Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A: Extending towards the β-exosite

Article abstract of DOI:10.1016/j.bmcl.2012.04.019

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to man, exposure to which results in flaccid paralysis. Given their extreme potency, these proteins have become studied as possible weapons of bioterrorism; however, effective treatments that function after intoxication have not progressed to the clinic. Here, we have reexamined one of the most effective inhibitors, 2,4-dichlorocinnamyl hydroxamate, in the context of the known plasticity of the BoNT/A light chain metalloprotease. Our studies have shown that modifications of this compound are tolerated and result in improved inhibitors, with the best compound having an IC₅₀ of 0.23 μM. Given the inconsistency of structure-activity relationship trends observed across similar compounds, this data argues for caution in extrapolating across structural series.

Full text of DOI:10.1016/j.bmcl.2012.04.019

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3754–3757
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A:
Extending towards the b-exosite
a
b
a
b
a
ˇ b
ˇ
Garry R. Smith , Dejan Caglic , Petr Capek , Yan Zhang , Sujata Godbole , Allen B. Reitz ,
Tobin J. Dickerson b,
⇑
a Fox Chase Chemical Diversity Center, Doylestown, PA 18902, USA
b Department of Chemistry and Worm Institute of Research and Medicine (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:
Botulinum neurotoxins (BoNTs) are the most toxic proteins known to man, exposure to which results in
flaccid paralysis. Given their extreme potency, these proteins have become studied as possible weapons
of bioterrorism; however, effective treatments that function after intoxication have not progressed to the
clinic. Here, we have reexamined one of the most effective inhibitors, 2,4-dichlorocinnamyl hydroxamate,
in the context of the known plasticity of the BoNT/A light chain metalloprotease. Our studies have shown
that modifications of this compound are tolerated and result in improved inhibitors, with the best com-
Received 9 February 2012
Accepted 3 April 2012
Available online 11 April 2012
Keywords:
Botulinum neurotoxin
Hydroxamic acids
Bioterrorism
pound having an IC50 of 0.23 lM. Given the inconsistency of structure–activity relationship trends
observed across similar compounds, this data argues for caution in extrapolating across structural series
Ó 2012 Elsevier Ltd. All rights reserved.
Rational inhibitor design
Botulinum neurotoxins (BoNTs), the proteins that cause the dis-
ease botulism, have become heavily studied in recent years due to
the dual nature of these proteins as both potent biopharmaceuti-
cals as well as possible weapons of bioterrorism.1 These proteins
are produced by the Gram-positive bacteria Clostridium botulinum
and consist of seven immunologically distinct serotypes (A–G)
with varying tropism. Estimates have been made that approxi-
mately 1,000 people are afflicted with botulism each year, with
the majority of cases being from BoNT/A, and a minority from
BoNT/B and /E.2 With an estimated lethality of 1–5 ng/kg, BoNT/A
is the most toxic protein known to man. Because of this risk, the
CDC has classified BoNTs as ‘category A’ agents, and recent reeval-
uation by a U.S. Federal panel of scientists and security experts has
recommended BoNT be designated a ‘Tier 1 select agent’, a cate-
gory subject to the highest possible security standards.3
While vaccine-based therapeutics designed to counteract the
extreme morbidity and mortality associated with BoNT intoxica-
tion have been reported, optimal efficacy is observed prior to toxin
exposure, limiting their use primarily to prophylactic measures.4
The biochemical mechanism of action of BoNTs has been closely
studied and three distinct stages of the intoxication process have
been characterized: neuronal cell surface receptor binding and
internalization, toxin translocation out of endosomes into the cyto-
sol, and light chain (LC) metalloprotease recognition and cleavage
of endogenous SNARE (soluble N-ethylmaleimide-sensitive factor
attachment protein receptor) complexes essential for vesicle fusion
and neurotransmitter exocytosis.5 The ultimate culmination of
these molecular events is a blockage of neurotransmitter exocyto-
sis, resulting in flaccid paralysis.
