Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor

Article abstract of DOI:10.1021/acs.biochem.8b00554

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N⁵-(1-imino-2-chloroethyl)-l-ornithine (K_I = 1.3 μM, k_inact = 0.34 min^-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline (K_i = 470 μM) and 2-chloroacetamidine (K_I = 310 μM, k_inact = 4.0 min^-1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N⁵-(1-imino-2-chloroisopropyl)-l-ornithine (k_inact/K_I = 208 M^-1 s^-1) and N⁵-(1-imino-2-chlorisopropyl)-l-lysine (k_inact/K_I = 440 M^-1 s^-1), and one that lengthens the linker beyond that found in the substrate, N⁵-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, K_I = 0.19 μM, k_inact = 0.22 min^-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 10⁴ M^-1 s^-1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 10² M^-1 s^-1), and has a partition ratio of 1 with a >100000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC₅₀ = 10 μM) with cytotoxicity appearing only at higher concentrations (ED₅₀ = 118 μM). A 1.91 ? resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.

Full text of DOI:10.1021/acs.biochem.8b00554

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Article
Dissection, Optimization and Structural Analysis
of a Covalent Irreversible DDAH1 Inhibitor
Gayle Burstein-Teitelbaum, Joyce Er, Arthur F. Monzingo, Alfred Tuley, and Walter Fast
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00554 • Publication Date (Web): 08 Jul 2018
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Biochemistry
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Dissection, Optimization and Structural Analysis of
a Covalent Irreversible DDAH1 Inhibitor
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Gayle Burstein-Teitelbaum, † Joyce A.V. Er, † Arthur F. Monzingo, ‡ Alfred Tuley, † Walter
†
,
Fast *
†Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, University of
Texas, Austin, TX 78712, United States
‡Center for Biomedical Research Support, University of Texas, Austin, TX 78712, United States
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ABSTRACT
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Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can
control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1,
N5-(1-imino-2-chloroethyl)-L-ornithine (KI = 1.3 μM, kinact = 0.34 min-1), was conceptually
dissected into two fragments and each characterized separately: L-norvaline (Ki = 470 μM) and
2-chloroacetamidine (KI = 310 μM, kinact = 4.0 min-1). This analysis suggested that the two
fragments were not linked in a manner that allows either to reach full affinity or reactivity,
prompting the synthesis and characterization of three analogs: two that mimic the dimethylation
status of the substrate, N5-(1-imino-2-chloroisopropyl)-L-ornithine (kinact/KI = 208 M-1s-1) and N5-
(1-imino-2-chlorisopropyl)-L-lysine (kinact/KI = 440 M-1s-1), and one that lengthens the linker
beyond that found in the substrate, N5-(1-imino-2-chloroethyl)-L-lysine (Cl-NIL, KI = 0.19 μM,
kinact = 0.22 min-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates
with a second order rate constant (1.9 × 104 M-1s-1) larger than the catalytic efficiency of DDAH1
for its endogenous substrate (1.6 × 102 M-1s-1), and has a partition ratio of 1 with a >100,000-fold
selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show
inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 μM), with cytotoxicity only
appearing at higher concentrations (ED50 = 118 μM). A 1.91 Å resolution X-ray crystal structure
reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL.
Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the
design and optimization of this potent and effective DDAH1 inhibitor.
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INTRODUCTION
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Nitric oxide biosynthesis is highly regulated to preserve the function of this second messenger in
biological signaling by neuronal and endothelial nitric oxide synthases, and to unleash the
toxicity of this radical when overproduced by inducible nitric oxide synthase.(1) One atypical
control mechanism used is the direct inhibition of these three nitric oxide synthase isoforms by
endogenously produced methylarginines.(2) Both asymmetric Nω,Nω-dimethyl-L-arginine
(ADMA) and Nω-monomethyl-L-arginine (NMMA) are released by proteolysis of
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posttranslationally modified proteins, and these methylarginines play functional roles in their
“second act” as a reversible inhibitor (ADMA, Figure 1) or an irreversible inactivator (NMMA)
of nitric oxide synthase.(3-10) Both ADMA and NMMA are present in plasma at low micromolar /
high nanomolar concentrations in healthy persons, are elevated in disease states such as in
patients with chronic renal disease, and regulate vascular tone through inhibition of nitric oxide
production in the endothelium.(11) The concentrations of these inhibitory methylarginines are
controlled by both renal excretion and metabolism, with the main catabolic enzyme for both
being dimethylarginine dimethylaminohydrolase-1 (DDAH1).(11, 12)
L
-Arg
ADMA
+H3N
-OOC
CH3
N
H
1.5 NADPH
2 O2
H
N
CH3
+
NH2
NOS
DDAH1
+H2O
H2
N
+
1.5 NADP+
2 H2O
H3C
CH3
O
+H3N
-OOC
H
H
N
N O
NH2
L-Cit
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Figure 1. Regulation of !NO Production by DDAH1. Conversion of L-Arg to !NO by isoforms
of NOS is inhibited by endogenously produced ADMA, the concentrations of which are
regulated by DDAH1 activity.
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Pharmacologically-useful inhibitors of DDAH1 can be of potential benefit to pathologies
with elevated nitric oxide production by preventing catabolism of methylarginines and enhancing
their ability to inhibit nitric oxide synthases.(2) Examples include DDAH1 inhibitors that regulate
vascular tone, that improve survival in models of septic shock, and that reduce collagen
deposition and epithelial proliferation in idiopathic pulmonary fibrosis.(13-15) DDAH1 is also
upregulated in melanomas (and other cancers) and likely serves to enhance nitric oxide
production, which correlates with poor patient survival.(16-19) Potent and selective inhibitors of
DDAH1 are of use as biochemical tools for elucidating the role of this enzyme in pathological
states, and for potential therapeutic development.(20) We previously reported a covalent
inactivator of DDAH1, N5-(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) and demonstrated its
ability to target DDAH1 expressed within cultured cells and to selectively inhibit nitric oxide
production in a melanoma cell line with upregulated DDAH1.(16) Herein, we describe the
optimization of this inhibitor by dissecting the compound into two fragments, and characterizing
each fragment individually. The stepwise characterization of these fragment components led to
non-obvious improvements resulting in reconstruction of a very potent and selective inactivator
with cellular availability as a promising probe for DDAH1 study.
