Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Article abstract of DOI:10.1016/j.bmcl.2011.02.115

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therape

Full text of DOI:10.1016/j.bmcl.2011.02.115

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2229–2231
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of library of betulin derivatives against
the botulinum neurotoxin A protease
Peter Šilhár a,b, , Sami Alakurtti c,d, , Katerina Capková a,b, Feng Xiaochuan e, Charles B. Shoemaker e,
ˇ
ˇ
a,b,f,
Jari Yli-Kauhaluoma c, , Kim D. Janda
⇑
⇑
a Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA
b Department of Immunology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA
c Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, PO Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland
d VTT Technical Research Centre of Finland, PO Box 1000 (Biologinkuja 7), FI-02044 VTT, Finland
e Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Road, NorthGrafton, MA 01536, USA
f Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Rd., 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 currently known. Current treatments for
botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT
light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it
may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was
synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC50
Received 20 January 2011
Revised 25 February 2011
Accepted 28 February 2011
Available online 4 March 2011
<100 lM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds
Keywords:
revealed a competitive model, with sub-micromolar Ki value for the best inhibitor (7). Unfortunately, an
in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxica-
tion by 7.
Betulin derivatives
Botulinum neurotoxin
Protease inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Botulinum neurotoxins (BoNTs), proteins produced by bacteria
of the genus Clostridium,1 are responsible for botulism, a disease
characterized by peripheral neuromuscular blockade and a charac-
teristic flaccid paralysis of humans. BoNTs are the most poisonous
substances known, with serotype A having a lethal dose for a 70 kg
as it may provide an effective post-exposure remedy. BoNTs are
synthesized as ꢀ150 kDa proteins that are post-translationally
activated by proteolytic cleavage to form mature di-chain proteins
consisting of a 100 kDa heavy chain (HC) and a 50 kDa light chain
(LC) linked by a disulfide bond.5 The HC is responsible for the neu-
rospecific binding, uptake, and translocation of the LC into the
cytosol of neuronal cells. The LC is a Zn2+-dependent metallopro-
tease that cleaves one of three intracellular soluble N-ethylmalei-
mide-sensitive factor attachment protein receptor (SNARE)
proteins: syntaxin, vesicle-associated membrane protein (VAMP)/
synaptobrevin, or synaptosomal-associated protein of 25 kDa
(SNAP-25) depending on the serotype. As a consequence of protein
cleavage, release of acetylcholine at the neuromuscular junction is
blunted resulting in the loss of neurotransmission. Small molecule
inhibitors of the LC may provide an opportunity for development of
both pre- and post-exposure therapeutics. In recent years, the LC of
serotype A (LC/A) has been a major focus, primarily due to its po-
tency and long duration of paralysis.4 A number of competitive
inhibitors of LC/A have been reported the most potent have Ki val-
human of approximately 0.09–0.15
lg intravenously or intramus-
cularly, and 0.7–0.9
l
g inhalationally.2 Despite their potentially
lethal toxicity, BoNTs have emerged as an extremely valuable ther-
apeutic tool for the treatment of a variety of maladies, including
strabismus, migraines, and even facial wrinkles.3 However, the po-
tential use of BoNT in a bioterrorist attack remains imminent and
the Center for Disease Control (CDC) now classifies this agent as
‘category A’, placing it among the six highest-priority agents. Cur-
rent treatments for botulinum poisoning are all antibody based
with a limited window of therapeutic effectiveness. A particular
drawback with these vaccine approaches is that they cannot re-
verse the effects after the toxin has reached its target inside the
cell.4
Inhibition of the BoNT light chain metalloprotease (LC) has sur-
faced as a new therapeutic strategy for the treatment of botulism
ues of 0.6–10
cation required for catalysis (Ki ranging from 0.3 to 12.3
More recently, the natural product -chicoric acid was discovered
l
M,6 several of which coordinate the active site zinc
l
M).7
D
⇑
Corresponding authors. Tel.: +1 858 784 2516, fax: +1 858 784 2595 (K.D.J.);
tel.: +358 9 19159170 (J.Y.-K.).
in our laboratory that putatively binds to an exosite region outside
the LC/A active site, displaying noncompetitive partial inhibition.8
Betulin is a naturally occurring, pentacyclic triterpene alcohol
belonging to the lupane series of compounds. Betulin is the
E-mail addresses: jari.yli-kauhaluoma@helsinki.fi (J. Yli-Kauhaluoma), kdjanda@
scripps.edu (K.D. Janda).
