Development of a Selective Inhibitor of Protein Arginine Deiminase 2

Article abstract of DOI:10.1021/acs.jmedchem.7b00274

Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the

Full text of DOI:10.1021/acs.jmedchem.7b00274

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Article
Development of a selective inhibitor of Protein Arginine Deiminase 2
Aaron Muth, Venkataraman Subramanian, Edward Beaumont, Mitesh Nagar, Philip Kerry, Paul
McEwan, Hema Srinath, Kathleen Wanda Clancy, Sangram S. Parelkar, and Paul R. Thompson
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Mar 2017
Downloaded from http://pubs.acs.org on March 24, 2017
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Development of a selective inhibitor of Protein Arginine Deiminase 2
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Aaron Muth , Venkataraman Subramanian , Edward Beaumont, Mitesh Nagar , Philip Kerry,
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Paul McEwan, Hema Srinath , Kathleen Clancy , Sangram Parelkar , Paul R. Thompson
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1,4*
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Department of Biochemistry and Molecular Pharmacology, UMass Medical School, 364
Plantation Street, Worcester, MA, 01605, USA.
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Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s
University, Queens, NY, 11439, USA.
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Evotec, Milton Park, Abingdon, Oxfordshire OX14 4RZ, UK.
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Program in Chemical Biology, UMass Medical School, 364 Plantation Street, Worcester, MA
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1605, USA
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Author to whom correspondence should be addressed: Paul R. Thompson
paul.thompson@umassmed.edu
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Abstract
Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of
multiple sclerosis, rheumatoid arthritis and breast cancer. To date, no PAD2-selective inhibitor
has been developed. Such a compound will be critical for elucidating the biological roles of this
isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is
dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives
of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective
inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the
N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and
selectivity (30a, 41a, and 49a) as well as cellular efficacy 30a. Notably, these compounds use
the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical
tool for understanding cellular PAD2 function and sets the stage for treating diseases in which
PAD2 activity is dysregulated.
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Introduction.
The protein arginine deiminase (PAD) family of enzymes, which catalyze protein
citrullination (Figure 1), have garnered significant recent interest because aberrantly upregulated
protein citrullination is associated with a variety of autoimmune diseases (e.g., rheumatoid
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arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) as well as certain cancers.
In
addition to playing an important role in human pathology, the deiminase activity of the PADs
regulates a diverse array of cellular processes including NET formation, the epigenetic control of
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, 3-7
gene transcription, differentiation, and the maintenance of pluripotency.
There are five PAD
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isozymes (PADs 1-4 and 6) that are uniquely distributed in various tissues. PADs 1-4 all
possess deiminase activity, whereas PAD6 has a number of mutations that render it inactive.
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, 9
The active PAD enzymes (i.e., PADs 1-4) are all regulated by calcium, wherein calcium binding
triggers a conformational change that moves a nucleophilic cysteine residue into the active site,
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resulting in a >10,000-fold increase in PAD activity.
Figure 1. The PAD catalyzed hydrolysis of peptidyl-arginine (1) to generate peptidyl-citrulline
2).
(
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PAD2, which is expressed in most tissue and cell types, contributes to the development
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of certain inflammatory diseases and cancers.
Specifically, PAD2 is upregulated in multiple
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sclerosis (MS) where it citrullinates myelin basic protein (MBP) leading to demyelination.
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PAD2 is also released into the synovial fluid of arthritic joints where it citrullinates a number of
extracellular targets, thereby promoting the development of anti-citrullinated antibodies (ACPA),
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which is a characteristic feature of RA.
Additionally, PAD2 is highly expressed in luminal
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breast cancers and its expression correlates with the levels of the HER2 protooncogene.
When PAD2 activity is inhibited in MCF10-DCIS cells, a breast cancer cell line, proliferation is
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decreased with a corresponding increase in apoptosis. This appears to be an epigenetic
phenomenon as PAD2 is recruited to ERα promoters where it citrullinates histone H3 at R26 to
trigger the localized decondensation of chromatin to facilitate ERα binding to its promoters to
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drive gene transcription.
st
Figure 2. Structures and kinact/K values for the 1 -Generation PAD inhibitors F-amidine (3a) and
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Cl-amidine (3b) and the 2 -Generation PAD inhibitors BB-F-amidine (4a) and BB-Cl-amidine
(4b).