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MATERIALS AND METHODS
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Expression and purification of human DDAH1
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Wild-type human DDAH1 bearing an N-terminal His6 affinity tag was expressed using an
expression plasmid encoding a reengineered N-terminus (pET28a-hDDAH-1re) to avoid N-
terminal nonenzymatic gluconylation, as described previously.(21) Briefly, recombinant His6-
DDAH1 was purified after expression in E. coli BL21(DE3) by affinity and hydrophobic
interaction chromatography according to the previous procedure(21) with the following
modifications: Elution Buffer (25 mL) (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole,
15% glycerol pH 8.0) was used to elute protein from the Ni-NTA affinity resin (8mL, Qiagen).
The eluent was diluted into Buffer A (50 mM NaH2PO4, 3.0 M NaCl, 15 % glycerol, pH 7.0) to a
final NaCl concentration of 1.8 M and loaded onto a 1.5 × 18 cm phenyl-sepharose column (GE
Healthcare, Piscataway, NJ). Proteins were eluted using a stepwise gradient of Buffer A
supplemented with 1.8 M NaCl, and subsequent steps with 1.6 M, 1.3 M, and 1M NaCl. As
gauged by SDS-PAGE, fractions containing DDAH1 were pooled and concentrated using an
Amicon Centrifugal Filter (Millipore, Billerica, MA) with a 10 kDa molecular weight cutoff. The
final protein was dialyzed overnight in Dialysis Buffer 1 (2 mM 1,10-phenanthroline, 100 mM
KCl, 10 % glycerol, pH 7.3) followed by 2 × 4 h dialyses in Chelex-100 (BioRad) treated
Dialysis Buffer 2 (50 mM KH2PO4, 100 mM KCl, 10 % glycerol, pH 7.3), flash frozen and
stored in aliquots at -80 oC. All buffers were made using deionized water.
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Synthetic Methods
A general synthetic scheme is provided in Supporting Information (Figure S1) to
illustrate the transformations described below.
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2-Chloroethanimidic acid, ethyl ester: Chloroacetonitrile (2.55 mL, 40.3 mmol) and
anhydrous ethanol (2.6 mL, 22.4 mmol) were added to 22 mL of anhydrous diethyl ether and
cooled to 0 °C. The solution was bubbled through with HCl(g) until a white precipitate formed.
The precipitate was washed with cold diethylether followed by cold hexanes to yield 2-
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chloroethanimidic acid, ethyl ester, HCl (1.8 g, 78 %). H-NMR (300 MHz, D2O): 4.66 (2 H,
overlapped with reference peak), 4.16 (2 H, s), 1.16 (3 H, d, J = 7.2 Hz).
2-Chloropropanimidic acid, ethyl ester: Chloropropionitrile (3.5 mL, 39.56 mmol) and
anhydrous ethanol (2.6 mL, 22.4 mmol) were added to 20 mL of anhydrous diethylether and
cooled to 0 °C. The solution was bubbled through with HCl(g) until a white precipitate formed.
The precipitate was washed with cold diethyl ether followed by cold hexanes to yield 2-
1
chloropropanimidic acid, ethyl ester, HCl. (1.4 g, 46 %). H-NMR (400 MHz, D2O): 4.10 (1 H,
q, J = 7.6 Hz), 3.19 (2 H,m), 1.48 (3 H, dd, J1 = 6.8 Hz, J2 = 22.8 Hz), 1.07-1.15 (3 H, m).
General methods for synthesis of chloroamidine inactivators: Nα-(Tert-butoxycarbonyl)-
L-ornithine or Nα-(t-butoxycarbonyl)-L-lysine (1 mmol of either) was dissolved in 5 mL water at
0 °C followed by the dropwise addition of 2.5 M NaOH to bring the pH to 10. Either 2-
chloroethanimidic acid methyl ester or 2-chloropropanimidic acid methyl ester was added in
portions (2.5 mmol final), keeping the reaction at pH 10 through dropwise addition of NaOH.
After addition of either of the imidic acids was completed, the reaction was stirred at 0 °C for 2 h
and at room temperature for 1 additional hour. The reaction was neutralized by the dropwise
addition of 1 M HCl and stirred at 4 °C for 1.5 days. Volatile solvents were removed by reduced
pressure rotary evaporation and the resulting compound was purified using a Teledyne/Isco
CombiFlash Rf200 RediSep Rf 26 g C18 Reverse Phase column (0.1 % TFA/H2O to 0.1 %
TFA/MeOH linear gradient) at the Texas Institute for Drug and Diagnostic Development
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(University of Texas at Austin). Volatile solvents were removed by reduced pressure rotary
evaporation and the remaining residue treated with 4 M HCl in 1,4-dioxane for 2 h to remove the
t-Boc protecting group. Volatile solvents were removed as above to yield the desired products.
