Toward the discovery of dual inhibitors for botulinum neurotoxin A: Concomitant targeting of endocytosis and light chain protease activity

Article abstract of DOI:10.1039/c5cc00677e

Dyngo-4a has been found to be an endocytic inhibitor of BoNT/A neurotoxicity through dynamin inhibition. Herein, we demonstrate this molecule to have a previously unrecognized dual activity against BoNT/A, dynamin-protease inhibition. To establish the imp

Full text of DOI:10.1039/c5cc00677e

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Toward the discovery of dual inhibitors for
botulinum neurotoxin A: concomitant targeting
of endocytosis and light chain protease activity†
Cite this: Chem. Commun., 2015,
1, 6226
5
Received 23rd January 2015,
Accepted 4th March 2015
a
a
b
c
d
Hajime Seki, Song Xue, Mark S. Hixon, Sabine Pellett, Marek Reme ˘s ,
c
ae
Eric A. Johnson and Kim D. Janda*
DOI: 10.1039/c5cc00677e
www.rsc.org/chemcomm
Dyngo-4at has been found to be an endocytic inhibitor of BoNT/A (synaptosome-associated protein-25 kDa), and B targets synapto-
neurotoxicity through dynamin inhibition. Herein, we demonstrate this brevin. In particular, intoxication from BoNT/A persists up to
molecule to have a previously unrecognized dual activity against BoNT/ several months and its potency is greater than any other serotypes
À1
4
A, dynamin–protease inhibition. To establish the importance of this (LD50: 1 ng kg ). BoNT/A is sold under the trade name of
s
dual activity, detailed kinetic analysis of Dyngo-4a’s inhibition of BoNT/ BOTOX and has been used for the treatment of medical condi-
A metalloprotease as well as cellular and animal toxicity studies have tions such as migraine and facial wrinkle reduction. Yet, it is also
been described. The research presented is the first polypharmacological recognized as a potential bioweapon by Center for Disease Control
5
approach to counteract BoNT/A intoxication.
and Prevention (CDC) as one of the six category A agents. Current
treatments for BoNT/A intoxication are limited to antitoxin and
Clostridium botulinum, an anaerobic bacterium, produces botulinum vaccination, which can readily remove the toxin from circulation;
neurotoxin (BoNT), which is considered to be the most lethal of however, such treatments become ineffective once cellular inter-
6
known toxins. BoNT is a 150 kDa protein consisting of a 100 kDa nalization of the toxin takes place.
heavy chain and a 50 kDa light chain. The heavy chain is responsible
1
With increasing threats of terrorism in the last two decades,
for cellular surface recognition, toxin internalization and translocation the discovery of therapeutic treatments for BoNT/A intoxication
of the light chain. The light chain (LC), a zinc metalloprotease, is the is of utmost importance and urgency, which cannot be over-
7
etiological agent responsible for neurotoxicity, namely, proteolysis of stated. Because BoNT/A’s metalloprotease is the agent respon-
SNARE (soluble N-ethylmaleimide sensitive factor attachment protein sible for its neurotoxicity our laboratory as well as others have
2
receptor) proteins. Upon the cleavage of SNAREs, vesicles containing been actively investigating the development of LC inhibitors
8
acetylcholine are unable to fuse with the presynaptic neuromuscular including active site and exosite inhibitors. In terms of active
junction. As a result, the release of acetylcholine is blocked, leading to site inhibitors, we have developed hydroxamic acids, taking
9
flaccid paralysis (botulism) and potential death.
advantage of their strong coordinating nature to zinc. We have
Among the eight serotypes for the BoNTs (A–H), A, B, and E also discovered exosite inhibitors from natural product screens:
3
cause botulism in humans. BoNT/A and E target SNAP-25 chicoric acid and lomofungin (1 & 3, Fig. 1) are prototypical
10
examples. Although, it has been a major challenge to translate
potent active site enzyme inhibitor efficacy to cellular and ultimately
animal lethality models, we recently demonstrated that a prodrug
approach has enabled hydroxamic acids to gain traction in order to
a
Departments of Chemistry and Immunology and Microbial Sciences, The Skaggs
Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA. E-mail: kdjanda@scripps.edu;
Tel: +1 858 784-2516
11
facilitate cellular uptake and attenuated LC activity.