Equal contribution.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.115

2230
P. Šilhár et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2229–2231
Table 1
Table 1 (continued)
Structures of betulin derivatives screened against BoNT/A protease
Compound
R1
R2
R3
R3
OMe
O
O
OAc
OH
R1
OAc
AcO
R2
25
O@
O
O
H
HO
AcO
2 -28
14
O
1
13
O
Compound
R1
R2
R3
26
O@
1
2
OH
OH
CH2OH
CH2OH
27
28
OH
CH@NOH
CH@NOH
@NOH
O
O
O
3
4
OH
OH
O
R3
N
O
OH
OMe
O
H
N
N
H
OR4
O
O
N
H
R4O
5
OH
29-40
Compound
R3
R4
O
O
29
30
31
32
33
34
35
36
37
38
39
40
Me
Me
Me
n-Bu
Ph
Bn
3-MeO-Ph
4-F-Ph
3-NO2-Ph
4-Ac-Ph
4-Cl-Ph
Indan-5-yl
Ac
6
7
OH
OH
CocHex
COPh
Ac
Ac
Ac
Ac
Ac
Ac
Ac
O
O
OH
O
8
OH
OH
O
O
O
Ac
Ac
9
10
11
OAc
OH
a
See structure above.
OAc
OAc
CH2OAc
CH2OAc
CH2OAc
principle extractive substance of outer birch bark, and it has been
extracted from white-barked birches (Betula sp.) in amounts up to
30% dry weight. Betulin 1 can be converted to betulinic acid 18,9
which has plethora of pharmacological properties, such as cyto-
toxic activity against several tumor cell lines by inducing apoptosis
in cells.10 Excitingly, some betulin derivatives have also shown
remarkable anti-HIV activity with new mechanisms of action.11
We have previously reported several new bioactivities for these
highly interesting compounds, such as anti-leishmanial,12 anti-
alphaviral13 and anti-chlamydial activity.14 Finally, structure–
activity relationship (SAR) studies and pharmacological properties
of betulin and its derivatives have been recently reviewed.15
Based on previous studies we were intrigued about potential
activity of such compounds against BoNT/A. Therefore, a library
of 40 betulin derivatives was tested for their inhibition of
BoNT/A protease. The chemical structures of the betulin-derived
triterpenoids are presented in Table 1; we note that we have pre-
viously reported these compounds,12a,13 with the exception of 4, 7,
12 and 26, which are now provided in Supplementary data. Thus,
O
12
OAc
13a
14a
OAc
OAc
CH2OAc
O
O
15
16
OAc
OAc
O
S
O
O
O
O
O
17
O
O
O
18
19
20
21
22
23
OH
CO2H
CO2Me
CHO
OH
O@
O@
O@
O@
compounds were tested at a single concentration (50
lM) by
LC/MS assay7b at 10
l
M concentration of optimized truncated
CO2H
CO2Me
CO2H
SNAP-substrate (66-mer; 141–206 aa) encompassing the key rec-
ognition elements of SNAP-25. From this lot, five positive hits 7,
8, 18, 19 and 21 (IC50 <100 lM) were uncovered and three of these,
7, 8 and 18 were further evaluated at various concentrations of
substrate and inhibitor. Obtained data were most consistent with
a competitive inhibition model (Supplementary data). The inhibi-
tion constants, Ki were determined by a non-linear least squares
global fit to the initial rates of product formation for matrixes of
substrate and inhibitor concentrations bracketing Km and Ki,
O
O
H
N
OMe
OMe
24
O@
O

P. Šilhár et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2229–2231
2231
Table 2
the Finnish Funding Agency for Technology and Innovation, Foun-
dation for Research of Natural Resources in Finland, Marjatta ja
Eino Kollin Säätiö, and the project by the European Commission
(Contract No. EU-KBBE-227239) are gratefully acknowledged
(J.Y.-K.).
