A click chemistry mediated in vivo activity probe for dimethylarginine dimethylaminohydrolase

Article abstract of DOI:10.1021/ja906432e

(Chemical Equation Presented) Asymmetric N^ω,N ^ω-dimethyl-L-arginine (ADMA) is an endogenously produced inhibitor of human nitric oxide synthase and an emerging biomarker for cardiovascular disease. Concentrations of ADMA are controlled by two isoforms of its catabolic enzyme dimethylarginine dimethylaminohydrolase (DDAH), the dysregulation of which has been studied as a mediating factor for endothelial dysfunction. A two-part, click-chemistry mediated activity-based probe, N-but-3-ynyl-2-chloroacetamidine, is shown to label myc-tagged DDAH-1 expressed in HEK 293T cells, but not an inactive mutant or inhibited enzyme. A two-color Western blotting technique is used to determine the in vivo IC₅₀ value for a reversible inhibitor of DDAH-1, N⁵-(1-iminopropyl)-L- ornithine, indicating this compound's bioavailability and its competition for binding to the active site. This probe provides a novel tool for the analysis of DDAH-1 activity in normal and pathophysiological states and should allow more meaningful studies of the etiology of endothelial dysfunction.

Full text of DOI:10.1021/ja906432e

Published on Web 10/05/2009
A Click Chemistry Mediated in Vivo Activity Probe for Dimethylarginine
Dimethylaminohydrolase
†
†,‡
,†,‡
Yun Wang, Shougang Hu, and Walter Fast*
DiVision of Medicinal Chemistry, College of Pharmacy, Texas Institute for Drug and Diagnostic DeVelopment,
UniVersity of Texas, Austin, Texas 78712
Received July 30, 2009; E-mail: WaltFast@mail.utexas.edu
ω
ω
Asymmetric N ,N -dimethyl-L-arginine (ADMA) is an endog-
enously produced inhibitor of human nitric oxide synthase and an
emerging biomarker for endothelial dysfunction in cardiovascular
1
disease. Concentrations of ADMA are tightly controlled by two
isoforms of its catabolic enzyme dimethylarginine dimethylami-
nohydrolase (DDAH-1 and DDAH-2) that have different tissue
2
distributions. Homozygous disruption of the Ddah1 gene in mice
Figure 1. In ViVo activity probe for DDAH-1. HEK 293T cells expressing
myc-tagged DDAH-1 are treated with 1, washed, lysed, and reacted with
results in embryonic lethality; heterozygous mice have elevated
concentrations of plasma ADMA and display endothelial dysfunc-
3
biotin-PEO -azide and catalysts to biotinylate the active fraction of DDAH-
3
tion. Accordingly, dysregulation of DDAH activity has been
1
. The syn/anti ratio of 3 has not been determined.
studied as a mediating factor for endothelial dysfunction in a number
4
5
of disease states including hyperhomocysteinemia, renal failure,
3
with biotin-PEO -azide (Supporting Information) and catalysts for
6
and diabetes. However, the existing methodology has shortcom-
ings. Measurements of transcript mRNAs and immunoblots by
DDAH-selective antibodies have been used to monitor changes in
DDAH expression, but these values do not always correlate well
with the activity of DDAH because the enzyme can be inhibited
the cycloaddition reaction followed by SDS-PAGE to resolve the
crude protein mixture. The presence of myc and biotin was
demonstrated by Western blot using rabbit antimyc and mouse
antibiotin primary antibodies and two near-infrared dye labeled
secondary antibodies with emissions at 680 nm (antirabbit, red)
and 800 nm (antimouse, green) (Figure 2, Supporting Information)
7
by physiologically relevant modulators such as homocysteine,
8
-10
11
12
19
S-nitroso-L-homocysteine,
nitric oxide,
zinc(II), 4-hydroxy-2-nonenal,
that were chosen for their linear concentration-dependent response.
Response to the myc tag (red) indicated consistent levels of
1
3,14
and other reactive nitrogen and oxygen species.
Likewise, enzyme activity assays are also problematic because they
are performed after cell lysis and homogenation, which removes
DDAH from its cellular environment and alters the concentrations
of modulators. A cell permeable probe to detect enzyme activity
would likely prove more relevant for the study of biological
function. Therefore, to better understand the regulation of DDAH
in normal and pathophysiology, we developed a two-part click-
chemistry mediated activity-based probe that labels active DDAH-1
