1. In Vitro Botulinum A Toxicity Inhibition by Small Molecule Inhibitors
Kathleen Housman, Joshua Emory, Raymond Sullivan, Matt Levit, Michael Adler and Jonathan Oyler
usamricd.apgea.army.mil
DISCUSSION/CONCLUSIONSDISCUSSION/CONCLUSIONS
Clostridium botulinum A toxin (BoNT-A) is a di-chain protein comprised of
single 100 kDa heavy (HC) and 50 kDa zinc-dependent light (LC) chain
metalloprotease. The HC induces toxin nerve terminus receptor binding and
intracellular LC translocation. Cytosolic LC cleaves synaptosomal-associated
protein 25 (SNAP-25) , one of the essential proteins comprising the SNARE
Complex, inhibiting acetylcholine release leading to flaccid muscular
paralysis. Although the structural and functional characteristics of BoNT-A
have been extensively examined, robust, efficacious in vivo inhibitors for
treatment of clinical intoxication have not been developed. The work
described evaluated the in vitro inhibition efficacy of candidate small
molecule inhibitors (SMIs). Five Hawaii Biosystems hydroxamates and three
quinolines were evaluated using SNAP25ac187-203ca (17mer) as substrate for
truncated LC 429 (LCA). Various SMI concentrations were evaluated, and the
17mer and two cleavage products were quantified by LC/ESI-MSMS.
Absolute reagent concentrations were briefly optimized and time course
studies were performed.
In addition, we characterized SNAP25141-206 (66mer) and its LCA N-
terminal cleavage product and two cyclic peptides with LC inhibition
potential using nominal and high resolution/mass accuracy methods. The
66mer contains the BoNT-A cleavage site and has been shown to have a
substantially lower Km for LCA than the 17mer using a Förster resonance
energy transfer (FRET) assay. A quantitative MS-based method for it and
one of its LCA digest products would substantially reduce analytical cost
since the recombinant LCA is the cost-limiting reagent. The two cyclic
peptides (Fig 1) are candidate BoNT-A SMIs: CPI-1 has been demonstrated to
be a LCA inhibitor in the FRET assay. CPI-3, identical to CPI-1 minus a C-
terminus L residue, has not been evaluated.
1.Christian T, Shine N, Eaton L and Crawford K. (2005) Comparison of Activity of Botulinum
Neurotoxin Type A Holotoxin and Light Chain Using SNAPtide FRET Substrates. 5th International
Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, Denver, CO, 23
June, 2005.
2.Thompson AA, Jiao GS, Kim S, Thai A, Cregar- Hernandez L, Margosiak SA, Johnson AT, Han GW,
O'Malley S, Stevens RC. (2011) Structural characterization of three novel hydroxamate-based
zinc chelating inhibitors of the Clostridium botulinum serotype A neurotoxin light chain
metalloprotease reveals a compact binding site resulting from 60/70 loop flexibility.
Biochemistry. 50 (19): 4019-28.
3.Moreira TH, O’Malley S, Levit M, Krebs M, Apland JP, Sweeney R, Smith LA, Adler M (2014)
New Potent Inhibitors of BoNT/A Light Chain Protease Activity. Proceedings of the 51st
Interagency Botulism Research Coordinating Committee Meeting, Philadelphia, PA, 27 Oct
2014.
4.Adler M, Levit M, Sweeny R, Moreira T, Krebs M, Kumaran D, Swaminathan S (2014) A novel
Cyclic Peptide inhibitor of Botulinum Neurotoxin A. Proceedings of the 51st Interagency
Botulism Research CoordinatingCommittee Meeting,Philadelphia,PA, 27 Oct 2014.
5.Barr J, Moura H, Boyer A, Woolfitt A, Kalb S, Pavlopoulos A, McWilliams L, Schmidt J, Martinez
R and Ashley D. (2005) Botulinum Neurotoxin Detection and Differentiation by Mass
Spectrometry. Emerging InfectiousDiseases. Vol 11, No. 10, 1578-1583.
Initially, appropriate substrate-to-LC stoichiometric ratios (S/LC) were
determined and the time at which complete cleavage occurred at those
concentrations. The following S/LC ratios were initially investigated: 100
µM:1 µM, 100 µM:100 nM and 100 µM:10 nM. The 100 µM:1 µM ratio
produced complete cleavage within a reasonable time span (30 min).