b
c
Modeling & Simulation Global DMPK, Takeda California, Inc., 10410 Science
Center Drive, San Diego, CA 92121, USA
An alternative rational approach to neutralize BoNT/A is to
prevent its internalization from the cellular surface. In this regard,
Meunier and co-workers reported how the dynamin inhibitor,
Dyngo-4at, thwarted BoNT/A internalization in hippocampal neu-
Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison,
Wisconsin 53706, USA
d
e
Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova
2
030/8 128 43 Praha 2, Czech Republic
Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute,
0550 North Torrey Pines Road, La Jolla, CA 92037, USA
Electronic supplementary information (ESI) available: Compound synthesis
rons and delayed the onset of botulism in an in vivo mouse model
12
(
4, Fig. 1). Dyngo-4a is a pharmacologically refined analogue of
1
13
Dynasore, which is a known inhibitor of dynamin. Dynamin is a
mechanochemical GTPase responsible for the vesicle scission step
in endocytosis. From the standpoint of BoNT/A neurotoxicity,
†
and characterization, kinetics data, cell assay and animal study data. See DOI:
0.1039/c5cc00677e
14
1
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Fig. 3 Mutually exclusive fit for Dyngo-4a and chicoric acid i-Pr ester.
Fig. 1 Structures and targets of exosite inhibitors (1–3) and endocytic
inhibitor (4).
Dyngo-4a’s mechanism of action was attributed to the fact that
BoNT/A mainly utilizes a dynamin-dependent endocytic pathway to
12
enter neuronal cells.
In the effort to search for molecules that could inhibit the
protease of BoNT/A, we turned our focus to the polyphenolic
scaffold embedded within Dyngo-4a. We suspected that this
Dyngo-4a’s phenolic architecture could serve as an exosite LC
inhibitor, based on its structural similarity to chicoric acid.
Thus, Dyngo-4a was tested in vitro employing both FRET-based
Fig. 4 Mutually exclusive fit for Dyngo-4a and lomofungin.
1
5
SNAPtide and LC/MS-based 66mer assays. As anticipated the
compound was inactive in the SNAPtide test, while it showed
inhibitory activity in the 66mer assay, indicating that Dyngo-4a’s
binding was exosite driven. To further define Dyngo-4a’s inhibition
profile, detailed kinetics of the compound was examined with varied
concentrations of the 66mer substrate (Fig. 2). Dyngo-4a showed a
mutually exclusive binding, indicating that the two molecules
cannot bind simultaneously (Fig. 3). A parallel assay now engaging
lomofungin, a presumed b-exosite inhibitor, also presented mutual
exclusivity (Fig. 4). Moreover, Dyngo-4a demonstrated complete
inhibition of the BoNT/A LC at 20 mM, by a competitive inhibition
competitive inhibition mechanism with K = 0.32 Æ 0.05 mM.
i
mechanism, whereas chicoric acid only displayed partial inhibition
As a means to understand Dyngo-4a’s mechanism of inhibition,
we investigated Dyngo-4a’s ability to access the a and b-exosites of
BoNT/A’s LC. To begin, a dual inhibition assay between Dyngo-4a
and chicoric acid i-Pr ester (2, Fig. 1), an a-exosite inhibitor, was
10a
with a non-competitive profile.
In a final attempt to further
define the BoNT/A LC and Dyngo-4a’s interactions, a competitive
assay between Dyngo-4a and an active site hydroxamate inhibitor,
previously validated from crystallographic analysis, was conduc-
16
undertaken. As a frame of reference, 2 presents a competitive
inhibition mechanism with K
= 1.8 Æ 0.3 mM. Here we observed
9b,d
ted. Here non-mutually exclusive binding was observed, i.e. both
i
can bind simultaneously with an enhancement factor (a) of 1.6 Æ 0.8
(ESI†). Although, we are currently unable to unambiguously assign
the location within the BoNT/A LC where Dyngo-4a resides, our data
obtained in total suggest that Dyngo-4a imparts itself within one or
both of the exosite regions of the BoNT/A LC.
Augmenting these kinetic studies we conducted an SAR exam-
ination of Dyngo-4a to probe what functional groups were essential
for its activity (Scheme 1). A variety of analogues were either
purchased or synthesized, the latter through a condensation
between a hydrazide and an aldehyde under acidic conditions
(Scheme 1), and tested in the 66mer assay. As expected, the three
phenolic groups were found to be important for the inhibitory
activity. Interestingly, 6, having an altered phenolic substitution
pattern exhibited virtually equal potency and a competitive inhibition
Fig. 2 Global fit using a competitive inhibition mechanism.
mechanism with K
= 0.46 Æ 0.08 mM (ESI†). Moreover, Dyngo-4a
i
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Fig. 5 Mouse lethality study of Dyngo-4a protection.
