl 2-chloroacetimidate then
provided inhibitors 6 and 7.
Replacement of the amide in 2 with carbamate (6) or urea
(7) functionality was detrimental to PAD3 inhibitory potency
with pronounced effects on the potency against other PAD
isozymes also observed (Table 1). Interestingly, while both
replacements resulted in 3-fold weaker PAD2 inhibition, urea
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inhibitor 18 according to the methods described previously.
Inhibitor 18, though somewhat less selective than parent
inhibitor 2, was 2-fold more potent as determined by kinact/KI.
This is an exciting result because inhibitor 18 introduces an
imidazole in place of the amide that has the potential to be
susceptible to hydrolases.
The chloroimidazole inhibitor 20 (Scheme 5) was prepared
due to the altered sterics and modulation of the pKa relative to
an unsubstituted imidazole as well as because of the reported
success of this type of imidazole halogenation in protease
inhibitors.23 Chlorination of the imidazole in intermediate 17
to give 19 was followed by replacement of the Boc protecting
group with the chloroacetamidine functionality to give inhibitor
20. Chloroimidazole inhibitor 20 proved to be less potent than
the corresponding imidazole inhibitor 18 and also was less
selective.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00215.
■
S
Complete experimental procedures and characterization
data for all compounds described as well as IC50 data and
kinact/KI data for inhibitors (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: jonathan.ellman@yale.edu.
■
Present Address
†(H.A.K.) AbbVie, 1 North Waukegan Road, North Chicago,
Illinois 60064, United States.
Finally, inhibitor 23 was prepared and evaluated because the
1,2,3-triazole functionality has also been used as an amide
surrogate24,25 (Scheme 6). Amine 21, prepared by literature
methods,26 was coupled with 3-biphenyl azide using Cu-
catalyzed Click chemistry to give 22. Installation of the
chloroacetamidine pharmacophore then provided inhibitor 23.
Although replacement of the amide by the 1,2,3-triazole was
reasonably well tolerated, inhibitor 23 proved to be less potent
than both parent inhibitor 2 and imidazole inhibitor 18, as
determined by IC50 and by kinact/KI.
At this stage we chose to evaluate the most potent PAD3
inhibitor 18 as well as the parent inhibitor 2 for activity in cells.
Feretti and co-workers have developed a cellular assay for the
assessment of PAD3 inhibition.9 In this assay, HEK293T cells
transfected with PAD3-containing vectors are treated with
thapsigargin, a natural product used to rapidly increase the
concentration of intracellular Ca2+. The authors showed that
rapid, dose-dependent cell death as a result of PAD3-activation
was the consequence of thapsigargin treatment. Significantly,
the authors also observed that PAD3 inhibition mediated by the
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support has been provided by the NIH (R01-GM054051). H.J.
also acknowledges support from the NIH Chemical Biology
training grant (5T32 GM7499-34). Paul Thompson is
gratefully acknowledged for providing constructs enabling the
recombinant expression of PADs. Corey E. Perez, for his
assistance with cloning, and Erin E. Duffy, for her help with cell
culture experiments, are also acknowledged.
ABBREVIATIONS
■
AIF, apoptosis-inducing factor; Boc-GABA−OH, γ-(tert-
butoxycarbonylamino)butyric acid; EDCI, 1-ethyl-3-(3-di
methylaminopropyl)carbodiimide; HDAC, human deacetylase;
HEK, human embryonic kidney; hNSC, human neural stem
cells; PAD, protein arginine deiminase; TBTU, O-(benzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
nonselective PAD inhibitor Cl-amidine with a kinact/KI of
14,16
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