An Arsenical-maleimide for the generation of new targeted biochemical reagents

Article abstract of DOI:10.1021/ja310553h

The finding that arsenic trioxide is an effective treatment for acute promyelocytic leukemia has renewed interest in the pharmacological uses of inorganic and organic arsenicals. Here we synthesized and characterized the reactivity of an arsenical-maleimide (As-Mal) that can be efficiently conjugated to exposed cysteine residues in peptides and proteins with the ultimate goal of directing these As(III) species to vicinal thiols in susceptible targets within cells and tissues. As-Mal conjugated to a surface cysteine in thioredoxin provides a more potent inhibitor for Escherichia coli thioredoxin reductase than comparable simple inorganic or organic arsenicals. As-Mal can be coupled to all of the eight cysteine residues of reduced unfolded ribonuclease A or to site-specific locations using appropriate cysteine mutations. We observed particularly strong binding to the two CxxC motifs of protein disulfide isomerase using a mutant RNase in which As-Mal was specifically incorporated at residues 26 and 110. As-Mal will serve as a facile reagent for the incorporation of As(III) species into a wide range of thiol-containing proteins, biomaterials, and surfaces.

Full text of DOI:10.1021/ja310553h

Communication
pubs.acs.org/JACS
An Arsenical−Maleimide for the Generation of New Targeted
Biochemical Reagents
Aparna Sapra and Colin Thorpe*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States
S
* Supporting Information
ABSTRACT: The finding that arsenic trioxide is an
effective treatment for acute promyelocytic leukemia has
renewed interest in the pharmacological uses of inorganic
and organic arsenicals. Here we synthesized and
characterized the reactivity of an arsenical−maleimide
(As-Mal) that can be efficiently conjugated to exposed
cysteine residues in peptides and proteins with the
ultimate goal of directing these As(III) species to vicinal
thiols in susceptible targets within cells and tissues. As-Mal
conjugated to a surface cysteine in thioredoxin provides a
more potent inhibitor for Escherichia coli thioredoxin
Figure 1. (top) Structures of melarsen oxide (1) and 4-(N-(S-
reductase than comparable simple inorganic or organic
arsenicals. As-Mal can be coupled to all of the eight
cysteine residues of reduced unfolded ribonuclease A or to
site-specific locations using appropriate cysteine mutations.
We observed particularly strong binding to the two CxxC
motifs of protein disulfide isomerase using a mutant RNase
in which As-Mal was specifically incorporated at residues
26 and 110. As-Mal will serve as a facile reagent for the
incorporation of As(III) species into a wide range of thiol-
containing proteins, biomaterials, and surfaces.
glutathionylacetyl)amino)phenylarsonous acid (2). (bottom) Syn-
thesis of the arsenical−maleimide 4-(2,5-dioxo-2,5-dihydro-1H-pyr-
rol-1-yl)phenylarsonous acid (5) from 3.
acid.2,7 However, the bromoacetyl function reacts rather slowly
under conditions typically used in thiol bioconjugation
reactions. Hence, we sought a simple arsenical reagent that
could be introduced rapidly and stoichiometrically at cysteine
residues on peptides and proteins. We chose maleimides
because they exhibit high specificity for thiols, react several
orders of magnitude more rapidly than bromoacetamide
derivatives,8,9 and have been widely used to conjugate cytotoxic
agents to cysteine residues on monoclonal antibodies.10−12
Here we show selective and efficient incorporation of an
arsenical−maleimide reagent into peptides and proteins and
demonstrate that these conjugates are more effective inhibitors
than simple arsenical derivatives. This new arsenical−maleimide
will also allow the facile preparation of designed arsenical
materials, including resins for thiol-based chromatography.