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Given the strong evidence linking dysregulated PAD2 activity to MS, RA and breast
cancer, we and others have focused on developing inhibitors targeting this enzyme, as well as
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PAD4, whose activity is also upregulated in a variety of inflammatory diseases including RA
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and lupus.
Notably, our first-generation pan-PAD inhibitor Cl-amidine (3b, Figure 2)
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demonstrates efficacy in animal models of RA, lupus, ulcerative colitis, spinal cord injury,
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breast cancer, and atherosclerosis. Despite these successes, many peptide-based inhibitors
similar to Cl-amidine (3b) have displayed less than optimal potency in cellulo, which has been
attributed to their poor metabolic stability and cell membrane permeability. In an effort to
address these issues, the C-terminal carboxamide was replaced with a benzimidazole moiety to
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yield BB-Cl-amidine (4b, Figure 2). This second-generation PAD inhibitor is at least 10-fold
more potent than Cl-amidine (3b) in cells and animal models of lupus and RA, which represents
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a significant advance over Cl-amidine (3b).
Interestingly, the fluoroacetamidine counterparts of Cl-amidine (3a, F-amidine) and BB-
Cl-amidine (4a, BB-F-amidine) have yet to find much utility in animal models as they display a
marked decrease in PAD inhibition as compared to their chloroacetamidine analogs. This has
been attributed to the less electrophilic nature of the fluoroacetamidine warhead, which reacts
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with an active site thiol conserved in all PAD isozymes.
It is also important to note that
while BB-Cl-amidine (4b) brought a significant advance to PAD inhibitor development, as a pan
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PAD inhibitor, it does not provide utility in investigating the roles of specific isozymes.
Prior to the development of BB-Cl-amidine (4b) advances had been made in improving
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isozyme-specific inhibition. Specifically, TDFA provided PAD4-selective inhibition ,
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-Cl-
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amidine provided PAD1-selective inhibition , while Cl4-amidine
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and a series of hydantoin-
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based inhibitors
provided PAD3-selective inhibition. While these inhibitors have brought
about significant advances in PAD inhibitor development, two main issues remain: 1) most of
these inhibitors exhibit poor efficacy in cell-based assays (potentially due to metabolic instability
and poor membrane permeability) and 2) a PAD2-selective inhibitor has yet to be developed.
The deficiencies of current PAD inhibitors underscore a pressing need for PAD2-selective
inhibitors with enhanced cellular efficacy. Herein, we report the development of benzimidazole-
based PAD2-selective inhibitors that inhibit cellular PAD2 activity.
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Results and Discussion.
Inhibitor Design
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Figure 3. (A) Co-crystal structure of BB-F-amidine (4a) bound to PAD4 (PDB ID: 5N0M). (B)
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PAD2 (PDB: 4N2C) was superimposed on the PAD4·BB-F-amidine (4a) co-crystal structure
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and then PAD4 was removed leaving BB-F-amidine. (C) Evolution of 2 -Generation PAD
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inhibitors BB-F-amidine (4a) and BB-Cl-amidine (4b) to 3 -Generation benzimidazole-based
PAD inhibitors.
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Given the favorable cellular efficacy observed with BB-Cl-amidine (4b), we
hypothesized that further elaboration of this scaffold would identify compounds with enhanced
potency, selectivity and cellular efficacy. To aid these efforts we first generated the fluoro-
containing analog, BB-F-amidine (Figure 2, 4a). We then determined the crystal structure of
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BB-F-amidine (4a) bound to PAD4 (Figure 3A). Similar to crystal structures of F-amidine,
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TDFA , and o-F-amidine bound to PAD4, the amidine forms an intricate hydrogen bonding
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network with D350, H471, and D473. Interestingly, while R374 forms a hydrogen bond with the
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C-terminal carboxamide of the 1 -Generation PAD inhibitors, it instead forms a cation-π
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interaction with the benzimidazole. Overlays of the PAD2 structure onto the BB-F-
amidine·PAD4 co-crystal structure (Figure 3B) shows a similar network of favorable interactions
with D473, D351, and H471. These interactions properly orient the warhead for covalent
modification by C647. By contrast to the PAD4 structure, R373, which corresponds to R374 in
PAD4, orients away from the benzimidazole moiety such that it does not form the favorable
cation-π interaction observed with PAD4.