Scheme 1 SAR study examining Dyngo-4a as the inhibitor-scaffold and
BoNT/A protease. The top panel presents the general synthetic route used
of dynamin, has now been found to be also a regulator of the
to prepare Dyngo-4a analogues. All assays were conducted as previously BoNT/A protease. This unrecognized BoNT/A LC inhibition by
1
5
described.
Dyngo-4a engenders this molecule to now be the first reported
pharmacological antagonist that can blunt multiple processes
associated with BoNT/A neurotoxicity. Kinetic examination of
Dyngo-4a and the BoNT/A LC though complex and challenging
posits Dyngo-4a’s mode of inhibition through the protease’s
exosite. Although the results from a cell-based assay using
hiPSC neurons suggest predominant endocytic inhibition, the
survival of mice was observed following a lethal injection of
BoNT/A, implicating protease inhibition by Dyngo-4a.
analogues presenting only one/two hydroxyl groups were inactive.
Strikingly, although the hydroxyl functionality displayed within the
naphthyl moiety was not crucial as a point of the inhibitory action,
naphthyl scaffolding was required for inhibition (12 & 13, Scheme 1).
Having established that Dyngo-4a is a promising BoNT/A
protease inhibitor, we evaluated the compound in a cellular assay
using human induced pluripotent stem cell (hiPSC)-derived
17
While unintended off-target activities can lead to toxicity
and jeopardize drug discovery efforts, in recent years, the
number of drugs targeting multiple targets has been increased,
and a number of polypharmacological strategies have been put
forth to address challenging goals, as represented by the
neurons. This platform was chosen due to the high sensitivity
to the BoNT, a steep dose–response curve, and species-relevant
assay with a more pure and defined population of neurons than
other cell-based assays. To distinguish between its endocytic and
protease inhibitory activities, the assay was conducted in two
different modes. In one assay, Dyngo-4a was added 1 h before
the toxin exposure to examine its endocytic inhibition; in the other,
the inhibitor was added 45 min after the toxin exposure to assess
its protease inhibition. Dyngo-4a showed SNAP-25 protection, albeit
at a relatively high concentration of 200 mM when it was added
before toxin exposure. Unfortunately, a similar inhibition was not
observed when added after toxin exposure. Post-exposure cellular
18
success of multikinase inhibitors. Making a case for Dyngo-4a
is tantalizing in that a single pharmacophore, a polyphenol, targets
two completely different cellular proteins, one endogenous and the
other exogenous. We believe that a rational dual inhibition design
strategy targeting endocytosis and the BoNT/A LC has enormous
potential to combat one of the most potent toxins known to man.
We are hopeful that this newly found dual inhibitor activity will
stimulate the discovery of optimized new generation endocytic-
protease inhibitors of not only BoNT/A but also of the other BoNT
serotypes.
8,11
rescue using protease inhibitors has been rare. We also hypothe-
size that a lack of inhibition might also be due to our inability to
examine higher concentrations of Dyngo-4a. This is especially
pertinent with the sensitive nature of hiPSC neurons to foreign
substances, which has been noted, and indeed Dyngo-4a showed
cellular toxicity above 250 mM.
With kinetic, SAR, and cellular studies unfolded, we summoned a
mouse lethality model, hoping to further probe the importance of
non-endocytic efficacy engendered within Dyngo-4a. Thus, Dyngo-4a
was injected 2.5–3 h post intoxication where in the mice begin to
labor (pinched abdomens and labored breathing), and survival time
was monitored and is summarized in Fig. 5. Remarkably, we
observed that multiple rodents survived the challenge. Although
other mechanisms that might come into play are currently unknown,
clearly Dyngo-4a has an effect on survival that would not be governed
in this time frame by dynamin inhibition of BoNT/A internalization.
In summary, Dyngo-4a, a heretofore inhibitor of BoNT/A
neurotoxicity previously thought to function solely via the
blocking of heavy chain internalization through the inhibition
We acknowledge the National Institute of Health (AI080671)
for funding, and Dr. Michael C. Goodnough (Metabiologics,
Madison, WI) for conducting the BoNT/A mouse lethality assay.
˘
´
The authors thank Dr. Peter Silhar for the synthesis of chicoric
acid i-Pr ester. M.R. acknowledges the Fulbright Commission
for the financial support. This is manuscript #29038 from The
Scripps Research Institute.
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