IC50 and Ki values of positive hits
Compound
IC50
(
lM)
Ki (lM)
7
8
18
1.5
10
20
0.8 0.2
13.3 2.3
14.3 2.2
Supplementary data
N.D. = not determined.
Supplementary data (experimental procedures, full character-
ization for compounds 4, 7, 12, 26 and biological assay conditions)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.02.115.
respectively (Table 2). These results revealed that 28-hemisucci-
nylbetulin 7 was the best inhibitor with Ki = 0.8 0.2 M. Thus,
l
making it interesting lead structure for iterative rounds of
structural modification in search of more potent small molecule
antagonists of BoNT/A. The other betulin derivatives, betulinyl
28-carboxymethoxycarvacrolate 8 and betulinic acid 18 (Table 1)
were about 16 and 18-fold less potent, respectively.
Potency of the best inhibitor was further investigated using an
in vitro cell-based assay that monitors intracellular cleavage of
SNAP-25. Thus, the cellular efficacy of compound 7 was tested
References and notes
1. (a) Johnson, E. A.; Bradshaw, M. Toxicon 2001, 39, 1703; (b) Roxas-Duncan, V.;
Enyedy, I.; Montgomery, V. A.; Eccard, V. S.; Carrington, M. A.; Lai, H.; Gul, N.;
Yang, D. C. H.; Smith, L. A. Antimicrob. Agents Chemother. 2009, 53, 3478.
2. Arnon, S. S.; Schechter, R.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.;
Ascher, M. S.; Eitzen, E.; Fine, A. D.; Hauer, J.; Layton, M.; Lillibridge, S.;
Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T. M.; Russell, P. K.; Swerdlow, D.
L.; Tonat, K. JAMA 2001, 285, 1059.
using primary rat cerebellar neurons at 30 and 80 lM concentra-
3. (a) Hackett, R.; Kam, P. C. Med. Chem. 2007, 3, 333; (b) Truong, D. D.; Jost, W. H.
Parkinsonism Relat. Disord. 2006, 12, 331.
4. Willis, B.; Eubanks, L. M.; Dickerson, T. J.; Janda, K. D. Angew. Chem., Int. Ed.
2008, 47, 8360. and references cited therein.
tions, respectively. However, this assay did not show inhibitory
activity of this derivative against BoNT/A. Clearly, cells are complex
biological systems and other factors such as permeability can influ-
ence the efficacy of small molecules within cellular models. Addi-
tionally, we highlight that betulin derivatives possess very low
solubility in aqueous buffers that can also influence cellular activ-
ity (see Supplementary data for calculated physicochemical data).
In summary, a small library of 40 betulin derivatives was pre-
pared and subsequently tested for potential inhibition of BoNT/A
protease. Interestingly, five compounds within this library were
discovered to exert low micromolar activities against the protease.
Further detailed evaluation of the mechanism of inhibition of the
three most active compounds revealed competitive inhibition,
with a sub-micromolar Ki value for the best inhibitor. Disappoint-
ingly, in vitro cell-based examination did not demonstrate protec-
tion of rat cerebellar neurons against BoNT/A intoxication by 7. It is
anticipated that additional modifications of the betulin structure to
endow better water solubility and cell permeability will allow the
discovery of betulins with cellular activity against BoNT/A.
