Benzylidene cyclopentenediones: First irreversible inhibitors against botulinum neurotoxin A's zinc endopeptidase

Article abstract of DOI:10.1016/j.bmcl.2009.10.129

A series of benzylidene cyclopentenedione-based inhibitors, acting through covalent modification of the active site of botulinum neurotoxin A light chain metalloprotease, are reported.

Full text of DOI:10.1016/j.bmcl.2009.10.129

Bioorganic & Medicinal Chemistry Letters 20 (2010) 206–208
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzylidene cyclopentenediones: First irreversible inhibitors against
botulinum neurotoxin A’s zinc endopeptidase
a
b
c
d
c
a,e,
*
ˇ
ˇ
Katerina Capková , Mark S. Hixon , Sabine Pellett , Joseph T. Barbieri , Eric A. Johnson , Kim D. Janda
a Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b Discovery Biology, Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA
c Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706, USA
d Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
e Department of Immunology, The Skaggs Institute for Chemical Biology and Worm Institute of Medical Research (WIRM), The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzylidene cyclopentenedione-based inhibitors, acting through covalent modification of the
active site of botulinum neurotoxin A light chain metalloprotease, are reported.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 October 2009
Accepted 29 October 2009
Available online 31 October 2009
Keywords:
Botulinum neurotoxin
Protease inhibitor
Irreversible inhibition
Botulinum neurotoxins (BoNTs), the etiological agents responsi-
ble for botulism, are the deadliest poisons known.1 Among the
seven serotypes, BoNT/A ranks as the most potent, with a lethal
dose for humans of ꢀ1 ng/kg of body weight. The intoxication
event itself may result from improperly stored food, an accidental
overdose during a therapeutic treatment or as a result of bioterror-
ism.2 For an extensive discussion on the etiology of the BoNTs see
Janda and co-workers;2 however, in brief, the toxin itself consists
of a disulfide-linked dimer comprised of a 100 kDa heavy chain
and 50 kDa light chain (LC). The heavy chain is responsible for neu-
ronal cell receptor binding and translocation while the light chain
is a Zn2+-dependent endopeptidase. The light chain (LC), exclu-
sively cleaves at specific sites of the intracellular soluble N-ethyl-
maleimide-sensitive factor attachment protein receptor (SNARE)
proteins; namely, synaptosomal-associated protein (SNAP-25) in
the case of BoNT/A LC. This cleavage results in the impairment of
neural vesicular docking–fusion machinery and inhibition of ace-
tylcholine release at neuromuscular junctions. At the somatic level,
this leads to progressive flaccid paralysis, and, in severe cases, to
respiratory failure and death.
vaccine is available, supplies are reserved for high-risk individu-
als.2 Alternate countermeasures consist of administration of
equine antibodies, yet, this can cause both serious side-effects
and has a limited window of application. Additionally, antitoxins
are ineffective once BoNT enters the neuronal cells, leaving
mechanical ventilation for life support as the only treatment op-
tion.2 In this light, small molecule inhibitors effective after toxin
internalization represent a promising strategy, and several efforts
have been made to uncover viable lead structures for clinical
development.2 To date, these efforts have been mostly focused
on competitive inhibitors targeting the BoNT/A LC. Unfortunately,
even if a tight binding inhibitor with the appropriate drug metab-
olism and pharmacokinetic, (DMPK), properties is achieved, this
approach will still suffer from the persistent nature of the toxin.
Reversible inhibition of BoNT/A will be transient, solely dependent
on the inhibitor’s intrinsic DMPK, while BoNT neurotoxin is known
to persist within a neuron for weeks to months, albeit by an un-
known mechanism.2
Although the persistent nature of BoNT/A neurotoxin’s protease
is a substantial hurdle to overcome, one favorable aspect of BoNT/A
intoxication is its nonregenerative nature. In almost all other dis-
ease states (infectious agents, cancer, metabolic disorders), the en-
zyme targeted for therapy is regenerated. In contrast, intoxication
with BoNT is a solitary event; BoNT/A LC need to be ‘‘killed” once,
as there will be no regeneration of the enzyme. This singular intox-
ication event along with the persistent nature of the enzyme itself
makes therapy with an irreversible inhibitor a very attractive
Effective therapy against BoNT intoxication is currently lacking.
Indeed, the persistence of the activated toxin within a neuron
remains the greatest obstacle. Thus, while a pentavalent toxoid
Abbreviations: MMP, matrix metalloprotease.
* Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.129