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N6-(1-Imino-2-chloroethyl)-L-lysine, HCl (Cl-NIL) 1H-NMR (400 MHz, D2O): 4.26 (2 H,
s), 3.87 (1 H, t, J = 6 Hz), 3.22 (2 H, t, J = 7.2 Hz), 1.80-1.84 (2 H, m), 1.57-1.60 (2 H, m), 1.38-
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1.47 (2H, m). C-NMR(D2O): 171.58, 162.19, 52.24, 41.64, 38.61, 28.75, 25.51, 21.0. ESI
(m/z) M + H+ calcd for C8H17ClN3O2, 222.10038; found 222.10029.
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N6-(1-Imino-2-chloroisopropyl)-L-lysine, HCl (Me-Cl-NIL) H-NMR (400 MHz, D2O):
4.68 (1 H, overlapped with reference peak), 3.831 (1 H, t, J = 6.2 Hz), 3.188 (2 H, t, J = 7.2 Hz),
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1.25-1.91(9 H, m). C-NMR(D2O): 172.54, 166.21, 53.07, 51.63, 42.06, 29.38, 26.0, 22.04,
21.53. ESI (m/z) M + H+ calcd for C9H19ClN3O2, 236.11603; found 236.11632.
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N5-(1-Imino-2-chloroisopropyl)-L-ornithine, HCl (Me-Cl-NIO) H-NMR (400 MHz,
D2O): 4.70 (1 H, overlapped with reference peak), 3.95 (1 H, t, J = 6.4 Hz), 3.241 (2 H, t, J = 6.8
Hz), 1.63-1.94 (6 H, m), 1.61 (3 H, d, J = 7.2 Hz). 13C-NMR(D2O): 172.44, 166.73, 53.06, 51.89,
41.95, 27.32, 22.63, 22.04. ESI (m/z) M + H+ calcd for C8H17ClN3O2, 222.10031; found
222.10023.
The activity-based protein profiling probe CAA-probe (N-but-3-ynyl-2-
chloroacetamidine) was synthesized as previously described, as was the inactivator Cl-NIO (N5-
(1-imino-2-chloroethyl)-L-ornithine).(16, 22) Unless specified otherwise, all other chemicals were
from the Sigma Aldrich Chemical Co. (St. Louis, MO).
Time-dependent inactivation kinetics
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Purified His6-DDAH1 (60 – 100 nM) was incubated with inactivators (Cl-NIL, Cl-NIO,
Me-Cl-NIL, or Me-Cl-NIO) (0-64 μM) in Reaction Buffer (100 mM KH2PO4, 100 mM KCl, 2
mM EDTA, 1 % Tween-20, pH 7.3). To test for time-dependent loss of activity, aliquots were
removed from the incubations at time points (0-60 min) and diluted two-fold into an assay
solution containing a large excess (300 μM) of the alternative substrate, S-methyl-L-thiocitrulline
(SMTC, Figure S2, KM = 15 μM).(23) The remaining enzyme activity was assayed by detecting
methanethiol release upon substrate hydrolysis using 7-diethylamino-3-(4'-maleimidylphenyl)-4-
methylcoumarin (CPM), as described elsewhere.(24-26) The percent remaining activity across the
experimental timescale was fitted to a single exponential to determine the observed inactivation
rate at each concentration (kobs). The plot of kobs values versus inhibitor concentration was fitted
to a quadratic equation for tight binding to give KI if the enzyme and inhibitor concentrations
were similar [kobs=R((([E]+[I]+KI)-(([E]+[I]+KI)2-(4[E][I]))1/2)/2[E]), letting R and KI float].(27, 28)
If the inhibitor concentrations were well above that of the enzyme, the kobs values versus inhibitor
concentration plot was fitted instead to a non-linear version of the Kitz and Wilson plot to
determine KI and kinact values.(29)
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IC50 determination with DDAH1
To determine whether purified DDAH-1 is inhibited by L-Nva, purified His6-DDAH1 (4
µM) was incubated with varying concentrations of L-Nva (50 µM − 10 mM) in the presence of
ADMA (340 µM), KCl (100 mM), K2HPO4 buffer (100 mM), EDTA (5 mM) and Tween-20
(0.02%) at pH 7.4, for 45 min at 25 °C. The reaction was quenched with 10 µL of trichloroacetic
acid (6 M) and the formation of the L-citrulline product was determined using a color developing
reagent (COLDER assay) which detects compounds containing urea functional groups, as
described previously.(30) The IC50 value and Hill coefficient (h) were fitted using
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Activity(%)=(100/(100/(1+(IC50/[L-Nva])h))) and the Ki value calculated by the method of Cheng
and Prusoff, assuming competitive inhibition and using a KM for ADMA of 170 μM.(23, 31)
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Partition ratio determination
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To determine the partition ratio of DDAH1 inactivation by Cl-NIL, purified His6-
DDAH1 (62 nM) was incubated with varying concentrations (substoichiometric to
suprastoichometric) of Cl-NIL (0-400 nM) for 1 h and then assayed for remaining activity by
dilution into SMTC as described above. The remaining fractional activity values were plotted
versus the ratio of Cl-NIL to DDAH1 concentrations and a linear fit of the initial points was
extrapolated to find the intercept, which gives the partition ratio.(32)
In cell target validation
To compare the efficacy of DDAH1 inactivators for modification of the active site in a
cultured cell environment, a competitive labeling strategy was used.(16, 22) Cultured HEK293T
cells were seeded in two 6-well polystyrene plates using complete growth medium containing
DMEM with 10 % FBS (Invitrogen, Carlsbad, CA) and grown to 80 % confluency. The pEF6a-
DDAH-1 plasmid, which encodes an N-terminally myc-tagged DDAH-1, was transiently
transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 24
h, spent medium was removed and the cells were each washed with 1 mL of 0.5 mL PBS at pH
7.2 (Invitrogen, Carlsbad, CA). To separate wells, the DDAH1 inactivators were added (10 μM
each of Cl-NIL, Cl-NIO, Me-Cl-NIL, or Me-Cl-NIO). The compounds were allowed to incubate
at 37 ºC in an atmosphere of 5 % CO2 for 15 min before the addition of CAA-probe (110 μM),
followed by an additional 10 min incubation. After treatment, cells were washed two times with
PBS (1 mL) to remove inhibitors remaining in the extracellular environment and harvested in 1
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mL PBS followed by centrifugation at 14000 g for 5 min at 4 ºC. Cell pellets were stored at – 80
ºC before analysis. The frozen cell pellets were thawed, lysed, labeled with biotin-PEO3-azide
using bioorthogonal chemistry and analyzed by two-color Western blot detection, as described
below.