Most small molecule studies to date have focused on inhibition
of the 50 kDa LC of BoNT/A,1 not only because it is this serotype
that is most lethal to humans but also because it possesses an
extremely long half-life in cell culture and in vivo.6 Indeed, the
duration of action of BoNT/A when used therapeutically can be
as long as 3–12 months.7 This serotype specifically recognizes
and cleaves SNAP-25 (synaptosomal-associated protein of
25 kDa), a critical component of the SNARE complex anchored onto
the intracellular face of motor neuron membrane. Small molecules
have been espoused as particularly suitable therapeutics for botu-
lism given that a successful therapeutic must be capable of treating
the patient after exposure to the toxin, meaning after internaliza-
tion of the toxin into peripheral motor neurons. While proteins
and other biological therapeutics frequently suffer from poor cellu-
lar permeability, small molecules can be designed such that they
have acceptable permeability profiles.
A number of small molecules have been reported to inhibit the
BoNT/A LC through a variety of mechanisms.8–14 Among these
compounds, cinnamyl hydroxamates have been particularly
successful inhibitors of BoNT/A due to their tight binding to metal
ions, and a variety of leads have been reported.8–10 One of the most
potent compounds in vitro, 2,4-dichlorocinnamyl hydroxamic acid
1,8 displayed marginal in vivo activity in a mouse model of BoNT/A
exposure and was the first to highlight the poor predictive value of
common cell models of intoxication.9 More recently, we have re-
ported a series of benzothiophene-2-yl hydroxamic acids that are
⇑
Corresponding author.
E-mail address: tobin@scripps.edu (T.J. Dickerson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.019

G. R. Smith et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3754–3757
3755
Table 1
among the most potent small molecule inhibitors discovered to
Inhibition of BoNT/A LC by 4-substituted 2-chlorocinnamyl hydroxamic acidsa
date and also display more favorable pharmacologic properties.10
In contrast to rational design efforts, there also has been recent
interest in the development of pharmacophore models for predict-
ing BoNT inhibitors in silico. In particular, a ‘three zone pharmaco-
phore’ has been reported and validated in a screen of the National
Cancer Institute Open Repository of compounds.12 Lead compounds
identified in this screen have been further improved using this mod-
el as a guide.13 Interestingly, one of the other motivations for the
development of an in silico screening model was the difficulty in
optimizing early lead candidates into more efficacious inhibitors.14
Indeed, the authors reported an inability to obtain crystals suitable
for crystallographic studies or improve lead candidates through
synthetic studies guided by molecular docking experiments.
This difficulty in further optimizing lead compounds for BoNT/A
inhibition is echoed in other studies where marginal improvement
in inhibition has been achieved through rational design.15 As a re-
sult, this study was conducted to examine the flexibility of the ac-
tive site of BoNT/A, particularly in the context of the plasticity
present at the b-exosite that is adjacent to the active site.16 By
designing compounds that reach further into the hydrophobic
pockets of this region and provide a ‘handle’ for the enzyme to rec-
ognize, we expected the potency of a given inhibitor would im-
prove. Also, while not designed to uncover better therapeutic
candidates per se, removal of the pharmacologically disfavored
aryl halides without sacrificing inhibitor potency could provide a
secondary benefit to this study.10
Compound
R
IC50 (lM)
1
2
3
0.67 0.04
0.69 0.05
13.4 1.1
4
5
6
7
1.23 0.57
9.49 4.11
31.3 4.6
0.98 0.52
8
0.64 0.17
9
0.55 0.10
32.3 2.6
The available structural data indicates that the 2-chloro moiety
of 1 makes contacts with the side chain of Arg363 in the BoNT/A LC,
filling a void that is observed in the structures of complexes miss-
ing this group.16 We speculated that by fixing the 2-chloro substi-
tuent and varying the para substituent of 1, the flexibility of the b-
exosite could be directly tested. Our initial studies commenced
with the preparation of a common intermediate from which a
number of analogs of 1 could be rapidly prepared via cross-cou-
pling chemistry. Interestingly, this compound, 2-chloro-4-bromoc-
innamyl hydroxamate 2, was an equipotent inhibitor of BoNT/A LC
10
11
12
13
14
24.9 3.0
60.7 3.8
>100
as 1 (IC50 = 0.69 lM, Table 1). Suzuki coupling of protected 2 with
phenylboronic acid followed by deprotection yielded biarylcinn-
amyl hydroxamate 4, which also was a comparable inhibitor of
38.2 7.0
BoNT/A (IC50 = 1.23 lM, Table 1). This was a particularly surprising
finding; the nonconservative change of a chloro substituent for a
phenyl group had almost no effect on the inhibitory potency of
the compound, despite the dramatic change in stereoelectronics.