Figure 1 shows the synthesis of the arsenical−maleimide 4-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenylarsonous acid
(As-Mal, 5) in an overall yield of 76% via the cyclization of
maleamic acid 4 [Figure S1 in the Supporting Information
(SI)]. As-Mal shows two modes of reaction with thiols: one via
Michael addition at the maleimide and the other through
coordination at the As(III) center. A titration of As-Mal with
GSH showed a decline in the absorbance at 320 nm (with a
sharp end point at GSH/As-Mal = 1.0; Figure S2). These data
are consistent with comparable experiments with N-phenyl-
maleimide (data not shown) and reflect rapid thiol−maleimide
conjugation without undue interference from reversible binding
to the arsenical moiety. The reaction between 100 μM As-Mal
and equimolar GSH at pH 7.5 and 25 °C was essentially
norganic arsenicals, including the sulfur-containing minerals
realgar and orpiment and the oxide, As2O3, have been used
I
for millennia to treat a wide range of medical conditions,
including leukemia, skin cancers, and solid tumors.1,2 In the
early 1900s, Ehrlich and Hata surveyed a range of organo-
arsenicals as antisyphilitics and introduced arsphenamine as the
first rational chemotherapeutic. The related melarsen oxide (1)
(Figure 1) and its derivatives have been widely used as
antitrypanosomals.1,2 While the medical applications of
arsenicals declined in the 1940s with the development of
antibiotics and other modern therapies, they are now the
subject of renewed interest following the demonstration that
As2O3 is remarkably effective as a treatment for acute
promyelocytic leukemia.3,4 In addition to inorganic arsenicals,
several organoarsenicals are now in clinical trials for the
treatment of leukemias and solid tumors.2,5
There is general agreement that the biological effects of
As(III) species largely reflect their coordination to vicinal
thiols.1,6 Hence a continuing challenge is the efficient targeting
of arsenicals while minimizing extraneous labeling and the
toxicity associated with the many off-target thiol-containing
proteins. For example, compound 2 (Figure 1) uses the
tripeptide glutathione (GSH) as a vehicle for arsenical delivery
via conjugation with 4-(2-bromoacetylamino)phenylarsonous
Received: October 25, 2012
Published: February 5, 2013
© 2013 American Chemical Society
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Communication
complete in 40 s (half complete in 2.2 s, comparable to the
behavior of N-phenylmaleimide under these second-order
conditions; Figure S3).
has recently been suggested as a target for antiviral,15
antithrombotic,16 and anticancer therapies.17 Human PDI is
composed of four thioredoxin domains, with the a and a′
domains carrying CxxC motifs that are responsible for the
varied oxidoreductase and isomerase activities of the
enzyme.18,19 PDI proteins have multiple intracellular roles in
eukaryotes. However, they are also found at the cell surface,
where they can modulate extracellular redox poise20−22 and are
involved in platelet activation,20,23,24 enhancing metastasis,25,26
and viral fusion22,27 and where they might be targeted by
protein-based inhibitors and antibodies. Typically, the sub-
strates of PDI family members are proteins that retain
disordered or conformationally mobile regions.18,19 A widely
employed substrate of PDI is reduced, or disulfide-mispaired,
pancreatic ribonuclease A (RNase), and hence, we used this
protein as an initial vehicle for arsenical conjugation. Reduction
of the four native disulfides of RNase leads to a tractable
unfolded protein that can be labeled with eight As-Mal moieties
(Figure S8). However, here we prepared a limited series of site-
directed mutants, including those in which only cysteines 1 and
4, 4 and 8, or 1 and 8 were retained in the sequence. These
were subsequently labeled with As-Mal (Figure 3A; also see the
SI and Figures S9 and S10).
These experiments suggested that As-Mal should react
rapidly with protein thiols provided that they are solvent-
accessible. We first demonstrated placement of a single As-Mal
group at the surface of a folded protein. Here, Escherichia coli
thioredoxin, an oxidoreductase containing a redox-active pair of
cysteine residues (C32 and C35), was mutated to leave the
surface-accessible C32 available for conjugation. Treatment of
this C35S mutant with 1 equiv of As-Mal rapidly generated a
monolabeled derivative (at 100 μM concentrations, the reaction
was half complete in <5 s; Figure S4). Monoalkylation was
confirmed by the mass spectrum (Figure S5), the loss of the
single 5,5′-dithiobis(2-nitrobenzoate) (DTNB)-reactive thiol
group, and titration of the conjugated arsenical reagent with
dithiothreitol (Figures S6 and S7).
