Identification of a potent botulinum neurotoxin A protease inhibitor using in situ lead identification chemistry

Article abstract of DOI:10.1021/ol0603211

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. By using in situ lead identification chemistry, we have uncovered the first class of inhibitors that displays nanomolar potency. From a 15 μM lead compound, structure-activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays an inhibition constant of 300 nM.

Full text of DOI:10.1021/ol0603211

ORGANIC
LETTERS
2
006
Vol. 8, No. 8
729-1732
Identification of a Potent Botulinum
Neurotoxin A Protease Inhibitor Using in
Situ Lead Identification Chemistry
1
Grant E. Boldt, Jack P. Kennedy, and Kim D. Janda*
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology
and Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
kdjanda@scripps.edu
Received February 6, 2006
ABSTRACT
Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known.
By using in situ lead identification chemistry, we have uncovered the first class of inhibitors that displays nanomolar potency. From a 15
µ
M
lead compound, structure-activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays
an inhibition constant of 300 nM.
Botulinum neurotoxin (BoNT), an agent responsible for the
deadly food poisoning disease botulism and a dreaded
biological weapon, is one of the most toxic proteins currently
cleavage sites of these SNARE proteins (SNAP-25, VAMP,
Sb-1) differ across the BoNT serotypes; however, any
degradation of these SNARE proteins disables the exocytosis
of acetylcholine, resulting in paralysis and potentially death.2
Current therapy for BoNT intoxication involves “passive
1
known (∼100 billion times more toxic than cyanide).
Clostridium botulinum is classified into seven strains (A-
G) each of which can cause flaccid muscle paralysis and
subsequent death by blocking the release of a neurotrans-
4
immunization” with equine antitoxin. Unfortunately, treat-
ment must start shortly after intoxication, and several safety
2
2
mitter, acetylcholine, at neuromuscular junctions. Structur-
concerns exist over the use of antitoxins in the general
5
ally, BoNT consists of three functional domains, catalytic,
translocation, and binding; BoNT toxicity results from the
catalytic activity of its light chain, a Zn(II) endopeptidase.
The catalytic domain of BoNT is a compact globule
consisting of a mixture of R-helices, â-sheets, and strands
population. Therefore, inhibition of the catalytic light chain
protease with a small molecule inhibitor may provide an
attractive approach to counter the effects of botulism
poisoning.
BoNT serotype A (BoNT/A) is the most toxic form of
BoNTs and is considered the most threatening for biological
attacks due to a prolonged half-life in vivo and ease of its
with a gorgelike zinc-containing metalloprotease active site
3
(
15-20 Å deep depending on serotype). The metalloprotease
4
activity is responsible for BoNT’s neurotoxicity through the
hydrolytic cleavage of one of three SNARE (soluble NSF-
attachment protein receptor) proteins that are involved in the
neuronal synaptic vesicle function. Moreover, the hydrolytic
production. Although there are reports of success treating
6
BoNT/A toxicity with multiple monoclonal antibodies as
(4) Hicks, R. P.; Hartell, M. G.; Nichols, D. A.; Bhattacharjee, A. K.;
Von Hamont, J. E.; Skillman, D. R. Curr. Med. Chem. 2005, 12, 667-
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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, 1059-1070.
(
1) Singh, B. R. Nature 2000, 7, 617-619.
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1
93.
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1
0.1021/ol0603211 CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/17/2006