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K. Capková et al. / Bioorg. Med. Chem. Lett. 20 (2010) 206–208
207
approach. Mechanistically, an irreversible inhibitor in theory
would destroy the cellularly internalized toxin, that is, the light
chain protease, as a function of inhibitor exposure (concentration
& time) and inhibitor specificity constant (kinact/KI). Details on the
kinetics of irreversible inhibitors are well described in Copeland.3
Briefly, as depicted in Figure 1, an initial (and reversible) enzyme
inhibitor complex forms (EI) that proceeds through a chemical step
to an irreversible enzyme inhibitor adduct (EI*). The observable
inactivation kinetics by this mechanism is presented in Eq. 1. The
method for ranking the potency of irreversible inhibitors is the
specificity constant kinact/KI.
Irreversible inhibitors acting through covalent modification of
the active site of the target enzyme have been generally shunned
by the pharmaceutical industry mainly due to perceived toxicity
resulting from off-target interactions of the drug or its metabo-
lites;4 however, it is very interesting to note that of the currently
marketed drugs within the United States that target enzymes
approximately 35% of those enzymes are irreversibly inhibited by
the drug.4
Inhibitors were evaluated (Supplementary data) via a two step
assay consisting of an enzyme–inhibitor pre-incubation period fol-
lowed by dilution with catalytic initiation via substrate addition.
Quantitative analysis of BoNT/A LC catalysis was determined as de-
scribed in Capková et al. All concentrations of the inhibitors exam-
ined produced first order inactivation profiles with respect to BoNT
9
ˇ
activity. From a secondary plot of the first order rate constant k(ob-
versus compound concentration we obtained the specificity
served)
parameter kinact/KI (Eq. 1):
m
E
½Iꢂ
¼
¼ eꢁk
where kobs ¼ kinact
t
obs
V0 E0
½Iꢂ þ kI
I
KI
kinact
but as
! 0 kobs
¼
½Iꢂ
KI
ð1Þ
Equation 1: Enzyme activity in the presence of an irreversible
inhibitor as a function of time and inhibitor concentration.
The results are summarized in Figure 2. Following the trend of the
hydroxamate series,7 1 and 2 display comparable affinities for the
active site of the enzyme, resulting in similar kinact/KI values of 520
and 580 Mꢁ1 sꢁ1, respectively. Compound 3, on the other hand,
displayed a decreased affinity for the active site of BoNT/A LC as re-
flected in the significantly lower kinact/KI value of 80 Mꢁ1 sꢁ1 and
consistent with its hydroxamate counterpart.
Continuing our research toward an effective therapy for BoNT
intoxication, herein we describe the synthesis and evaluation of a
series of BoNT/A LC covalent inhibitors. In our previous work, by
use of a hydroxamic acid warhead, we had developed a series of
competitive small molecule inhibitors that exploit the relatively
shallow P1 binding pocket of BoNT/A LC.5–7 In our current tactic,
we make use of these same binding elements to produce relatively
potent affinity labels by replacing the hydroxamate with a cyclop-
entenedione warhead. Thus, we designed benzylidene cyclopen-
tenedione derivatives 1–3 depicted in Figure 2. In the original
hydroxamate series, analogs of both 1 and 2 (1a and 2a, respec-
tively) displayed comparable submicromolar potencies, while the
hydroxamate analog of 3 (3a) was significantly weaker with an
In order to establish the persistence of enzyme inactivation,
*
that is, susceptibility of the EI complex to hydrolytic rescue,
BoNT/A LC was inactivated with 1 (Supplementary data). Following
inactivation, the enzyme was exhaustively dialyzed for 16 h, then
assayed for recovered activity. Within our limit of quantitation
no activity was recovered thus any rescue mechanism must have
a half-life of more than 7 h.
We attempted to identify which nucleophilic amino acid within
the active site generates the covalent adduct (we suspected Tyr366
given its location within the active site and proximity based on
crystal structure6 to the pentenedione warhead). After pre-expo-
sure of 1 with the BoNT/A light chain sufficient to inactivate the
enzyme, the mixture was subjected to tryptic digestion followed
by mass spectral analysis via Matrix Assisted Laser Desorption Ion-
ization (MALDI). A series of tryptic digest conditions were exam-
ined as well as MALDI conditions, however, no strong fragment
IC50 of 41 l
M when assayed with BoNT/A LC (Fig. 2).7 Compounds
1–3 were synthesized from the corresponding benzaldehydes in a
single step, albeit in moderate yields (Supplementary data).8
Figure 1. The kinetic mechanism of inhibition by an irreversible inhibitor.
Figure 2. Structure and activity of BoNT/A LC irreversible inhibitors and their hydroxamate counterparts.