Arsenical−peptide/protein conjugates might be expected to
provide more specificity toward their targets than the parent
arsenical reagent alone. The first confirmation of this was
obtained using a thioredoxin (Trx) reductase from E. coli.13
The flow of reducing equivalents from NADPH to Trx is
schematically depicted in Figure 2A. After the CxxC motif on
The widely used insulin reductase assay was chosen to assess
these inhibitors.28,29 Here PDI catalyzes the reduction of insulin
disulfides driven by the water-soluble phosphine TCEP, and the
accumulation of the isolated B chain is followed by light
scattering. In the absence of an As-Mal-RNase derivative, the
onset of turbidity occurred at ∼230 s, whereas it was strongly
suppressed by the 1,8-bis-As-Mal-RNase and to a lesser extent
with 1,4- and 4,8-bis-As-Mal-RNase (Figure 3E). For
perspective, the monoarsenical 4-As-Mal-RNase was consid-
erably less effective than 1,8-bis-As-Mal RNase, and the 1,4,8-
tris-As-Mal RNase was roughly comparable to the 1,8 derivative
(Figure 3E). To confirm that inhibition reflected the
conjugation of arsenicals to RNase, we evaluated the effect of
the parent unconjugated cysteine RNase mutant proteins and
found no significant inhibition of insulin reduction (Figure
S11). Furthermore, the simple monoarsenical PSAO failed to
delay the onset of turbidity significantly at a concentration of 10
μM (Figure 3E). Thus, the placement and spacing of arsenicals
along a protein chain can be exploited to modulate the
inhibitory potency.
Figure 3C shows a spectrophotometric titration of 1,8 bis-As-
Mal-RNase with reduced PDI, in which the increase in
absorbance at 300 nm accompanying thiol coordination to
hydrated As(III) species was followed. The data were fit to a Kd
of 22 7 nM with a stoichiometry of 1.21 0.16 RNase per
PDI. These data are consistent with the complexation of both
the a and a′ CxxC motifs of reduced PDI with 1,8-bis-As-Mal-
RNase (Figure 3B). For comparison, a direct assessment of the
binding of PSAO to reduced PDI showed much weaker binding
with a Kd of 1.1 μM,29 consistent with failure to impact the
insulin reductase assay significantly. Finally, since the levels of
GSH in the extracellular matrix reach ∼10 μM,30,31 we included
GSH in the assay at this concentration and found that it did not
significantly attenuate the inhibition observed with 1,8-bis-As-
Mal-RNase. In contrast, intracellular concentrations of GSH are
in the 0.5−10 mM range,32 and we found that inclusion of 5
mM GSH provides insignificant inhibition of PDI by these
arsenicals (data not shown).
Figure 2. Inhibition of thioredoxin reductase by arsenicals. (A)
Reduction of Trx by NADPH catalyzed by thioredoxin reductase,
together with the arsenicals tested at 1 μM. (B) DTNB reoxidation of
reduced Trx formed in the absence or presence of arsenicals (see the
SI).
the reductase has received a pair of reducing equivalents from
the FAD moiety, a conformational change leads to formation of
a mixed disulfide with Trx docked against the reductase and
then to the eventual release of reduced Trx.13,14 Figure 2B
shows that at a concentration of 1 μM, the simple arsenicals
sodium arsenite, monomethylarsenous acid (MMA), and p-
succinylamidophenylarsenoxide (PSAO) provided only modest
inhibition of this bacterial thioredoxin reductase. However, at
the same concentration, the As-Mal−Trx conjugate was
considerably more potent, with a rapid and progressive loss
of enzymatic activity.
We next wanted to test arsenical−protein conjugates as
potential inhibitors of protein disulfide isomerase (PDI). PDI
In summary, this work provides a simple way to conjugate
arsenicals to cysteine-bearing peptides and proteins and
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for targeted, controlled release of cytotoxic species.35 Finally,
the facility of the conjugation chemistry described here could
allow multiple peptidic and protein scaffolds to be screened
prior to manipulation of linker stability.11,36
ASSOCIATED CONTENT
■
S
* Supporting Information
Materials and Methods, synthesis of As-Mal, and character-
ization of As-Mal-derivatized proteins. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
cthorpe@udel.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH Grant GM26643. We thank
Jennifer Codding and Benjamin Israel for gifts of materials and
Drs. Melissa Blackman, Joseph Fox, Danny Ramadan, Ronak
Rughani, and Sharon Rozovsky for helpful discussions.
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