In contrast, 2-chlorocinnamyl hydroxamate 3 displays a greater
than 10-fold loss in potency.
Assuming compounds such as 4 would bind to BoNT/A LC in a
manner similar to the parent hydroxamate 1, one would anticipate
a binding orientation with the cinnamyl moiety oriented towards
the 370 loop that makes up the b-exosite of the light chain.16 In
the case of 1, the sharp boundary created by the 370 loop creates
a pocket that has been reported to be entirely filled by the 2,4-di-
chloro-substituted phenyl ring. Furthermore, it has been reported
that bulky substituents such as in 4-tert-butylcinnamyl hydroxa-
mate result in a dramatic loss of potency, arguing that changes in
this position will result in poor inhibitor candidates.8 Yet, biaryl
compound 4 is a comparable inhibitor to 1, refuting this hypothesis
as a general rule within this series.
With this data in hand, we further explored this series of biarylc-
innamyl hydroxamates to determine the extent of substitution that
would be tolerated without compromising inhibition. Using com-
mon intermediate 2, a series of biaryl cinnamyl hydroxamates were
synthesized. All compounds were prepared using Suzuki or Heck
cross-couplings to generate the desired hydroxamates as final prod-
ucts in good yield and excellent purity. This initial set of compounds
was subsequently screened in a high-throughput SNAPtideÒ assay
15
16
17
35.7 3.4
33.1 5.4
15.4 1.7
18
>100
19
20
21
68.4 5.4
42.7 2.8
1.13 0.29
22
23
35 6.3
>100
IC50 values were determined using the SNAPtideÒ assay.17
a

3756
G. R. Smith et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3754–3757
Table 2
widely used by our laboratory as well as others as a primary triage
for the discovery of BoNT/A inhibitors.17 This assay relies on the
appearance of fluorescence upon cleavage of the FRET-quenched
SNAPtideÒ substrate, allowing relative IC50 values to be calculated.
Surprisingly, many of the 4-aryl-cinnamyl hydroxamates were
potent inhibitors of the BoNT/A LC, with five compounds (4, 7, 8,
9, and 21) being comparable or better inhibitors than the parent
compound 1. More importantly, some SAR can be clearly seen from
the data. First, while the biphenyl moiety is tolerated (1 vs 4), meta
substitution of the newly installed phenyl ring results in poor inhi-
bition (i.e., 6, 11, 13, 14, 16). Also, the binding pocket seems not to
tolerate additional steric bulk at the para position (10, 12, 15, 17,
and 18), but does tolerate electronic modulation of the aromatic
ring (4 vs 7, 8). Interestingly, not only is the steric bulk of an aro-
matic ring tolerated, but also other groups such as the piperidyl
moiety of 21. Additional SAR can be gleaned from compounds
21–23. In 21, the piperidine group is tolerated similar to the phenyl
Inhibition of BoNT/A LC by second generation pyridyl-cinnamyl hydroxamic acidsa
Compound
R
IC50 (lM)
24
0.23 0.02
27.3 7.1
25
26
27
35.3 9.6
group of 4 (IC50 = 1.13 and 1.23 lM, respectively), yet changing the
0.56 0.15
piperidine to a morpholine or piperazine group (22 and 23) results
in very poor inhibitors, likely indicating that the lone pairs of the
heteroatoms have disfavored interactions with the enzyme.
Given the structures of these compounds, a concern regarding
their ability to interfere with the fluorescence assay and result in
significant inner-filter effect quenching18 could be raised. To ad-
dress this, a set of compounds were tested for intrinsic fluores-
cence, as well as their ability to quench the fluorescence of the
product of SNAPtideÒ cleavage. Both potent inhibitors as well as
compounds that showed poor to no inhibition were included in
this testing. In all cases, no effect on SNAPtideÒ cleavage product
fluorescence was observed, indicating that the observed inhibition
is not an artifact of the assay.