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In cell apparent IC50 determination
HEK293T cells were grown, seeded, and transfected with pEF6a-DDAH-1 as described
above. Stock solutions of Cl-NIL (10 mM) were diluted into complete growth medium in the
cultures (1000 μL each well) to give final concentrations of 0.078, 0.156, 0.313, 0.625, 1.25, 2.5,
5, 10, 20, 40, 80, and 0 μM. The resulting cultures were subsequently incubated for 15 min at 37
ºC in an atmosphere of 5 % CO2 before addition of CAA-probe (110 μM), followed by an
additional 10 min incubation. After treatment, cells were washed two times with PBS (1 mL) to
remove the media, and harvested in 1 mL PBS followed by centrifugation at 14000 g for 5 min at
4 ºC. Cell pellets were stored at – 80 ºC prior to analysis.
Frozen cell pellets were lysed and labeled with biotin-PEO3-azide using bioorthogonal
chemistry as described earlier.(16, 22) Briefly, cell pellets were resuspended in PB Buffer (10 mM
sodium / potassium phosphate buffer at pH 8.0 containing the complete mini EDTA-free
protease cocktail inhibitor (Roche, Indianapolis, IN) and Triton X-100 (1%)) and then were
subjected to three cycles of vortexing, freezing in liquid N2, and thawing at room temperature.
The resulting solution was centrifuged at 14000 g for 5 min at 4 ºC to pellet insoluble cell debris,
which was then discarded. The remaining supernatant was analyzed for total protein
concentration using the Bradford assay. Biotin-PEO3-azide was appended to alkyne functional
groups in the resulting mixture using the Cu(I) catalyzed 1,3 dipolar cycloaddition reaction as
follows: 25 μg total protein in 50 μL PB buffer was mixed with 0.5 μL of biotin-PEO3-azide (2.5
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mM), 0.5 μL CuSO4 (50 mM), 0.5 μL tris(2-carboxyethyl)phosphine (TCEP, 50 mM) and 1.65
μL tris[(1-benzyl-1H-1,2,3-trazol-4-yl)methyl]amine (TBTA, 1.5 mM), vortexed and incubated
at 25° C for 1 h. Reactions were quenched by addition of 2 × SDS loading buffer and heat
inactivated at 100 °C for 10 min.
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Samples were directly used for SDS-PAGE or stored at -80 °C before analysis. Two-
color Western blot detection was used to detect the expression levels of DDAH1 through
recognition of the genetically encoded myc-tag, and detection of any biotin-labeled proteins, as
described previously.(21) Fluorescent emission images were captured using an Odyssey Infrared
Imaging System (Li-Cor Biosciences, Lincoln, NE) at the Core DNA Facility (University of
Texas, Austin). Fluorescence intensities (I) for both 680 nm and 800 nm channels were
integrated. The response to the myc-tag (I680, displayed in red) was used normalize the response
to the biotin tag (I800, displayed in green) for any loading differences. The resulting I680 values,
expressed as a percentage of response to the sample with no Cl-NIL (100%), were plotted versus
Cl-NIL concentration and fitted (Fluorescence Intensity (%) = 100% - (100%/(1+(IC50/[Cl-
NIL])h))) to determine IC50 and the Hill coefficient (h).
X-ray crystallography
Prior to crystallization set-up, protein was incubated with Cl-NIL in the following
mixture: 12 mg/mL purified recombinant human His6-DDAH1 in 100 mM KCl, 100 mM
potassium phosphate, pH 7.3, 10 % glycerol, 2 mM dithiothreitol, 0.7 mM Cl-NIL. At the
Macromolecular Crystallography Facility (University of Texas at Austin) co-crystals were grown
at 4 ˚C of the Cl-NIL:His6-DDAH1 complex using the sitting drop method from the
inactivator:enzyme mixture and 20 % PEG 3350, 0.2 M NaCl. Prior to data collection, a crystal
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was transferred briefly to a drop of 20 % PEG 3350, 0.2 M NaCl, 0.1 M HEPES at pH 7.2, 10 %
propylene glycol for cryoprotection. The crystal was mounted in a cryoloop (Hampton
Research, Laguna Niguel, CA), flash cooled in liquid nitrogen, and mounted in the cold stream
on the goniostat.
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X-ray diffraction data were collected from the co-crystal at 100 K at the Advanced Light
Source (ALS) beamline 5.0.3 at the Lawrence Berkeley National Laboratory. Diffraction images
were processed and data reduced using HKL2000.(33) The structure of the complex was solved by
molecular replacement with MOLREP(34) using the structure of human DDAH1 (PDB accession
code 3I2E)(21) as the search model. Model building was carried out using Coot.(35) Refinement of
models was done using PHENIX.(36) There were several rounds of refinement followed by
manual rebuilding of the model. To facilitate manual rebuilding, a difference map and a 2Fo-Fc
map, σA-weighted to eliminate bias from the model(37), were prepared. A portion (5 %) of the
diffraction data was set aside throughout refinement for cross-validation.(38) MolProbity(39) was
used to determine areas of poor geometry and to make Ramachandran plots. The final model
does not include side chain atoms for which there was no observed electron density. Coordinates
and structure factors were deposited in the Protein Data Bank (accession code 6DGE).