antitoxins, this is of limited therapeutic utility because the
antibodies must be administered prior to, or shortly after,
toxin exposure (<12 h).
carboxylic acid library. Namely, 150 carboxylic acids
purchased from Aldrich Chemical Co. were randomly chosen
and converted to their ester by treatment with diazomethane
(Scheme 1). After removal of the solvent, reactions were
Presently, there are only modest small molecule, nonpep-
tidic, protease inhibitors for BoNT/A with IC50 values in the
7
range of >20 µM. We established a high-throughput screen
8
for the identification of inhibitors of BoNT/A LC protease.
Scheme 1. Synthesis and Screening of the in Situ
Using this screen, we have analyzed a library of hydroxam-
ate-based compounds generated using in situ chemistry to
reveal the lead structure 1 (Figure 1). Herein, we report on
Hydroxamate Library
subjected to a mixture of THF:MeOH:50% aqueous HONH
:2:1 with a catalytic amount of KCN overnight. Again, the
2
2
solvent was removed and the crude products were reconsti-
tuted with DMSO to prepare stock solutions of a final
concentration of 10 mM for screening. To check the quality
of the library, 30 compounds were randomly selected and
analyzed by ES-MS, and in all cases, the expected masses
corresponding to the products were found (data not shown).
8
Using a high-throughput screen, namely, a 13 amino acid
substrate fluorescence resonance energy transfer (FRET)
assay developed in our laboratory, we analyzed the library
of hydroxamates at a concentration of 50 µM. Any inhibitors
found to display 50% inhibition were considered “hits” and
evaluated further. From the initial screen, five compounds
were found to give 50% or more inhibition in the FRET-
based assay. These compounds were resynthesized, purified,
and validated using the FRET-based assay. Of the five lead
compounds, only compound 1 showed potent activity; para-
chloro-cinnamic hydroxamate (1) displayed an IC50 value
of 15 µM, and thus, this simple high-throughput screen
uncovered an interesting lead for further development.
To refine this positive lead, we synthesized a series of 12
compounds so as to further explore the structure-activity
relationship of compound 1. However, the method outlined
in Scheme 1 was found to be inefficient for the isolation of
the desired compounds because of purification difficulties.
Therefore, we sought out a more convenient method for the
expeditious generation and purification of the hydroxamate
compounds. Solid-phase organic synthesis (SPOS) lends
itself to these requirements nicely due to reactions proceeding
in high yield and purification being significantly simplified.
Several groups have demonstrated successful hydroxamate
Figure 1. Structures of lead compounds identified from in situ
screening.
the synthesis and structure-activity relationship studies of
these BoNT/A inhibitors.
Recently, a convenient method for the preparation of
9
hydroxamates from readily available esters was reported.
In this general procedure, a diverse array of acids can easily
be converted to hydroxamates with hydroxylamine in the
presence of a catalytic amount of potassium cyanide. In
addition, several reports have been disclosed for the prepara-
10
tion of hydroxamates; however, these procedures have strict
substrate requirements.
With these thoughts in mind, we set out to generate a
library of diverse hydroxamates from a readily available
(6) (a) Amersdorfer, P.; Wong, C.; Smith, T.; Chen, S.; Deshpande, S.;
Sheridan, R.; Marks, J. D. Vaccine 2002, 20, 1640-1648. (b) Nowakowski,
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11
syntheses using solid-phase routes and hydroxyamine
T. L.; Hermone, A. R.; Panchal, R. G.; Vennerstrom, J. L.; Kodukula, K.;
Zaharevitz, D. W.; Gussio, R.; Bavari, S. Bioorg. Med. Chem. 2005, 13,
1
2
resins. However, we found these procedures to be insuf-
ficient for our purposes. 2-Chlorotrityl resin was purchased
3
33-341. (b) Burnett, J. C.; Schmidt, J. J.; Stafford, R. G.; Panchal, R. G.;
Nguyen, T. L.; Hermone, A. R.; Vennerstrom, J. L.; McGrath, C. F.; Lane,
D. J.; Sausville, E. A.; Zaharevitz, D. W.; Gussio, R.; Bavari, S. Biochem.
Biophys. Res. Commun. 2003, 310, 84-93.
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Billedeau, R. J.; Lawley, L. K.; Abbot, S. C.; Martin, R. L.; Chan, C. S.;
Van Wart, H. E.; Walker, K. E. Bioorg. Med. Chem. Lett. 2000, 10, 2513-
2516.
(12) (a) Barlaam, B.; Koza, P.; Berriot, J. Tetrahedron 1999, 55, 7221-
7232. (b) Mellor, S. L.; McGuire, C.; Chan, W. C. Tetrahedron Lett. 1997,
38, 3311-3314. (c) Floyd, C. D.; Lewis, C. N.; Patel, S. R.; Whittaker, M.
Tetrahedron Lett. 1996, 37, 8045-8048. (d) Ede, N. J.; James, I. W.;
Krywuth, B. M.; Griffiths, R. M.; Eagle, S. N.; Gubbins, B.; Leitch, J. A.;
Sampson, W. R.; Bray, A. M. Lett. Pept. Sci. 1999, 6, 157-163. (e) Bauer,
U.; Ho, W.-B.; Koskinen, A. M. P. Tetrahedron Lett. 1997, 7233-7236.
(8) Boldt, G. E.; Kennedy, J. P.; Hixon, M. S.; McAllister, L. A.; Barbieri,
J. T.; Tzipori, S.; Janda, K. D. J. Comb. Chem. 2006, in press.
9) Ho, C. Y.; Strobel, E.; Ralbovsky, J.; Galemmo, R. A. J. Org. Chem.
005, 70, 4873-4875.
10) (a) Burns, C. J.; Groneberg, R. D.; Salvino, J. M.; McGeehan, G.;
(
2
(
Condon, S. M.; Morris, R.; Morrissette, M.; Mathew, R.; Darnbrough, S.;
Neuenschwander, K.; Scotese, A.; Djuric, S.; Ullrich, J.; Labaudiniere, R.
Angew. Chem., Int. Ed. 1998, 37, 2848-2850. (b) Mori, K.; Koseki, K.
Tetrahedron 1988, 44, 6013-6020. (c) Spengler, J.; Burger, K. Synthesis
1
998, 1, 67-70.
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Org. Lett., Vol. 8, No. 8, 2006

