Discovery of a structurally novel, drug-like and potent inhibitor of peptidylarginine deiminase

Article abstract of DOI:10.1039/c3md00091e

The synthesis and biological properties of a structurally novel, potent and non-peptidic inhibitor of peptidylarginine deiminase are described. The novel drug-like PAD inhibitor contains a 3,5-dihydroimidazol-4-one ring that replaces the acyclic guanidine-binding unit present in arginine residues. This new drug-like PAD inhibitor was effective at 100 nM or below and could have relevance to diseases in which PAD expression is up-regulated, including rheumatoid arthritis, cancer, multiple sclerosis, and neural injury.

Full text of DOI:10.1039/c3md00091e

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Discovery of a structurally novel, drug-like and potent
inhibitor of peptidylarginine deiminase
Cite this: Med. Chem. Commun., 2013,
4, 1109
Patrizia Ferretti,a Kin Pong U,a Barbora Vagaska,a Rohan Merchant,b
b
b
*
Christopher J. Matthews and Charles M. Marson
The synthesis and biological properties of a structurally novel, potent and non-peptidic inhibitor
of peptidylarginine deiminase are described. The novel drug-like PAD inhibitor contains 3,5-
a
Received 26th March 2013
Accepted 29th May 2013
dihydroimidazol-4-one ring that replaces the acyclic guanidine-binding unit present in arginine residues.
This new drug-like PAD inhibitor was effective at 100 nM or below and could have relevance to diseases
in which PAD expression is up-regulated, including rheumatoid arthritis, cancer, multiple sclerosis, and
neural injury.
DOI: 10.1039/c3md00091e
www.rsc.org/medchemcomm
The family of calcium ion-dependent peptidylarginine deimi- calcium ion concentration;1,16,17 additionally, reduced deimi-
nases (PADs, EC 3.5.3.15),1 including the ve known mamma- nation of PAD targets, including histone H3, is observed
lian isoforms, PAD1–4 and PAD6, catalyse the formation of following treatment of injured chick spinal cords with Cl-ami-
citrulline residues in proteins from arginine residues.1–3 Such dine.16 The putative role of PAD3 in spinal cord tissue loss
deimination (Scheme 1), also referred to as citrullination, following injury makes novel compounds that can effectively
results in loss of positive charge that can induce conformational inhibit the activity of this isozyme of particular interest for
changes in PAD protein structure,2 and hence alters its inter- neuroprotection.
actions with other proteins. Elevated levels of citrullination,
Given the rapidly emerging importance of PADs in several
owing to up-regulation of PAD enzymes, have been associated physiological and pathological processes, targeting these
with several major types of disease, including rheumatoid enzymes could provide an attractive therapeutic strategy for
arthritis (mainly PAD4 involvement), in which antibodies to several chronic diseases and also for traumatic neural injury.
citrullinated proteins and anti-PAD autoantibody levels can act New PAD inhibitors are needed, given that PAD inhibitors
as important diagnostic aids.4,5 Recently, in vivo efficacy of a are being increasingly investigated against a wide range of
PAD inhibitor against rheumatoid arthritis has been demon-
strated.6 Up-regulation of PAD2 has been implicated in the
development of glaucoma7 and autoimmune diseases such as
multiple sclerosis, for which PAD inhibition in an animal model
prevented onset of the disease.8–10 Increased protein deimina-
tion has also been reported in patients with Alzheimer's
disease11 and in other neurodegenerative diseases.12
A role for PADs has also been demonstrated in traumatic
injury. Tissue loss following spinal cord injury is associated
with up-regulation of PAD3, but has been ameliorated using a
non-selective PAD inhibitor. Cl-amidine,13–15 an alkylating
agent, decreases deimination, apoptosis and consequently
tissue loss in injured chick spinal cords,16 and ameliorates
tissue damage following perinatal ischaemia.1 Those effects can
be modeled in cultures of human neural cells in which Cl-
amidine inhibits apoptosis induced by increased cytoplasmic
aInstitute of Child Health, University College London, 30 Guildford Street, London
WC1N 1EH, UK
Scheme 1 (a) PAD-catalysed citrullination of a peptidylarginine residue. (b) The
structure and synthesis of a novel potent synthetic inhibitor of PAD. Reagents and
conditions: (i) MeI, K2CO3, acetonitrile, 20 ꢀC, 3 h, 54%; (ii) tryptamine, ethanol,
reflux 4 days, 55%.
bDepartment of Chemistry, University College London, Christopher Ingold
Laboratories, 20 Gordon Street, London WC1H 0AJ, UK. E-mail: c.m.marson@ucl.
ac.uk; Fax: +(44) (0)207 679 7463; Tel: +(44) (0)207 679 4712
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biological targets,10,16,18–20 and that PAD inhibitors are limited in inhibitor, can reduce human PAD3 activity, and appears to be
number and chemical structure, being mainly halogenated more potent than Cl-amidine in the BAEE assay.