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We previously reported a covalent inactivator of human DDAH1 that was designed to
mimic the asymmetric dimethylarginine substrate (ADMA) of the enzyme (Figure 2):
Substitution of the guanidine in the substrate with a 2-chloroacetamidine moiety resulted in an
irreversible inhibitor, Cl-NIO (KI = 1.3 μM, kinact = 0.34 min-1).(16) Herein, we further characterize
and improve upon this strategy for designing covalent inhibitors for DDAH1. Optimizing
covalent inhibitors often requires balancing non-covalent affinity and reactivity to achieve
inhibition that is both potent and selective.(40) To assess both of these contributions separately,
we conceptually dissected Cl-NIO into two parts and analyzed them separately: L-norvaline (L-
Nva), which was expected to provide non-covalent binding affinity, and 2-chloroacetamidine
(CAA), which was expected to provide the reactive group that enables covalent modification
(Figure 2).(41)
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+H3N
-OOC
CH3
N
H
H
N
CH3
+
ADMA
NH2
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mimic
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+H3N
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-OOC
Cl
+
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dissect
NH2
noncovalent
covalent
H2N
+H3N
H
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-OOC
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-Nva
CAA
probe
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-OOC
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H
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CAA-probe
Cl-NIL
methylation
+H3N
-OOC
CH3
H
H
n = 1: Me-Cl-NIO
n = 2: Me-Cl-NIL
N
Cl
n
+
NH2
Figure 2. Structure of DDAH1 Ligands and Design Intent. A previously reported substrate
(ADMA) mimic and irreversible DDAH1 inactivator (Cl-NIO) is shown with its constitutive
fragments (CAA and L-Nva), an activity-based protein profiling reagent (CAA-probe), an
extended-linker analog (CL-NIL) and two analogs designed to better mimic the methylation of
ADMA (Me-CL-NIO, Me-CL-NIL).
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L-Nva is a rapid reversible inhibitor of DDAH1 with an IC50 value of 1.42 ± 0.06 mM, which
was used to calculate a Ki of 470 μM, assuming competitive inhibition (Figure 3, Table 1).
Somewhat surprisingly, we found that the CAA reactive group also provides significant
noncovalent binding to DDAH1 (KI = 310 uM), reflecting a noncovalent affinity more potent
than L-Nva (Figure 3). CAA binds approximately 10-fold more tightly to human DDAH1 than
to the Pseudomonas aeruginosa homolog PaDDAH (KI = 3 mM), despite high active-site
similarity.(41) The human DDAH1 active site is slightly smaller than that of PaDDAH due to a
neighboring Leu271 residue (Gly246 in PaDDAH), which may improve selective interactions
made with ligands to human DDAH1 and provide an opportunity for isoform selective inhibitor
design.(13, 42)
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A)
B)
C)
Figure 3. DDAH1 Inhibition by CAA and L-Nva. A) Preincubations of human DDAH1 with
CAA ( 5 (!), 10("), 20 (#), 50 (!), 100 ("), 200 ($), 500 (%) μM) are diluted into excess
substrate at various time points to measure activity remaining and fit as described in
Experimental Procedures. B) The concentration dependence of kobs values taken from A) is fit as
described in Methods to determine kinact (4.0 ± 0.2 min-1) and KI (310 ± 30 μM) values for CAA
inactivation of DDAH1. C) Reversible L-Nva inhibition of DDAH1 was fit as described in
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Methods to determine IC50 (1.42 ± 0.06 mM), Hill coefficient (0.97 ± 0.3) and a calculated Ki
(470 ± 20 μM).
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Table 1. Kinetic parameters of DDAH1 inactivation / inhibition.
kinact
(min-1)
KI
kinact / KI
(M-1s-1)
Compound
Cl-NIOa
CAA
(μM)
0.34 ± 0.07
4.0 ± 0.2
N.A.b
1.3 ± 0.6
310 ± 30
470 ± 20c
0.19 ± 0.04
N.D.
(4.4 ± 0.3) × 103
(2.2 ± 0.3) × 102
N.A.
L-Nva
Cl-NIL
0.22 ± 0.02
N.D.d
(1.9 ± 0.6) × 104
208 ± 3
Me-Cl-NIO
Me-Cl-NIL
N.D.
N.D.
440 ± 20
a Values taken from reference (16).
b Not Applicable
c Ki value
d Not determined. Inactivation kobs values are linear up to 32 μM inactivator.
To estimate the total noncovalent affinity afforded by both fragments, we used the relationship
between Gibbs free energy and equilibrium constants, here the inhibition constants given by Ki
and KI, to calculate the free energy for binding of each fragment, added the two values, and back-
calculated the resulting theoretical Kd to be approximately 150 nM, a value somewhat lower than
the KI for Cl-NIO (1.3 μM).(16) However, linking inhibitory fragments can result in affinity
improvements much greater than that predicted by additive binding due to the entropic advantage
of unimolecularity.(43, 44) The observation that the affinity of Cl-NIO is even poorer than just the
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additive values of its constituent fragments suggests that the two fragments may not be optimally
linked.
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In addition to noncovalent affinity, the CAA fragment also causes irreversible inactivation of
DDAH1 (kinact = 4 min-1) (Figure 3, Table 1). We determined the rate constant for inactivation to
be > 10-fold larger than that of Cl-NIO, indicating that the fragment-sized CAA can bind in a
more reactive orientation when not constrained by the tethered amino acid L-Nva moiety. As
was seen with noncovalent affinity above, this analysis also suggests that the CAA and L-Nva
fragments are not optimally linked to allow either their binding or reactivity to be fully realized.