28
0.75 0.25
29
30
0.66 0.19
8.01 1.7
31
>100
We next chose to replace the phenyl ring with various six- (24–
38) or five-membered (39–44) heterocyclic analogs both in an at-
tempt to improve on the interactions between the inhibitor and
enzyme, as well as to test our SAR-derived hypotheses (Table 2).
After screening this second-generation set, some interesting trends
emerged. First, of the 21 compounds, 8 compounds had improved
inhibition relative to the parent hydroxamate (Table 2). Indeed, 3-
pyridyl substitution resulted in compound 24 which displayed an
IC50 of 230 nM, a threefold improvement in potency over 1.
Interestingly, some of the SAR trends observed in the biphenyl
series remained valid in this series (Table 2). In particular, meta
substitution relative to the second aromatic ring was disfavored
and resulted in poor inhibition (25, 26, and 31), as did bulky sub-
stitution at the para position (36, 37, and 38). Ortho substitution
was tolerated and resulted in good inhibitors in both series (9
and 29). However, addition of a p-methoxy moiety resulted in po-
tent inhibition within the pyridyl series, in contrast to the first gen-
eration biphenyls (27 vs 10).
32
33
34
10 1.2
0.61 0.12
2.79 0.35
35
36
1.21 0.2
73.2 4.2
37
12.3 1.6
Indeed, in the pyridyl series, p-methoxy substitution had little
effect on the IC50 (24 vs 27 or 28), while this same substitution
in the biphenyl series attenuated the inhibitory activity (4 vs 10).
The reason for this discrepancy could be that because of the change
in the electronics of the aromatic moiety by inclusion of the het-
eroatom, the enzyme can now tolerate the additional steric bulk
at this position. Put another way, we hypothesize that the inconsis-
tent SAR across compound series is best explained through the
known plasticity of this enzyme;16 addition of the hydrogen
bond-accepting pyridyl ring leads to a structural change in the en-
zyme that induces a pocket capable of accommodating additional
substitution that was not tolerated in the initial biphenyl series
(i.e., 4 vs 12). A further example that supports this hypothesis is
given in the comparison of compounds 34 and 35; here, inclusion
of two nitrogen atoms is comparable to the parent 4 and in the
presence of both heteroatoms, the addition of the p-methoxy group
in 35 leads to improved inhibition.
38
39
21.3 5.4
1.32 0.27
40
41
24.9 6.5
22.3 8.1
42
1.16 0.38

G. R. Smith et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3754–3757
3757
lM), and an additional nine compounds had comparable potency
Table 2 (continued)
to the parent hydroxamate (IC50 6 2 M), a ‘hit’ rate of 35%. This
l
Compound
R
IC50 (lM)
is a surprisingly large number of inhibitors, particularly given that
the structural data has suggested that certain modifications (e.g.,
removal of the 4-chloro moiety of 1) are not tolerated. In total, this
study highlights the difficulty in establishing structure–activity
relationships with BoNT/A LC and cautions against making gener-
alizations across compound series, even when there is significant
structural homology present.
43
66.2 5.8
44
9.11 0.87
IC50 values were determined using the SNAPtideÒ assay.17
a
Acknowledgments
This work was supported by the National Institutes of Health
(AI082190 to T.J.D.). S.G. was supported by an internship from
the California Institute for Regenerative Medicine (TB1-01186).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.019.
References and notes
1. Willis, B.; Eubanks, L. M.; Dickerson, T. J.; Janda, K. D. Angew. Chem., Intl. Ed.
2008, 47, 8360.
2. Foran, P. G.; Davletov, B.; Meunier, F. A. Trends Mol. Med. 2003, 9, 291.
3. Bhattacharjee, Y. Science 2011, 322, 1491–1492.
4. Aoki, K. R.; Smith, L. A.; Atassi, M. Z. Crit. Rev. Immunol. 2010, 30, 167.
5. Schiavo, G.; Matteoli, M.; Montecucco, C. Physiol. Rev. 2000, 80, 717.
6. Wang, J.; Zurawski, T. H.; Meng, J.; Lawrence, G.; Olango, W. M.; Finn, D. P.;
Wheeler, L.; Dolly, J. O. J. Biol. Chem. 2011, 286, 6375.