amidine derivatives13,14 and some guanidine derivatives.21
In a kinetic study using BAEE as the substrate for HEK293T
Another general current limitation is that PAD isoform selec- cell extracts overexpressing human recombinant PAD3, the
tivity remains to be achieved. Mindful of those limitations, we initial rates at various concentrations of imidazolin-4-one 3 (0,
sought to develop non-peptidic, potent PAD inhibitors with 10 and 100 mM) were determined. In the presence of compound
drug-like properties that cannot alkylate protein residues, and 3, the observed Km increased with increasing concentration of 3,
that bind to PAD only through non-covalent interactions. whereas Vmax remained constant, thereby displaying Michaelis–
Herein is described the rst potent reversible PAD inhibitor Menten kinetics consistent with competitive reversible inhibi-
that, to our knowledge, fulls those criteria.
tion (Fig. 2).
Our hypothesis was that a potent and drug-like PAD inhibitor
The effect of the novel PAD inhibitor 2-[2-(1H-indol-3-yl)-
could be designed that lacked both a peptide unit and a chiral ethylamino]-3,5-dihydroimidazol-4-one (3) on human neural
centre; those are both present in almost all previously reported cells (methylene blue assay, Fig. 3) showed that compound 3 did
PAD inhibitors including benzoyl L-arginine amide which has not decrease the percentage of viable cells (Fig. 3) but rather
appreciable potency but is not CNS-active. Additionally, we increased it, indicating as little toxicity towards those cells as
considered that inhibitors containing a guanidine terminus, that of Cl-amidine.
although likely to induce PAD inhibition by analogy with the
We then further examined the effect of imidazolin-4-one 3 on
natural substrate, would ultimately prove unsatisfactory, in part human neural stem cells in culture in order to establish whether
because of susceptibility to citrullination. All of those disad- it was also more potent than Cl-amidine in live cells. Cell death
vantageous structural features were absent in the imidazolin-4- was induced in human neural cells by treatment with 5 mM
2
˚
one 3 (clogP ¼ 1.0, polar surface area ¼ 69 A , compared with Cl- thapsigargin, and their pre-treatment with different concentra-
2
˚
amidine: clogP ¼ 0.2, polar surface area ¼ 108 A ) that was tions of imidazolin-4-one 3 and Cl-amidine was examined for
proposed: formal annulation of the guanidine terminus to give their ability to antagonise cell death (Fig. 4). Thapsigargin-
an imidazolin-4-one was expected to hinder deimination, and induced cell death was not altered by Cl-amidine at or below
yet still permit extensive hydrogen bonding, in which two Asp concentrations of 1 mM, and a signicant, though partial,
residues near the active site of PAD enzymes have been increase in cell survival was observed only at concentrations
invoked;22 the peptide linkage and side-chain was replaced by a above 10 mM (Fig. 4). In marked contrast, imidazolin-4-one 3
(1H-indol-3-yl)ethyl unit, isosteric in regard to its partial
framework (as depicted in blue in Scheme 1).
N-Acetyl thiohydantoin (1), prepared by heating a mixture of
glycine, ammonium thiocyanate and acetic anhydride at 100
ꢀC,23 was reacted with methyl iodide in the presence of potas-
sium carbonate in acetonitrile to give 1-acetyl-2-methyl-
thiohydantoin (2). Heating 1-acetyl-2-methylthiohydantoin (2)
with tryptamine in ethanol initially gave the 1-acetyl derivative
of 3 but on prolonged heating, deacetylation occurred to give
imidazolin-4-one 3.
The effect of imidazolin-4-one 3 on PAD activity in protein
extracts from HEK293T cells over-expressing human recombi-
nant PAD3 was measured using the BAEE assay. In this in vitro
assay, imidazolin-4-one 3 showed comparable potency to Cl-
amidine up to 1 mM, and markedly superior potency at 100 mM
(Fig. 1). These results show that imidazolin-4-one 3 is a PAD
Fig. 2 Effect of imidazolin-4-one (3) on the rate of citrullination of BAEE by
extracts from human HEK293T cells overexpressing recombinant PAD3.
Fig. 3 Percentage of human neural stem cells surviving (detected by methylene
Fig. 1 Inhibition of PAD activity by Cl-amidine and imidazolin-4-one 3 assessed by
the BAEE assay using extracts of HEK293Tcells overexpressing human recombinant
PAD3. Both compounds potently inhibit PAD activity in vitro (** p # 0.01).
blue assay) after treatment with either imidazolin-4-one 3 or Cl-amidine (Cl-am).
No reduction in the percentage of live cells after 24 h treatment was observed at
any of the concentrations tested.
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Fig. 4 Inhibition of thapsigargin-induced cell death by imidazolin-4-one 3 as
compared to Cl-amidine. Controls and thapsigargin (5 mM) alone (no inhibitor)
contain either ethanol (controls for Cl-amidine, grey bar) or both ethanol and
DMSO (controls for imidazolin-4-one 3, striped bar). Imidazolin-4-one 3 is more
powerful than Cl-amidine in rescuing cells from thapsigargin-induced cell death
as assessed by the methylene blue assay. *p # 0.05, **p # 0.01 (as compared to
thapsigargin treated cells), > p # 0.01 (as compared to corresponding control)
and ^ p # 0.01 (as compared to Cl-amidine at the same concentration).