These findings prompted us to alter the linker to optimize noncovalent binding affinity, so
we considered changing the length between the amino acid and amidine moieties of Cl-NIO.
Substrate analogs with shorter linkers between the amino acid and methylguanidine groups, such
as 4124W (Figure S2), are DDAH inhibitors, but are not attacked by the active-site Cys
residue.(45) Therefore, we did not shorten the linker length because reaction with Cys is required
for inactivation. Other studies have shown that Arg analogs with long Nω-substituents on the
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guanidine can be well accommodated by the human DDAH1 active site and result in reversible
46)
inhibition, as demonstrated by L-257 (Figure S2) and other analogs.(13,
Therefore, we
anticipated that DDAH1 could accommodate compounds larger than ADMA, and synthesized a
lengthened version of Cl-NIO that has a linker one additional methylene in length, N5-(1-imino-
2-chloroethyl)-L-lysine (Cl-NIL) (Figure 2).
We determined that Cl-NIL is a time- and concentration-dependent inhibitor of DDAH1,
with KI and kinact values of 0.19 μM and 0.22 min-1, respectively (Figure 4, Table 1). Inhibition
cannot be reversed upon dilution into excess substrate, and only 1 equivalent of Cl-NIL is
required to inactivate DDAH1. The longer linker was well accommodated by the enzyme,
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despite being larger than the substrate ADMA, and increased the noncovalent affinity by
approximately 5-fold over that of Cl-NIO. The Cl-NIL kinact is similar to that of Cl-NIO,
suggesting that changing the linker length, but not the position of substitution (the same position
on CAA is derivatized), is not sufficient to allow the most reactive binding orientation to be
achieved. Although a significant improvement in noncovalent affinity is measured, a huge gain,
as is sometimes observed in fragment linking, is not achieved. The free energy difference
calculated from the difference between Cl-NIO and Cl-NIL KI values is approximately 1
kcal/mol (ΔΔG=-RTln(KI,Cl-NIL/KI,Cl-NIO). This value is close to the 0.7 kcal/mol increase in
binding due to hydrophobic interactions typically estimated for addition of a methyl group,(47) so
the increase in binding affinity may be derived solely from increased hydrophobic interactions
rather than allowing optimal positioning of the fragments.
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A)
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Figure 4. DDAH1 Inactivation by Cl-NIL. A) Time- and Concentration-dependent DDAH1
(0.06 μM) inactivation is observed with increasing concentrations of Cl-NIL: 0 ($), 0.0125 (%),
0.025 (#), 0.05 ("), 0.1 (+), 0.2 (#), 0.4 ("), 0.8 (!), 1.6 (!), 3.2 ($) μM in the
preincubation mixture prior to dilution into excess substrate. B) A plot of kobs values for
inactivation derived from A) is fit by a non-linear version of the Kitz and Wilson method (dashed
line, omitting points where [CL-NIL] ≤ 0.2 μM) to derive KI (0.2 ± 0.1 μM) and kinact (0.22 ± 0.02
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min-1) values, and is also fit by a quadratic equation (solid line, no omissions) to derive KI (0.19
± 0.04 μM), as described in Methods. C) A plot of the fraction of remaining DDAH1 activity at
extended incubation times with increasing molar equivalents of Cl-NIL is used to determine the
partition ratio (0.98 ± 0.03 equivalents of Cl-NIL : equivalents of DDAH1) for inactivation by
fitting the initial three points to a linear fit and solving for the x-intercept.
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In an attempt to further improve positioning of the reactive CAA moiety and to more closely
mimic positioning of the substrate, Nω-methylated analogs of both Cl-NIO and Cl-NIL were
synthesized (Figure 2). These analogs, Me-Cl-NIO and Me-Cl-NIL, were designed to more
closely mimic the dimethylated status of the substrate ADMA (Figures S3, S4, Table 1).
Although both compounds were irreversible time-dependent inactivators, they had much weaker
noncovalent affinity, with neither showing saturation kinetics at concentrations ≤ 30 μM. The
second order inactivation rate constants (kinact/KI) for these methylated derivatives were similar to
that of the CAA fragment, but 1 or 2 orders of magnitude lower than the unmethylated
congeners. Methylation also lowers the non-enzymatic reactivity of these chloroacetamidines by
approximately 7-fold (Figure S5). Therefore, methylation decreases the reactivity of this
electrophile and likely exceeds the ability of the active site to readily accommodate substituted
amidines.
It is not easy to predict how well these inactivators will gain access to the targeted cytoplasmic
DDAH1 enzyme because accessibility is likely mediated by cationic amino acid transporters.(10,
48)
To assay for target engagement between Cl-NIL and DDAH-1 within cultured human cells,
we used an activity-based protein profiling probe consisting of the CAA fragment appended to
an alkyne (CAA-probe) (Figure 2).(22) When this probe is incubated with cultured HEK293T
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cells that are episomally overexpressing DDAH1 (the probe is not sensitive enough to detect
endogenous DDAH1 in these cells), bioorthogonal chemistry can be used to append a biotin
label to the probe and enable visualization of labeled proteins. Two color western blotting
enables the separate monitoring of biotin and a myc-tag that is genetically incorporated into the
expressed DDAH1. At the times and CAA-probe concentrations used herein, the only
significant band detected during imaging is overexpressed DDAH1. Previously, we used this
system to demonstrate target engagement of DDAH1 by Cl-NIO within cultured cells, and using
the relative fluorescence of each signal, estimated an apparent cellular IC50 of approximately 7
μM.(16) In this case, apparent IC50 values provide experimental guidelines for use of these
inhibitors rather than reflect the true affinity of the interaction since inhibition is both time-
dependent and irreversible and will be strongly dependent on the particular experimental
conditions used.