Figure 1. Docking of 1 (red) and 24 (blue) onto BoNT/A LC (PDB ID: 2IMA). The
white arrow indicates the severe steric clash with the wall of the binding pocket
and the yellow sphere represents the Zn2+ ion.
7. Fernandez-Salas, E.; Ho, H.; Garay, P.; Steward, L. E.; Aoki, K. R. Mov. Disord.
2004, 19, 23.
8. Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Org. Lett. 2006, 8, 1729.
9. Eubanks, L. M.; Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.; Tepp, W. H.;
Baldwin, M. R.; Malizio, C. J.; Goodnough, M. C.; Barbieri, J. T.; Johnson, E. A.;
Boger, D. L.; Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
2602.
In order to explore the nature of the binding interactions be-
tween the biaryl cinnamate inhibitors and the BoNT/A LC, we per-
formed molecular modeling experiments in which compounds
were docked onto the active site of the protease (Glide). Starting
from the published crystal structure of 1 with BoNT/A (PDB ID:
2IMA),16 it was possible to dock compound 1 in the proper position
relative to the crystal structure; however, when using this struc-
ture for the biaryl inhibitors, significant rotational strain was intro-
duced in the inhibitor about the cinnamate double bond and the
aryl substituent extended through the back of the pocket, resulting
in poor binding energies ( Fig. 1). We speculated that this poor
docking could be corrected by allowing flexibility in the enzyme
and consequently, we attempted to dock the biaryl compounds
using induced fit parameters to allow for additional enzyme flexi-
bility. Under these conditions, the compounds could be accommo-
dated as reflected by Glide scores that indicated that these
compounds would be comparable or better inhibitors to 1.
10. Capek, P.; Zhang, Y.; Barlow, D. J.; Houseknecht, K. L.; Smith, G. R.; Dickerson, T.
J. ACS Chem. Neurosci. 2011, 2, 288.
11. Ruthel, G.; Burnett, J. C.; Nuss, J. E.; Wanner, L. M.; Tressler, L. E.; Torres-
Melendez, E.; Sandwick, S. J.; Retterer, C. J.; Bavari, S. Toxins 2011, 3, 207.
12. Hermone, A. R.; Burnett, J. C.; Nuss, J. E.; Tresler, L. E.; Nguyen, T. L.; Solaja, B. A.;
Vennerstrom, J. L.; Schmidt, J. J.; Wipf, P.; Bavari, S.; Gussio, R. ChemMedChem
1905, 2008, 3.
13. Burnett, J. C.; Wang, C.; Nuss, J. E.; Nguyen, T. L.; Hermone, A. R.; Schmidt, J. J.;
Gussio, R.; Wipf, P.; Bavari, S. Bioorg. Med. Chem. Lett. 2009, 19, 5811.
14. Wang, C.; Widom, J.; Petronijevic, F.; Burnett, J. C.; Nuss, J. E.; Bavari, S.; Gussio,
R.; Wipf, P. Heterocycles 2009, 79, 487.
15. Capkova, K.; Yoneda, Y.; Dickerson, T. J.; Janda, K. D. Bioorg. Med. Chem. Lett.
2007, 17, 6463.
16. Silvaggi, N. R.; Boldt, G. E.; Hixon, M. S.; Kennedy, J. P.; Tzipori, S.; Janda, K. D.;
Allen, K. N. Chem. Biol. 2007, 14, 533.
17. Boldt, G. E.; Kennedy, J. P.; Hixon, M. S.; McAllister, L. A.; Barbieri, J. T.; Tzipori,
S.; Janda, K. D. J. Comb. Chem. 2006, 8, 513.
18. Liu, Y.; Kati, W.; Chen, C.-H.; Tripathi, R.; Molla, A.; Kohlbrenner, W. Anal.
Biochem. 1999, 267, 331.
The results of this screen show that 6 of the 43 compounds
tested had better potency than the parent compound (IC50 <0.67