Fig. 6 Molecular modeling of the binding of imidazolin-4-one 3 (turquoise) to
PAD4 using AutoDock3.0 (ref. 24) and viewed in PyMOL, with depiction of resi-
dues in and near the catalytic site.
PAD3 activity in live cells. This is of particular interest since PAD3
has been found to be up-regulated following spinal cord injury in
which extensive neural tissue loss has occurred, and such loss
was signicantly reduced by Cl-amidine treatment.16
Using AutoDock3.0,24 modeling of the binding of imidazolin-
4-one 3 in PAD4 (prepared from PDB code 3B1U) showed a
strong preference for a pose (Fig. 6) of the imidazolin-4-one ring
in proximity to PAD4 residues involved in hydrolytic deimina-
tion,22 including His471, the nucleophilic Cys645, and espe-
cially to Asp350 and Asp473 participating in hydrogen bonding.
Even an unsubstituted indole ring proves to be an acceptable
replacement for the amide chain present in BAA. This modeling
conrms the efficacy of a 2-amino-3,5-dihydroimidazol-4-one
moiety as a surrogate for a guanidine terminus, and also
suggests that even more potent, and perhaps selective, inhibi-
tion of PADs should result from the addition of appropriate
polar substituents to the indole ring present in 3.
maximally restored cell survival at 100 nM, and was highly
effective at all the concentrations tested (up to 1 mM). The
greater difference in potency between imidazolin-4-one 3 and Cl-
amidine in the cell assay as compared to the enzymatic assay in
protein extracts may be at least in part due to a higher concen-
tration of the imidazolin-4-one 3 in the cell, given its greater
lipophilicity.
Finally, weassessed whetherthe extent towhichimidazolin-4-
one 3 inhibited cell death in the HEK293T cells over-expressing
the human recombinant PAD3. In PAD3-expressing HEK293T
cells 1 mM thapsigargin was sufficient to induce cell death. A
signicant increase in cell survival, as assessed by the methylene
blue assay, was achieved by treatment with both imidazolin-
4-one 3 and Cl-amidine (Fig. 5). However, whereas imidazolin-
4-one 3 was effective at 10 mM, the same extent of inhibition
required Cl-amidine at a concentration of 100 mM. This further
supports the view that imidazolin-4-one 3 is more potent than Cl-
amidine in human cell-based assays and can inhibit human
Conclusions
The structure-based design and synthesis of a novel non-
peptidic drug-like PAD inhibitor 3 has been achieved, in which a
heterocyclic ring replaces the acyclic guanidine-binding unit
present in arginine residues. In human cell-based assays, imi-
dazolin-4-one 3 showed signicantly more potency than Cl-
amidine, a benchmark PAD inhibitor, and in some cases
between 10 and 100 times more potency. At all concentrations
tested, the novel PAD inhibitor 3 exhibited no toxicity towards
human neural stem cells and appeared to increase the
percentage of live cells as compared to controls. This proof-of-
principle of a new and drug-like PAD inhibitor effective on
human cells, and with potencies at a therapeutic level is timely,
given that PADs have been suggested as novel therapeutic
targets for several diseases, including cancer, multiple sclerosis,
rheumatoid arthritis and neural injury.10,16–19
Materials and methods
Fig. 5 Inhibition of cell death induced by thapsigargin (Th) in HEK293T cells Synthesis of 2-[2-(1H-indol-3-yl)-ethylamino]-3,5-
expressing human recombinant PAD3 by Cl-amidine (Cl-am, 100 mM) and imi-
dihydroimidazol-4-one (3)
dazolin-4-one 3 (Imid, 10 mM). Imidazolin-4-one 3 is effective at a 10 fold lower
A mixture of 1-acetyl-2-methylthiohydantoin (0.469 g, 2.73
mmol) and tryptamine (0.438 g, 2.74 mmol) in ethanol (4 mL)
concentration (*p < 0.51; **p < 0.01). All samples including control and 1 mM
thapsigargin alone (striped bars) contain both ethanol and DMSO.
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was heated at reux until t.l.c. showed that the initially formed
1-acetyl-2-[2-(1H-indol-3-yl)-ethylamino]-3,5-dihydroimidazol-4-
one (about 4 days) had been consumed. Aer allowing to cool,
the precipitate was ltered and washed with cold ethanol to
give 2-[2-(1H-indol-3-yl)-ethylamino]-3,5-dihydroimidazol-4-one
3 (0.36 g, 55%) as a yellow solid, mp 222–223 ꢀC; IR (nmax) (thin
Acknowledgements
We acknowledge support from the BBSRC and the Child
Research Appeal Trust.
Notes and references
lm) 3292, 1693, 1633, 1564 cmꢁ1 1H NMR (2 : 1 mixture of
;
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