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K. Capková et al. / Bioorg. Med. Chem. Lett. 20 (2010) 206–208
208
will require further studies including the examination of a series
of N-ethylmaleimides to sort out this interesting observation.
Finally, as an initial examination of the pharmacological stabil-
ity of our compounds, we examined the stability of one of our
affinity labels in the presence of 1 mM glutathione (approximately
physiologic concentration) and in bovine serum. Once again, 1 was
chosen as a representative of the series. Unfortunately, subsequent
LC–MS analysis showed complete disappearance of 1 within 5 min
of incubation with both glutathione and bovine serum. Hence, this
would most likely preclude this structure for additional testing in
the mouse lethality assay for BoNT/A.
In summation, the properties of BoNT/A intoxication argue for a
therapeutic approach engaging irreversible inhibitors. In contrast
to the extensive body of knowledge with reversible inhibitors of
metalloproteases, and numerous studies with irreversible inhibi-
tors of serine proteases, very little work has appeared on the irre-
versible inhibition of metalloproteases.13 We report the first
examples of irreversible inhibitors specifically designed against
BoNT/A’s zinc metalloprotease. Excitingly, these compounds pres-
ent selectivity against BoNT/A light chain over closely related
MMPs, but lack the necessary stability toward serum and glutathi-
one. Interestingly, through these studies we have also uncovered a
possible new lead, 4, providing relatively potent BoNT protection
in a cellular assay via a yet to be determined new mechanism of ac-
tion. Lastly, these studies demonstrate a proof of concept—an
important first step for future efforts striving to treat botulinum
intoxication.
Figure 3. Structure of maleimide derivative 4.
signals were uncovered, that is, none of the ionized peptide frag-
ments observed were consistent with cyclopentenedione adduct
addition. On the other hand, only 40% of the enzyme peptide se-
quence was identified by MALDI and Tyr366 was not within this
region. Most likely the inhibitor adduct resided in the remaining
poorly ionizing 60% of the protein sequence.
In order to evaluate enzyme selectivity, 1 and 2 were screened for
off-target inhibition of MMP-2 and MMP-9, both zinc proteases
bearing structural similarity to BoNT/A LC.10 Neither 1 nor 2 showed
any inhibitory activity towards MMP-2 (kinact/KI <33 Mꢁ1 sꢁ1);
however, both derivatives displayed modest inhibition of MMP-9
(kinact/KI of 61 and 58 Mꢁ1 sꢁ1, respectively) making it ninefold more
selective for BoNT/A LC over MMP-9.
To gain some insight into the specificity of interaction of inhib-
itors 1–3 with BoNT/A LC and to expand our understanding of the
enzyme’s active site, we synthesized 4 containing the 2,4-dichloro-
benzene scaffold, but bearing now a maleimide warhead. (Fig. 3).
Compound 4’s synthesis was straightforward, condensing
2,4-dichlorobenzylamine with maleic anhydride.11 When assayed
with BoNT/A LC, 4 lacked an irreversible step; instead it behaved
Acknowledgments
This project has been funded with federal funds from The Na-
tional Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, Department of Health and Human Services,
under Contract Nos. N01-AI30050, and AI080671.
as a weak competitive inhibitor with an IC50 of 80 lM. The inactiv-
ity of 4 demonstrates that BoNT/A LC is not generically sensitive to
any Michael acceptor even when optimized for active site binding.
Intrigued with the activity of our lead structures against the
enzyme, we examined compounds 1 and 4 for their potential to in-
hibit the biologic activity of BoNT/A1 holotoxin in primary rat
spinal cord neurons12 (Supplementary data). This assay measures
target protein (SNAP-25) cleavage inside the cell cytosol as an end-
point. Since it requires all steps of the cellular intoxication process
to take place in order to observe SNAP-25 cleavage (i.e., receptor
binding, internalization, channel formation, LC translocation into
the cytosol, and cleavage of the substrate inside the cytosol), it is
sensitive to inhibition at any of these steps. SNAP-25 cleavage
Supplementary data
Supplementary data (full experimental details: synthesis, inac-
tivation assays, EI* persistence assay, MMP assays, RSC assays)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.10.129.
References and notes
was significantly decreased at concentrations of 1 above 600
and was nearly completely inhibited at concentrations above
900 M (Supplementary data). Unexpectedly, the addition of 4 re-
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inhibition at 125 M (Supplementary data). Both 1 and 4 inhibit
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