5. Oguma, K.; Fujinaga, Y.; Inoue, K. Microbiol. Immunol. 1995, 39, 161.
6. (a) Burnett, J. C.; Ruthel, G.; Stegmann, C. M.; Panchal, R. G.; Nguyen, T. L.;
Hermone, A. R.; Stafford, R. G.; Lane, D. J.; Kenny, T. A.; McGrath, C. F.; Wipf, P.;
Stahl, A. M.; Schmidt, J. J.; Gussio, R.; Brunger, A. T.; Bavari, S. J. Biol. Chem. 2007,
282, 5004; (b) 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; (c) Pang, Y. P.; Vummenthala, A.; Mishra, R. K.; Park, J. G.; Wang, S.;
Davis, J.; Millard, C. B.; Schmidt, J. J. PLoS One 2009, 4, e7730.
7. (a) Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Org. Lett. 2006, 8, 1729; (b) Capkova,
K.; Hixon, M. S.; McAllister, L. A.; Janda, K. D. Chem. Commun. 2008, 3525; (c)
Park, J. G.; Sill, P. C.; Makiyi, E. F.; Garcia-Sosa, A. T.; Millard, C. B.; Schmidt, J. J.;
Pang, Y. P. Bioorg. Med. Chem. 2006, 14, 395; (d) Tang, J.; Park, J. G.; Millard, C.
B.; Schmidt, J. J.; Pang, Y. P. PLoS One 2007, 2, e761; (e) Stowe, G. N.; Silhar, P.;
Hixon, M. S.; Silvaggi, N. R.; Allen, K. N.; Moe, S. T.; Jacobson, A. R.; Barbieri, J. T.;
Janda, K. D. Org. Lett. 2010, 12, 756.
8. Silhar, P.; Capkova, K.; Salzameda, N. T.; Barbieri, J. T.; Hixon, M. S.; Janda, K. D.
J. Am. Chem. Soc. 2010, 132, 2868.
9. Kim, D. S. H. L.; Chen, Z.; Nguyen, T.; Pezzuto, J. M.; Qiu, S.; Lu, Z.-Z. Synth.
Commun. 1997, 27, 1607.
10. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, G. A.;
Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani, M. C.;
Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.; Pezzuto, J. M. Nat. Med. 1995, 1, 1046.
11. Soler, F.; Poujade, C.; Evers, M.; Carry, J. C.; Henin, Y.; Bousseau, A.; Huet, T.;
Pauwels, R.; De Clercq, E.; Mayaux, J. F.; Le Pecq, J. B.; Dereu, N. J. Med. Chem.
1996, 39, 1069.
Acknowledgments
12. (a) Alakurtti, S.; Heiska, T.; Kiriazis, A.; Sacerdoti-Sierra, N.; Jaffe, C. L.; Yli-
Kauhaluoma, J. Bioorg. Med. Chem. 2010, 18, 1573; (b) Alakurtti, S.; Bergström,
P.; Sacerdoti-Sierra, N.; Jaffe, C. L.; Yli-Kauhaluoma, J. J. Antibiot. 2010, 63, 123.
13. Pohjala, L.; Alakurtti, S.; Ahola, T.; Yli-Kauhaluoma, J.; Tammela, P. J. Nat. Prod.
2009, 72, 1917.
14. Salin, O.; Alakurtti, S.; Pohjala, L.; Siiskonen, A.; Maass, V.; Maass, M.; Yli-
Kauhaluoma, J.; Vuorela, P. Biochem. Pharmacol. 2010, 80, 1141.
15. (a) Yogeeswari, P.; Sriram, D. Curr. Med. Chem. 2005, 12, 657; (b) Alakurtti, S.;
Mäkelä, T.; Koskimies, S.; Yli-Kauhaluoma, J. Eur. J. Pharm. Sci. 2006, 29, 1.
We thank Dr. Mark S. Hixon and Adjunct Professor Salme Koski-
mies for very helpful discussions. This project was supported
through the Skaggs Institute for Chemical Biology as well as with
federal funds from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Department of Health and
Human Services, under contract number N01-AI-30050 (C.B.S.)
and award number AI080671 (K.D.J.). The financial support from