Angewandte
Communications
Chemie
Drug Discovery
A Boronic Acid Conjugate of Angiogenin that Shows ROS-Responsive
Neuroprotective Activity
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[
2a,8]
Abstract: Angiogenin (ANG) is a human ribonuclease that is
compromised in patients with amyotrophic lateral sclerosis
(ROS), which are cytotoxic.
Moreover, ALS is linked to
[
9]
the hyperactivity of superoxide dismutase (SOD1). This
enzyme catalyzes the conversion of a superoxide ion (O ) to
À
(
ALS). ANG also promotes neovascularization, and can
2
induce hemorrhage and encourage tumor growth. The causal
neurodegeneration of ALS is associated with reactive oxygen
species, which are also known to elicit the oxidative cleavage of
carbon–boron bonds. We have developed a synthetic boronic
acid mask that restrains the ribonucleolytic activity of ANG.
The masked ANG does not stimulate endothelial cell prolif-
eration but protects astrocytes from oxidative stress. By
differentiating between the two dichotomous biological activ-
ities of ANG, this strategy could provide a viable pharmaco-
logical approach for the treatment of ALS.
hydrogen peroxide (H O ), which is the major physiological
ROS.
2
2
The chemical reactivity of H O2 can be exploited in
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a physiological context. For example, H O has long been
2
2
known to effect the oxidative cleavage of the boron–carbon
bond in phenylboronic acid to give phenol and boric acid
[10]
(B(OH) ). This reaction has served as the basis of chemo-
3
selective probes for H O and in cancer prodrug strat-
2
2
[
11,12]
egies.
ANG relies on the intracellular manifestation of its
[
13]
ribonucleolytic activity to mediate neuroprotection.
We
A
myotrophic lateral sclerosis (ALS) is an aggressive, fatal
envisioned the oxidative cleavage of a boronic acid as a means
to generate active ANG only in cells suffering from ROS-
mediated toxicity. To install an ROS-sensitive trigger in ANG,
disease that is characterized by the selective destruction of
motor neurons in the motor cortex, brain stem, and spinal
[
1]
cord. Although the fundamental cause of ALS is not clear,
its pathogenesis arises from several mechanisms, including
oxidative stress. The only approved chemotherapeutic agent
for ALS is the sodium-channel-blocking agent riluzole
we chose to target a key active-site residue: Lys40
(Scheme 1). This residue is essential for the ribonucleolytic
[
2]
[14]
activity of ANG. We used recombinant DNA methods to
g
replace Lys40 with a cysteine residue. We reasoned that its S
(
Rilutek), which was approved for human use in 1995,
atom could serve as a reactive handle for the conjugation of
a boronic acid containing a latent amino group that is poised
to reconstitute catalytic activity. We synthesized boronic acid
1, as well as a control molecule lacking the boronic acid
moiety (2), from an azide precursor through a Curtius
rearrangement.
extends survival by only 2–3 months, and does not improve
motor function.
Loss-of-function mutations in the human gene encoding
the secretory ribonuclease angiogenin (ANG) are associated
with the progression of ALS. Accordingly, the administra-
tion of human ANG increases lifespan and improves the
motor function in ALS-like transgenic mice. Nevertheless,
ANG has a well-known adverse effect as a potential chemo-
therapeutic agent for ALS. As its name implies, ANG induces
the proliferation of endothelial cells to form new blood
vessels by a mechanism uncovered recently. Accordingly,
long-term treatment with ANG could cause hemorrhage and
[
3]
[4]
A ROS-activatable phenylboronic acid conjugate (B-
thiaK40 ANG) and a nonactivatable phenyl conjugate (P-
thiaK40 ANG) were made by employing a radical-initiated
[5]
[15]
thiol–ene reaction (Scheme 1). Notably, the classic method
of generating g-thialysine derivatives through S-alkylation
[6]
[16]
with a haloethylamine
failed with K40C ANG, despite
working with ribonuclease A, which is an ANG homo-
[
7]
[17,18]
tumor growth.
logue.
The integrity of the K40C variant and its
The neurodegeneration that is characteristic of ALS
correlates with an abundance of reactive oxygen species
conjugation products were confirmed by LC–MS/MS
after trypsin digestion (Figure S1 in the Supporting Informa-
tion).
Exposure to an ROS reconstitutes the enzymatic activity
of B-thiaK40 ANG in vitro. A zymogram assay of ribonu-
cleolytic activity revealed that P-thiaK40 ANG was inactive,
even after treatment with H O (Figure 1A). In contrast, this
[
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[
*] Dr. T. T. Hoang, Prof. R. T. Raines
Department of Biochemistry, University of Wisconsin-Madison
33 Babcock Drive, Madison, WI 53706-1544 (USA)
E-mail: rtraines@wisc.edu
Homepage: https://biochem.wisc.edu/labs/raines
4
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ROS did elicit activity from B-thiaK40 ANG (Figure 1A),
which was quantified to be (21 Æ 6)% that of the wild-type
enzyme (Figure 1B). This value is indistinguishable from the
relative value of k /K , which was determined to be (16 Æ
[
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T. P. Smith, Prof. R. T. Raines
Department of Chemistry, University of Wisconsin-Madison
101 University Avenue, Madison, WI 53706-1322 (USA)
1
cat
M
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4)% in solution through a fluorescence-based assay (Fig-
[
] These authors contributed equally to this work.
[19]
ure 1C).
[
20]
The intrinsic catalytic activity of ANG is low. In the
three-dimensional structure of ANG, the side chain of Gln117
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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