Ortho-carboxylate Series
Using these structures as a guide, we hypothesized that further elaboration of the
benzimidazole scaffold could lead to compounds with enhanced potency. Given our previous
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demonstration that the installation of an ortho-carboxylate (o-F-amidine and o-Cl-amidine)
enhanced potency by ≥10-fold, due to the formation of a key hydrogen bond with W348 in
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PAD2 or W347 in PAD4, we sought to combine the benzimidazole scaffold with the ortho-
carboxylate functionality, expecting that the ortho-carboxylate would enhance in vitro potency
and the benzimidazole would promote cellular uptake. This approach had previously been
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highly successful in developing highly potent tetrazole-based inhibitors. Combining these two
features (compounds 9a and 9b, Figure 4) increased PAD1 (18- and 1.8-fold, respectively) and
PAD4 (6.5- and 2.3-fold, respectively) inhibition as compared to BB-F-amidine (4a) and BB-Cl-
amidine (4b), however, PAD2 potency was relatively unaffected.
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N-alkyl Series
We next explored other aspects of the benzimidazole scaffold to improve PAD2 potency
and selectivity. Focusing on the nitrogen atoms of the benzimidazole scaffold, we noted that
when BB-F-amidine (4a) is bound to PAD4, each nitrogen atom is involved in a water-mediated
hydrogen bonding network; similar interactions are also observed with F-amidine (3a). While no
crystal structure of BB-F-amidine (4a) bound to PAD1 exists, these water molecules are
conserved in its structure. However, this hydrogen bonding network is absent in PAD2 and is
replaced with a unique hydrophobic binding region created by V469, L467, L642, and F573
(Figure S1). This insight was intriguing and we hypothesized that N-alkylation of the
benzimidazole could target this binding region for potent and selective PAD2 inhibition. N-
alkylation would also increase the overall hydrophobicity of the inhibitor, which we expected to
enhance cell permeability.
As discussed above, the ortho-carboxylic acid containing series (compounds 9a and 9b)
improved PAD1 and PAD4 inhibition as compared to BB-Cl-amidine (4b) and BB-F-amidine
(4a), but surprisingly did not improve PAD2 inhibition. By contrast, N-alkylation of the
benzimidazole exhibited a marked increase in PAD2 inhibition and selectivity (Figure 4).
Specifically, the N-ethyl derivative (11a) showed a 26-fold increase in PAD2 inhibition as
compared to BB-F-amidine (4a) while also exhibiting 31-fold selectivity for PAD2 over PAD4
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and 18-fold selectivity for PAD2 over PAD3. Also noteworthy is the fact that the
fluoroacetamidine warhead (9a-12a) demonstrated similar or better PAD2 inhibition than its
chloroacetamidine counterpart (9b-12b).
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Figure 4. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 1
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compounds 9-12. (B) Summary of isozyme selectivity for series 1 compounds 9-12 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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While this improvement in PAD2 potency and selectivity is not completely understood,
the co-crystal structure of BB-F-amidine (4a) with PAD4 and the subsequent PAD2 overlay do
provide some insight (Figure 3). These structures suggest that there is a key water-mediated
hydrogen bond that exists for PAD4 and not PAD2 with the nitrogen atom of the benzimidazole
moiety. Introduction of an N-alkyl group may disrupt this hydrogen bond causing a decrease in
PAD4 inhibition. The PAD2 overlay also shows a hydrophobic binding region unique to PAD2
which the N-alkyl group could be oriented toward. This gained hydrophobic interaction could
explain the improved PAD2 inhibition of compounds 10-12 with the N-ethyl substitution (11a)
proving to be optimal for this binding region. The fluoroacetamidine warhead demonstrates
similar (and in some instances improved) PAD2 inhibition as compared to the chloroacetamidine
warhead potentially due to a smaller binding region near C647, as fluorine is smaller than
chlorine. The differences in PAD2 selectivity, may also be attributed to the less electrophilic
nature of the fluoroacetamidine warhead as compared to the chloroacetamidine warhead leading
to a higher degree of specificity for the intended C647 target.