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Here, we used a competition assay to detect the ability of DDAH1 inhibitors, preincubated for
15 min at 10 μM each, to prevent labeling of DDAH1 by the CAA-probe in cultured HEK293T
cells, as described above (Figure 5). Both Cl-NIO and Cl-NIL effectively inhibited labeling of
DDAH1 by the CAA-probe, but the methylated analogs, Me-Cl-NIO and Me-Cl-NIL did not.
These results are consistent with our biochemical characterization of purified DDAH1 inhibition,
which revealed the methylated analogs as much less potent inhibitors. An apparent cellular IC50
for Cl-NIL was determined to be 10 ± 2 μM. Even though the noncovalent affinity of Cl-NIL is
approximately 5-fold more potent than Cl-NIO, this advantage is not conferred on the relative
apparent cellular IC50 values, indicating that under these particular experimental conditions, other
factors (e.g. transport or kinact) may be limiting. Nonetheless, these experiments provide evidence
that Cl-NIL can block active-site labeling of DDAH1 expressed within cultured cells.
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Figure 5. DDAH1 Labeling Within Cultured HEK293T Cells. A) Cultured human HEK293T
cells episomally expressing myc-tagged DDAH1 were treated with 1) no inactivator, 2) Cl-NIL,
3) Cl-NIO, 4) Me-Cl-NIL, 5) Me-Cl-NIO, 6) no CAA-probe, with each inactivator treatment at
10 μM. Cell cultures were incubated for the same lengths of time, treated with CAA-probe
(except for 6, which is a ‘no probe’ control) to label available DDAH1 active sites, and assayed
as described in the Experimental Section. Two-color western blotting is used to give a red signal
in response to the genetically encoded myc tag and to serve as a loading control. A green signal
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is a response to DDAH1 active sites covalently labeled by CAA-Probe. Yellow indicates both
signals are present. B) Cultured cells are treated with various concentrations of Cl-NIL (shown
in μM above each lane) and treated as described in A). C) Using the intensity of the red
fluorescent signal to normalize loading, the intensities of the green fluorescent signals are plotted
for each concentration and fitted (omitting the outlier at 0.16 μM) to give an apparent in cell IC50
10 ± 2 μM, with a Hill coefficient of 0.8 ± 0.2 under these experimental conditions and
timeframe.
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One potential off-target of particular interest is arginase, because the substrate of this enzyme
(L-Arg) is quite similar to that of DDAH1 (ADMA), and because the activity of arginase can also
impact nitric oxide production through L-Arg depletion.(49) Using purified enzyme, we
determined that Cl-NIL is not a very effective inhibitor of human dimanganese (II) arginase, and
measured an IC50 value of 20 mM, which represents >100,000-fold selectivity (based on
comparison of IC50 and KI values) (Figure S6). Likely the ω-carbon and chloro substituent in Cl-
NIL prevents coordination to, or sufficient proximity to, the metal center, as is seen with the
endogenous L-Arg substrate during turnover.(49) Off-target inhibition by covalent inactivators
can lead to cytotoxicity, so we also evaluated the cell viability of cultured HEK293T cells and
found the concentration at half maximum for cell killing to be 118 ± 2 μM, which provides a
wide window between the concentrations effective for blocking the active-site of DDAH1 and
the concentrations that are cytotoxic (Figure S7).
Because Cl-NIL appears to be a promising tool for the study of DDAH1, we chose this
inhibitor for structural determination. A 1.9 Å resolution X-ray co-crystal structure of the Cl-
NIL:DDAH1 complex was determined (Table 2). The overall protein fold is very similar to that
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of product (L-citrulline)-bound DDAH1 reported earlier (2JAI), with a RMSD between 271 α-
carbon atom pairs of 0.5 Å.(13) However, the active sites are different. The Fo-Fc omit map,
omitting the ligand and Cys274, shows strong electron density at the active site of DDAH1,
consistent with active site binding of a ligand, and continuous density is observed between this
bound ligand and the active-site Cys274 residue (Figure 6). The covalent adduct can be well fit
by a modification of the active site Cys thiol with N5-(1-iminoethyl)-L-lysine (Cl-NIL after loss
of the chloro substituent), covalently linked through a thioether bond with the ω-carbon of the
amidine. This adduct has the same thioether connection observed when P. aeruginosa DDAH is
inactivated by the CAA fragment, suggesting similar inactivation mechanisms.(41) The longer
linker found in Cl-NIL can be accommodated with only rotomeric changes in comparison to a
DDAH1 structure determined with bound product, with only two obvious changes apart from
Cys modification. First, the catalytic His173 residue is rotated to create extra volume in the
active-site cavity. A similar rotation is seen when DDAH1 binds Arg-based inhibitors bearing
long Nω-substitutents, and appears to be a general way for this enzyme to accommodate larger
ligands.(2, 46) The second change is instead seen in the ligand where the extreme ends of the
inactivator (the amidine ω-nitrogen and the amino acid moiety) bind in identical positions to that
of product, but the linker region in between bows out like an accordion to accommodate the extra
methylene group (Figure 6). A structural consequence of this bowing is loss of one of the two
potential H-bonds formed between the amidine nitrogens and the active-site Asp79. In
comparison, a bidentate interaction is observed between the urea nitrogens of the product L-
citrulline and Asp79, presumably through two H-bonds. This observation suggests that if further
optimization of Cl-NIL is pursued, strategies to recapture this second interaction with Asp79
may lead to further increases in potency.