from Chemical and Biopharmaceutical Laboratories of Patras
S. A. (CBL) and used in the synthesis of hydroxamate
inhibitors as outlined in Scheme 2. It is important to note
Table 1. IC50 Valuesa
Scheme 2. Solid-Phase Synthesis of Hydroxamic Acids
that several resins were explored using this methodology;
however, the CBL was the only resin found to give
satisfactory results. This resin was treated with N-hydroxy-
phthalimide for 2 days in DMF at room temperature for the
quantitative conversion to 6. The phthalimide was success-
fully removed with exposure to anhydrous hydrazine to give
the hydroxylamine resin 7, ready for the coupling of a variety
of acids. Next, the desired carboxylic acids were coupled to
the resin 7 using standard diisopropylcarbodiimide and
6-chlorohydroxybenzotriazole protocols. The desired hy-
droxamates were recovered by treatment of the resin with
5% TFA and removal of the solvent.
These compounds were further individually purified via
silica gel and then reconstituted in DMSO for evaluation of
BoNT/A LC inhibition.
To investigate the structure-activity relationship of com-
pound 1, we first synthesized a group of para-substituted
cinnamic hydroxamates. Unexpectedly, the chloro substitu-
tion in the para position appears to be highly conserved as
seen in Table 1, compounds 10-16. For example, when a
bulky functionality such as a tert-butyl, compound 14, is
introduced to the para position, inhibition is completely lost.
Moreover, inhibition is compromised when introducing
electron-withdrawing or donating groups (compounds 10-
16). Thus, we next examined other possible chloro substitu-
tion patterns of the lead compound 1. Gratifyingly, when
evaluating different chloro substitution patterns (compounds
17 and 18), the ortho-para-cinnamic hydroxamate, 18,
displayed an IC50 value of 0.41 µM. Furthermore, it appears
that the trans-olefin motif is also conserved as seen in
compounds 19-21. It is important to note that the cis-olefin
form of 1 was not examined because of instability in
solution.13 However, strikingly, when the trans-olefin was
substituted for an alkyne, 20, inhibition was attenuated by
greater than 175-fold. The reduced form of 18 to the alkane,
compound 19, also reduced potency significantly.
a
Assays were conducted at various inhibitor concentrations at 22.5 °C,
pH 7.4 in 40 mM HEPES 0.01% (W/V) Tween 20, 5 µM SNAPtide
substrate, and 200 nM enzyme.
To further kinetically characterize 18, we evaluated it in
an assay system with the native SNAP-25 (141-206) substrate
using HPLC for analysis. It is important to note that SNAP-
2
5 (141-206) has no structural modification, thus the
(13) cis-2,4,Dichloro-cinnamic acid was found to be unstable during
coupling.
structural integrity of the molecule has not been compro-
Org. Lett., Vol. 8, No. 8, 2006
1731

mised. Using this substrate, a K
i
of 300 ( 12 nM was found
Acknowledgment. This work was supported by the
National Institute of Health BT010-04 and The Skaggs
Institute for Chemical Biology.
and also displayed the expected competitive mode of
inhibition (data not shown). Moreover, these findings
demonstrate that a 10 contiguous carbon chain previously
thought to be a strict requirement for BoNT/A LC inhibition
is in fact not a prerequisite and small organic molecules can
be used for possible drug leads.
In total, a simple in situ synthesis and screen was
developed for the identification of nonpeptidic protease
inhibitors of BoNT/A LC. The power of using this strategy
was that no prior bias was placed on the selection of input
acids, yet a desirable lead was uncovered. We note that
compound 18 (Table 1) is the most potent protease inhibitor
described to date for BoNT/A and is currently being
investigated in cell and mouse assays for antibotulism effects
and will be reported in due course.
14
Note Added after ASAP Publication. A 13 amino acid
substrate FRET assay was used, not a 17 amino acid sub-
strate as indicated in the version published ASAP March 17,
2
2
006; the corrected version was published ASAP March 23,
006.
Supporting Information Available: Experimental syn-
thetic procedures and NMR and HRMS data for the library
members and intermediates and screening procedures. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(
14) 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, 395-408.
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