Lactam Series
Hypothesizing that the hydrophilic nature of the ortho-carboxylate might limit cellular
uptake, we next replaced this moiety with a lactam, yielding series 2 compounds (14-17). We
further hypothesized that the enhanced conformational constraint of this amide would further
improve PAD2 inhibition. Rewardingly, introduction of the lactam moiety resulted in a ~7-fold
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increase in PAD2 inhibition for 14a as compared to both 9a and ~9-fold compared to BB-F-
amidine (4a). By contrast, there was only a minor increase in PAD4 inhibition observed for 14a
as compared to 9a (~1.4-fold). Similar to the series 1 compounds, N-alkylation of the
benzimidazole caused a marked increase in PAD2 inhibition and selectivity. Specifically, the N-
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ethyl benzimidazole (16a) displayed the most potent PAD2 inhibition (kinact/K = 61,600 M min
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utilizing the fluoroacetamidine warhead, representing a ~41-fold increase in potency compared
to 9a and ~51-fold increase compared to BB-F-amidine (4a). Furthermore, 16a has the greatest
degree of PAD2 selectivity (56-fold over PAD3 and ~33-fold over PAD4). The
fluoroacetamidine warhead again proved superior with an optimal combination of PAD2 potency
and selectivity.
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Figure 5. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 2
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compounds 14-17. (B) Summary of isozyme selectivity for series 2 compounds 14-17 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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A co-crystal structure of 14a bound to PAD4 was then determined (Figure 6A); we used
PAD4 because efforts to co-crystallize PAD2 with 14a as well as other inhibitors proved
unsuccessful. The PAD4•14a complex is highly similar to the PAD4•o-F-amidine structure
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(Figure 6B). Notably, these structures show that the carbonyl of either the ortho-carboxylic acid
or the lactam form favorable hydrogen bonds with the indole nitrogen of W347. This interaction
is likely a key determinant of the enhanced potency observed with this series. A potential
hydrogen bond (either direct or water mediated) between R639 and the ortho-carboxylate or the
nitrogen of the lactam may also contribute to the observed potency. Overlays of these PAD4
39
structures with PAD1 (Figures 6C-D) suggest similar hydrogen bonding networks with W349
and R348 also led to the marked increase in PAD1 inhibition observed for these compounds.
1
2
However, modeling studies with PAD2 suggest that the increased potency for PAD2 is due
solely to a hydrogen bond with W348 as an arginine residue corresponding to R639 in PAD4 is
far removed from this binding region. The improved PAD2 inhibition for series 2 compounds
(14-17) as compared to series 1 compounds (9-12) may be attributed to the replacement of the
carboxylic acid with an amide functionality, the introduction of conformational constraint, or a
synergistic effect of the two changes. Efforts to fully understand this are underway through the
pursuit of a PAD2 co-crystal structure.
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Figure 6. (A) Co-crystal structure of AFM-14a bound to PAD4 (PDB: 5N1B). (B) Co-crystal
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structure of o-F-amidine bound to PAD4 (PDB: 3B1T). (C) PAD1 (PDB: 5HP5) overlay with
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PAD4•AFM-14a co-crystal structure in PyMol. (D) PAD1 (PDB: 5HP5) overlay with
1
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PAD4•o-F-amidine co-crystal structure in PyMol. (E) PAD2 (PDB: 4N2C) overlay with
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PAD4•AFM-14a co-crystal structure in PyMol. (F) PAD2 (PDB: 4N2C) overlay with
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PAD4•o-F-amidine co-crystal structure in PyMol.
N-alkyl Lactams
To further optimize this scaffold, we next focused on N-alkylation of the lactam moiety
(Figure 7) because the PAD4•14a co-crystal, as well as our PAD1 modeling studies, suggested
that modification of this position would disrupt the hydrogen bonding network with R639 and
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with R348 in PAD1, neither of which appear to aid in PAD2 binding. Furthermore, this
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substitution would further increase the overall hydrophobicity (19a ClogP = 1.61 and 14a ClogP
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1.00), which we expected to further improve the cell permeability.
Interestingly, this series of compounds (19-22) demonstrated similar PAD2 potencies and
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PAD2-selectivity as compared to series 2 compounds (14-17). Specifically, compounds 21a and
16a exhibited identical PAD2 vs PAD4 selectivity (33-fold), PAD2 vs PAD1 selectivity (~2-
fold) and similar PAD2 vs PAD3 selectivity (>33-fold). While the N-ethyl lactam does not
influence PAD2 potency or selectivity, it does improve the overall hydrophobicity of the
compounds.