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Table 2. Crystallographic Data
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CL-NIL:DDAH1
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Space group
P41
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Cell constants
a=b=55.2, c=89.5 Ǻ
Resolution (Å)a
50.-1.91 (1.94-1.91)
Rmerge (%)
0.072 (0.500)
10.3 (3.4)
100.0 (100.0)
20,872
7.5 (6.7)
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<I/σI>
Completeness (%)
Unique reflections
Redundancy
# of residues
# of protein atoms
# of ligand atoms
2024
13
# of solvent atoms
Rworking
83
0.215
Rfree
0.251
Average B factor for protein atoms (Å2)
Average B factor for ligand atoms (Å2)
Average B factor for solvent atoms (Å2)
rms deviation from ideality
bonds (Å)
24.7
22.0
24.2
0.004
0.864
angles (º)
Ramachandran plot
% of residues in favored region
% of residues in additional allowed region
96.7
3.3
a Values in parentheses correspond to highest resolution shell
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B)
A)
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C274
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C274
F76
H173
R145
D79
L
-Cit
L
-IEL
L-IEL
Figure 6. Structural Analysis of DDAH1 Inactivation by Cl-NIL. A) After inactivation of
DDAH1 by Cl-NIL, the Fo-Fc omit map for Cys274 and the covalently-bound ligand is shown in
blue, contoured at 2σ. There is continuous density from Cys274 to the covalently bound
inhibitor. B) X-Ray structures of the Cl-NIL:DDAH1 complex (grey) and the L-citrulline (L-
Cit):DDAH1 complex (light blue, from PDB accession code 2JAI) are superimposed with the
ligands of each centered in the image. Loss of chloride from Cl-NIL results in the covalent
attachment of N5-(1-iminoethyl)-L-lysine (L-IEL). This comparison highlights a covalent linkage
formed between Cys274 and L-IEL, one-fewer H-bond between L-IEL and Asp79 (center, back)
in comparison with L-Cit, and rotation of His173 to accommodate the extended length of the
inactivator. In both panels, heteroatoms are colored in blue, red or yellow, for nitrogen, oxygen
or sulfur, respectively.
SUMMARY AND CONCLUSIONS
The origins of noncovalent binding affinity and reactivity were studied for the DDAH1
inactivator Cl-NIO by dissecting the compound into two fragments expected to correspond
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roughly to the “binding” fragment (L-Nva) and the “reactive” fragment (CAA). Surprisingly, the
CAA fragment provided more noncovalent affinity than the other fragment, and the additive
properties of each suggested that their linkage did not enable the full potential of either fragment
to be expressed in Cl-NIO. In response, we used a longer linker in Cl-NIL, which was not an
obvious modification since Cl-NIO more closely resembles the size of the enzyme’s substrate.
Despite its larger size, Cl-NIL is well accommodated at the active-site through rotation of a
catalytic His173 residue, which seems to be a common method for DDAH1 to accommodate
larger ligands, and by a bowing out of the ligand with loss of one of potential H-bond from the
amidine to an acid site Asp79 residue. Although the longer linker still does not allow the
fragments to achieve the optimal binding, the observed ligand:enzyme structure does suggest
possible ways to further improve noncovalent affinity. Regardless, to the best of our knowledge,
the KI of Cl-NIL is more potent than any KI, Ki or IC50 value reported for extant DDAH1
inhibitors.(20) In addition, the kinact/KI value (1.9 × 104 M-1s-1) exceeds the catalytic efficiency of
DDAH1 for its endogenous substrate ADMA (kcat/KM = 1.6 × 102 M-1s-1) by approximately 100-
fold.(23) We would have not undertaken this linker modification unless prompted by the separate
analysis of the constitutive fragments. The type of covalent inhibition by Cl-NIL is likely best
classified(40) as either affinity labeling, due to the inherent reactivity of the CAA fragment, or as
mechanism-based inactivation, through comparison to mechanistic studies that characterized the
inactivation of the homologous enzyme protein arginine deiminase by the related compound Cl-
amidine.(50) Cl-NIL is selective for inhibition of DDAH1 instead of arginase, and shows little or
no toxicity to cultured HEK293T cells at concentrations used to validate target engagement of
cytoplasmic DDAH1. Therefore, Cl-NIL is a promising biochemical tool for the study of
DDAH1-related pathways. Dissecting a ligand to parse the contributions of its constituent
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fragments has proved helpful in the past to characterize interactions between ligand and
protein,(43, 44) and has proved useful here to optimize the most potent DDAH1 inactivator reported
to date.
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ASSOCIATED CONTENT
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Supporting Information.
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The following files are available free of charge:
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Supporting figures for synthesis, DDAH1 inhibitors, time-dependent DDAH1 inactivation by
Me-Cl-NIL, Me-Cl-NIL, nonenzymic reactivity of substituted chloroacetamidines, inhibition of
arginase by Cl-NIL, and HEK293T cytotoxicity assay for Cl-NIL (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: walt.fast@austin.utexas.edu (W.F.).
ORCID
Walter Fast: 0000-0001-7567-2213
NOTES
The authors declare no competing financial interest.
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ACKNOWLEDGEMENT
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For purified human arginase, we thank Everett Stone (University of Texas, Austin). This work
was supported in part by grants from the National Institutes of Health (GM69754) and the Robert
A. Welch Foundation (F-1572). A.T. acknowledges a postdoctoral fellowship from the National
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Institutes of Health (1K12 GM102745).
Additional assistance was provided by the
Macromolecular Crystallography Facility, with financial support from the College of Natural
Sciences, the Office of the Executive Vice President and Provost, and the Institute for Cellular
and Molecular Biology at the University of Texas at Austin. The Berkeley Center for Structural
Biology is supported in part by the National Institutes of Health, National Institute of General
Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is
supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under Contract No. DE-AC02-05CH11231.
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