Range finding with a single hydroxamate inhibitor, HB 33, was performed
at concentrations ranging from 10 nM-10 µM incubated for 30 minutes;
complete inhibition occurred at 1 µM. Using the same stoichio-metric
ratios, absolute concentrations were reduced to 14 µM substrate and 100
nM LCA. A time course study without inhibitors was performed at these
concentrations with quenching carried out at time points up to 240
minutes. Complete cleavage occurred at 120 min. Five hydroxamate
inhibitors (HB 33, 36, 37, 39, and 40) were tested across a concentration
range of 0.1 nM - 100 µM at 120 minutes. It appeared that with these S/LC
concentration ratios, a 1 µM concentration of each inhibitor induced
complete inhibition (Fig 2). Experiments were repeated with three
quinolines. Though observed, inhibition was much reduced with these
compounds compared to the hydroxamates; complete inhibition was not
observed even at 400 µM (Fig 3).
A SNAP-25 66mer containing the LC cleavage site and a HisTag/linkers
was characterized using nominal and high mass accuracy/resolution full
scan and product ion MSMS analyses (see sequence in Fig 4A with 66mer
sequence highlighted in green). The 66mer was initially extracted using 3
kDa filters to remove buffers and salts. Purified 66mer was characterized
by RP-LC/MSMS, was incubated with LC, and an N-terminal 57mer was
identified and characterized (Fig 4 & 5). It should be noted that only 6nM
LC/A 429 was required to induce ̴50% cleavage of 91 µM 66-mer within 15
min.
A cyclic peptide that has been demonstrated to possess in vitro
potential as an inhibitor and a cyclic peptide analog with the same
sequence minus the C-terminal L residue were also characterized (Fig 1).
The peptides were analyzed in their native cyclic and linear forms
(reduced with TCEP). Nominal mass full scan and MSMS spectra were
acquired confirming peptide sequences and cyclic conformation (Fig 6).
RESULTS
DISCLAIMERS. The views expressed in this poster are those of the author(s) and do not reflect the official policy of the Department of Army,
Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the
United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated
in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89-544), as
amended. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.
US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400
METHODS
Five hydroxamates & three quinolines were evaluated using 17mer
substrate and LCA.
– Reaction conditions included 5 mM TCEP; 50 mM HEPES-NaOH, pH 7.1;
5 mM NaCl; 0.1% Tween-20 & 10 µM ZnCl2.
– Samples were incubated at 37°C until quenched with 60 µL 0.7% TFA.
– A Shimadzu Prominence UPLC/Sciex ESI-QTrap 4000 was used. The
injection volume was 35 µL with reversed phase chromatography (RP)
on a Phenomenex Aeries C18 column (50mm x 2.1mm x 2.6µm) using a
2-40% 0.1% FA/ACN gradient over 4 minutes at 0.6 mL/min.
– Samples were diluted within the dynamic range of the calibration curve
(20-2000 nM).
– Two MRM transitions each were used to monitor substrate & cleavage
products (6-mer and 11-mer).
CPI-1, CPI-3 & the 66mer were analyzed by C18 RP-LC/+ESI-MSMS.
– Commercially available 66mer was extracted using 3kDa filters.
– Native and LCA-digested 66mer were characterized using RP LC/MSMS
on a Shimadzu Prominence UPLC/Sciex ESI-QTrap 4000 and a
nanoLC/Thermo QExactive Plus.
– Native and reduced forms of CPI-1 & CPI-3 were analyzed on a
Shimadzu Prominence UPLC/Sciex ESI-QTrap 4000 .