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Figure 7. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 3
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compounds 19-22. (B) Summary of isozyme selectivity for series 3 compounds 19-22 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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Alkoxy-substituted Benzimidazoles
Having demonstrated that the lactam enhanced potency and the N-ethyl benzimidazole
modification enhanced PAD2 selectivity, we next explored substitutions of the 6-membered ring
of the benzimidazole. Specifically, alkoxy substitutions were introduced to probe potential
interactions with D351, R373 and H471 in PAD2, all of which surround the 6-membered ring of
the benzimidazole.
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This investigation began with a methoxy substitution at the 4-position giving series 4
compounds 29a and 29b which both showed improvements in PAD2 inhibition and selectivity
over PAD4 as compared to their parent compounds 14a and 14b. This improvement was then
coupled with N-alkylation of the benzimidazole to give series 4 compounds 30-31. Rewardingly,
compound 30a (N-methyl benzimidazole) showed a dramatic increase in PAD2 inhibition
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(kinact/K = 210,300 M min ) while also displaying very good selectivity for PAD2 (1.6-fold
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over PAD1, 47-fold over PAD3 and ~15-fold over PAD4). Interestingly, the N-ethyl
benzimidazole (compound 31a) displayed a 4-fold decrease in PAD2 potency (compared to 30a)
while improving PAD2 selectivity over PAD4 to 32-fold. This result suggests that introduction
of the 4-methoxy group shifts the position of the benzimidazole so that the smaller N-methyl
group would better access the unique hydrophobic binding region of PAD2 than the larger N-
ethyl group. This is in contrast to series 1, 2, and 3 compounds which showed that the N-ethyl
group was optimal at this position. Utilization of a 4-ethoxy group (32a and 32b) also showed
excellent PAD2 inhibition, albeit ~2-fold less than 30a. However, 32a did demonstrate superior
selectivity for PAD2 over PAD4 (95-fold) and PAD2 over PAD3 (79-fold).
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Figure 8. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 4
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compounds 29-32. (B) Summary of isozyme selectivity for series 4 compounds 29-32 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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Further SAR of the benzimidazole moiety then explored a 5-methoxy substitution. As
seen in Figure 9, compounds 36a and 36b showed an increase in PAD2 inhibition compared to
series 2 compounds 14a and 14b. Similar to series 4 compounds, N-methyl alkylation of the
benzimidazole resulted in a significant increase in PAD2 inhibition (albeit ~2-fold lower than
30a) as well as excellent PAD2 selectivity over PAD4 (95-fold) and PAD3 (114-fold). The N-
methyl substitution again displayed a greater degree of PAD2 inhibition and PAD2 selectivity
than the N-ethyl substitution (38a and 38b).
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Figure 9. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 5
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compounds 36-38. (B) Summary of isozyme selectivity for series 5 compounds 36-38 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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In order to improve the hydrophobic nature of these compounds, we then reintroduced the
N-ethyl lactam moiety in conjunction with the 4-alkoxy substitutions on the benzimidazole (30a
ClogP=1.26 and 41a=1.62) to give series 6 compounds. Similar to 29a, compound 40a showed
enhanced PAD2 potency and selectivity as compared to 14a (Figure 10). Series 6 compounds
also demonstrated that the N-methyl benzimidazole substitution (41a) was optimal for PAD2
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inhibition resulting in the most potent PAD2 inhibitor to date. Notably, the kinact/K for this
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-1
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compound, which is 365,400 M min , represents a >300-fold improvement over F-amidine
(3a) and BB-F-amidine (4a). Compound 41a also demonstrated excellent selectivity for PAD2
over PAD4 (85-fold) and PAD3 (85-fold), while improving selectivity for PAD2 over PAD1
(~5-fold). The 4-methoxy benzimidazole substitution also proved superior over the 4-ethoxy and
5-methoxy substitutions utilized by series 6 compounds 43a and 43b (Figure 10) and series 7
compounds 45-47.
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Figure 10. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 6
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compounds 40-43. (B) Summary of isozyme selectivity for series 6 compounds 40-43 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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Figure 11. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 7
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compounds 45-47. (B) Summary of isozyme selectivity for series 1 compounds 45-47 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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Series 6 compound 41a demonstrated that a combination of a 4-methoxy N-methyl
benzimidazole with an N-ethyl lactam presented an ideal combination for potent and selective
PAD2-inhibition. However, further investigation of lactam N-alkylation was needed to fully
understand this phenomenon as well as finding a good balance between optimal potency and
hydrophobicity. Therefore, series 8 compounds 49-51 were synthesized to explore N-alkylation
of the lactam moiety. These data show that an N-methyl substitution of 49a was similar in PAD2
potency to the unsubstituted lactam (30a) while also exhibiting similar PAD2-selectivity to the
N-ethyl lactam, 41a. N-isopropyl and N-cyclopropyl lactams (50a and 51a, respectively) also
proved to be highly potent and selective PAD2 inhibitors, although neither were as potent and
selective as 30a, 41a or 49a. Despite this, 50a and 51a are more hydrophobic than other lactam
N-alkyl substitutions.