in intact mammalian cells but does not label an inactive mutant or
inhibited enzyme.
In prior work, we discovered that 2-chloroacetamidine (CAA)
1
5
selectively modifies the active site Cys of DDAH. Subsequently,
a similar 2-haloamidine activity probe specific for a related enzyme,
peptidylarginine deiminase-4, was demonstrated in Escherichia coli
1
6
cell lysates. As a candidate DDAH probe for in ViVo use with
mammalian cell cultures, we synthesized N-but-3-ynyl-2-chloro-
acetamidine (1, Supporting Information). The simple butynyl chain
was appended to CAA to minimize its impact on selectivity and
bioavailability yet still provide a handle for variable functional-
ization using a click reaction after cell lysis, here a Cu (I) catalyzed
Figure 2. Two-color imaging for DDAH-1 activity. (A) Red and green
coloring correspond to antimyc and antibiotin Western blot signals,
respectively. (B) Coomassie staining of the same samples used for (A).
The MWcalcd of myc-DDAH-1 is 35 894 Da.
17
1
,3-dipolar cycloaddition with an azide labeled-biotin (Figure 1),
DDAH-1 expression and loading. Response to the biotin tag (green)
was observed when cells were treated by 1, but not when 1 was
omitted. An inactive DDAH-1 in which the active-site Cys274 is
mutated to Ala is also not labeled by 1, despite the presence of
five other Cys residues in its primary sequence. Addition of CAA
(25 µM) prior to addition of 1 blocks biotinylation, indicating that
CAA has sufficient bioavailability to react with DDAH-1 in 293T
cells and that CAA and 1 compete for the same site. Mock
transfection with an empty expression vector or 293T cells alone
did not result in a labeled band at the expected position for DDAH-
1
8
a strategy proven successful with other enzyme superfamilies.
By transient transfection, human DDAH-1 bearing an N-terminal
Myc-tag was expressed in HEK 293T cells. Addition of 1 (150
µM) to the growth medium was followed by a short incubation,
cell washing, and harvesting. Cells were subsequently lysed by
multiple freeze/thaw cycles, and the resulting lysate was incubated
†
Division of Medicinal Chemistry.
Texas Institute for Drug and Diagnostic Development.
‡
1
5096 9 J. AM. CHEM. SOC. 2009, 131, 15096–15097
10.1021/ja906432e CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
1
. These experiments clearly demonstrate that 1 can label active
IC50 values of inhibitors. Additionally, the small size and simplicity
of 1 suggest its use as a broad-specificity probe for labeling
endogenous DDAH isoforms and enzymes with similar pharma-
cophores, the subject of ongoing studies. This probe provides a
novel tool for the analysis of DDAH-1 activity in normal and
pathophysiological states relevant to cardiovascular disorders and
should allow more meaningful studies of the etiology of endothelial
dysfunction.
DDAH-1 in cultured mammalian cells.
To determine whether 1 is capable of detecting reversible
2
0
inhibition of DDAH-1 in cells, a challenge experiment was
performed using a competitive reversible inhibitor of DDAH-1, N -
5
(
1-iminopropyl)-L-ornithine (4) (K
i
) 50 µM), that we previously
ω
developed as a nonhydrolyzable analogue of the substrate N -
2
1
methyl-L-arginine. Cells were preincubated with varying con-
centrations of 4, followed by addition of 1, washing, harvesting,
and lysis. For dose dependent studies, response to the biotin tag
was normalized for expression levels of myc-DDAH-1. In the
absence of inhibitor, DDAH-1 was effectively biotinylated, indicat-
ing that the active site Cys274 was available to react with 1 (Figure
Acknowledgment. This work was supported in part by grants
from the National Institutes of Health (GM69754), the Robert A.
Welch Foundation (F-1572), and a seed grant from the Texas
Institute for Drug and Diagnostic Development (Welch Foundation
Grant No. H-F-0032).
3
).
Supporting Information Available: Synthetic procedure for (1) and
biotin-PEO
3
-azide, cloning procedure for myc-tagged human DDAH-
1
, and conditions for biotinylation and Western blot analysis. This
material is available free of charge via the Internet at http://pubs.acs.org.
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JA906432E
J. AM. CHEM. SOC. 9 VOL. 131, NO. 42, 2009 15097