0
2
4
6
8
10
12
14
0 50 100 150 200 250 300 350 400 450
Quinoline 28 LC/A Inhibition
17-mer=14 µM & LC/A 429=100 nM
0
2
4
6
8
10
12
14
0 50 100 150 200 250 300 350 400 450
Quinoline 32 LC/A Inhibition
17-mer=14 µM & LC/A 429=100 nM
PeptideConcentration(µM)
0
2
4
6
8
10
12
14
0 50 100 150 200 250 300 350 400 450
6-mer
11-mer
17-mer
Inhibitor Concentration (µM)
Quinoline 36 LC/A Inhibition
17-mer=14 µM & LC/A 429=100 nM
CPI-1
C(DAB)-RWTKCL-amide[s-s]
N
H
O
NH
NH2
NH NH2
NH
NH
O
NH
O
OH
N
H
O
NH2
NH
O
O
NH
NH2
O
S
S
NH2
O
N
H
N
H
O
NH
NH2
NH NH2
NH
NH
O
NH
O
OH
N
H
O
NH2
NH
O
O
NH2
S
S
NH2
O
N
H
CPI-3
C(DAB)-RWTKC-amide[s-s]
Figure 1
4
8
12
16
20
HB 36 LC/A Inhibition
17-mer=15 µM & LC/A 429=100 nM
4
8
12
16
PeptideConcentration(µM)
HB 37 LC/A Inhibition
17-mer=15 µM & LC/A 429=100 nM
4
8
12
16
HB 40 LC/A Inhibition
17-mer=15 µM & LC/A 429=100 nM
4
8
12
16
4
8
12
16
0 nM 0.1 nM 1 nM 10 nM 100 nM 1 µM 10 µM 100 µM
Inhibitor Concentration
HB 39 LC/A Inhibition
17-mer=15 µM & LC/A 429=100 nM
HB 33 LC/A Inhibition
17-mer=15 µM & LC/A 429=100 nM
INTRODUCTION Figure 5
CPI-3 Spectrum
Cyclic CPI-3
915.7
893.7447.6
[M+2H]+2 [M+H]+
[M+Na]+
[M+3H]+3
336.1
503.5
[M+2H]+2
[M+H]+
[M+Na]+
Cyclic CPI-1
1028.8
1006.5
[M+H]+
[M+2H]+2
[M+3H]+3
Linear CPI-1
1008.5
505.5
337.1
[M+2H]+2
[M+H]+
447.6
895.4
m/z
Linear CPI-3
Figure 2 Figure 3
66-mer 4000QT
Full Scan Spectrum
GGSHHHHHHGMASSGLRSRARE
NEMDENLEQVSGIIGNLRHMALD
MGNEIDTQNRQIDRIMEKADSN
KTRIDEANQRATKMLGSGSNSGG
SWSHPQFEK
[M+9H]9+
[M+8H]8+
[M+11H]11+
[M+13H]13+
[M+14H]14+
[M+15H]15+
[M+16H]16+
[M+17H]17+
[M+18H]18+
[M+10H]10+
N-terminal
Cleavage Product
4000QT Full Scan
Spectrum
GGSHHHHHHGMASSGLRSR
ARENEMDENLEQVSGIIGNL
RHMALDMGNEIDTQNRQID
RIMEKADSNKTRIDEANQ
[M+8H]8+
[M+9H]9+
[M+7H]7+
[M+10H]10+
[M+11H]11+
[M+12H]12+
[M+13H]13+
Figure 4
66-mer N-terminal
cleavageproduct
Figure 6
 BoNT-A inhibition by five hydroxamate, SMIs previously shown to have BoNT-A inhibition
properties, was characterized by RP-LC/MSMS. The data directly confirmed data previously
collected using the FRET assay. The five hydroxamates exhibited similar high potencies
under experimental conditions.
 BoNT-A inhibition by three quinolines, a second class of SMIs was briefly investigated. All
three quinoline SMIs exhibited much reduced inhibition potencies as compared with the
hydroxamate SMIs. Using 100 nM LCA and 15 µM 17-mer as substrate indicated that
hydroxamate SMIs are at least 400 times more efficacious than quinolone SMIs (complete
inhibitionobservedat 1 µM and >400µM, respectively).
 SNAP25141-206, a SNAP25 66mer containing the LCA cleavage sequence and reported to have
a much lower Km (similar to intact SNAP25) for LCA than the 17mer, was characterized by
LC/+ESI-MSMS. In addition, the N-terminal LCA cleavage product, a 57mer, was also
identified and characterized by LC/MSMS. A quantitative MRM method is currently being
optimized and validated for use in SMI kinetics studies. Since much less LCA will be
required to perform experiments, and since LCA is the cost limiting reagent, potential SMIs
can be evaluatedin a much more cost effective way.
 A cyclic peptide previously reported to possess BoNT-A inhibitory properties, as well as a
second analog missing the C-terminal lysine residue were characterized by LC/+ESI-MSMS.
Reduction of both native peptides with TSEP induced a +2 Da shift for the (M+H)+ of both
proteoforms indicative of peptide bond reduction. A quantitative LC/MSMS method for
these peptides will be validated, and LCA inhibition by these peptides will be investigated
using both 17mer and 66mer methods in the future.
 LCA will be immobilized on a LC column as an affinity probe in an integrated, on-column
digest/LC/MSMSmethod.
REFERENCESREFERENCES
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