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Figure 12. (A) kinact/K values for BB-F-amidine (4a), BB-Cl-amidine (4b), and series 8
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compounds 45-47. (B) Summary of isozyme selectivity for series 1 compounds 45-47 based on
their respective kinact/K ratios setting the enzyme with the lowest value as 1.
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PAD1, PAD2 and PAD4 binding of 30a and 41a
Co-crystal structures of 30a and 41a bound to PAD4 were determined to shed light on
how these compounds bind PAD4 (Figure 13). Overlays of these structures with those
determined for PADs 1 and 2 further highlight common and unique interactions. For example,
these compounds interact with PAD1 (D474, H472, W349, D352 and C645), PAD2 (D473,
H471, D351, W348 and C647) and PAD4 (D473, H471, W347, D350 and C645) in a similar
fashion to 14a. Close inspection of these structures reveal three main reasons for the PAD2-
selectivity versus both PAD1 and PAD4. First, the N-methyl benzimidazole appears to orient
into a unique hydrophobic binding region of PAD2, while simultaneously eliminating a water
mediated hydrogen bonding network that is important for PAD4 binding. Second, the 4-
methoxy group of the benzimidazole appears to be ideal for this binding region in PAD2 and
tolerated for PAD1. However, the PAD4 cation-π with R374 is no longer optimal as the
methoxy group forces the benzimidazole moiety to move and it is not able to properly interact
with R374. Third, the N-ethyl lactam appears to cause R639 in PAD4 to orient away from the
lactam functionality. Since the corresponding residue in PAD2 is not an arginine, this likely
explains the higher selectivity for PAD2. Similarly, the enhanced PAD2-selectivity over PAD1
is likely explained by the fact that PAD1 does place an arginine residue close to this position.
Despite these findings, selective inhibition of PAD2 over PAD1 proved difficult and will be a
focus for future inhibitor development.
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Figure 13. (A) Co-crystal structure of AFM-30a bound to PAD4 (PDB: 5N0Y). (B) Co-crystal
3
structure of AFM-41a bound to PAD4 (PDB: 5N0Z). (C) PAD1 (PDB: 5HP5) overlay with
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PAD4•AFM-30a co-crystal structure in PyMol. (D) PAD1 (PDB: 5HP5) overlay with
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PAD4•AFM-41a co-crystal structure in PyMol. (E) PAD2 (PDB: 4N2C) overlay with
2
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PAD4•AFM-30a co-crystal structure in PyMol. (F) PAD2 (PDB: 4N2C) overlay with
PAD4•AFM-41a co-crystal structure in PyMol.
Target Engagement Assays
To measure target engagement, we used a stably transfected HEK293T cell line that
overexpresses PAD2 (HEK293T/PAD2). Briefly, cells were incubated with an inhibitor of
interest for 15 min, ionomycin (a calcium ionophore) for an additional 15 min, and calcium
chloride was then added and the cells incubated for 1 h. Cells were then harvested, lysed, and
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cell lysates incubated with RIBFA, 55, a fluorescently tagged PAD probe (see Supporting
Information) and calcium chloride for 1 h. Proteins were then resolved by SDS-PAGE and
fluorescently tagged proteins imaged. For proof of concept, BB-Cl-amidine (4b) was first
evaluated in this assay due to its high cell permeability and efficacy in other cell-based assays.
As seen in Figure 14, BB-Cl-amidine (4b) was effective at entering the cell and covalently
modifying PAD2, giving an EC of 7.5 µM in this target engagement assay. Figure 14 also
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shows that BB-F-amidine (4a) was ~3-fold less potent in the target engagement assay than BB-
Cl-amidine (4b), which was expected as it was less potent in vitro as well as less hydrophobic.
Figure 14. (A) Target engagement assay in HEK293/PAD2 cells for BB-Cl-amidine (4b). (B)
Quantified results of target engagement assay plotted as % occupancy of PAD2 by BB-Cl-
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