Newly Designed Quinolinol Inhibitors Mitigate the Effects of Botulinum Neurotoxin A in Enzymatic, Cell-Based, and ex Vivo Assays

Article abstract of DOI:10.1021/acs.jmedchem.6b01393

Botulinum neurotoxin A (BoNT/A) is one of the most deadly toxins and is the etiological agent of the potentially fatal condition, botulism. Herein, we investigated 8-hydroxyquinoline (quinolin-8-ol) as a potential inhibitor scaffold for preventing the deadly neurochemical effects of the toxin. Quinolinols are known chelators that can disrupt the BoNT/A metalloprotease zinc-containing active site, thus impeding its proteolysis of the endogenous protein substrate, synaptosomal-associated protein 25 (SNAP-25). By use of this information, the structure-activity relationship (SAR) of the quinolinol-5-sulfonamide scaffold was explored through preparation of a crude sulfonamide library and evaluation of the library in a BoNT/A LC enzymatic assay. Potency optimization of the sulfonamide hit compounds was undertaken as informed by docking studies, granting a lead compound with a submicromolar K_i. These quinolinol analogues demonstrated inhibitory activity in a cell-based model for SNAP-25 cleavage and an ex vivo assay for BoNT/A-mediated muscle paralysis.

Full text of DOI:10.1021/acs.jmedchem.6b01393

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Article
Newly Designed Quinolinol Inhibitors Mitigate the Effects of
Botulinum Neurotoxin A in Enzymatic, Cell-Based and Ex Vivo Assays
Paul T. Bremer, Michael Adler, Cecilia H. Phung, Ajay K. Singh, and Kim D Janda
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01393 • Publication Date (Web): 14 Dec 2016
Downloaded from http://pubs.acs.org on December 14, 2016
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Newly Designed Quinolinol Inhibitors Mitigate the Effects of Botulinum Neurotoxin A in Enzymatic,
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Cell-Based and Ex Vivo Assays
a
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Paul T. Bremer, Michael Adler, Cecilia H. Phung, Ajay K. Singh and Kim D. Janda *
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Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of
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Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA
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2037.
b
Neurobehavioral Toxicology Branch, Analytical Toxicology Division, U.S. Army Medical Research Institute
of Chemical Defense, 2900 Ricketts Point Road, APG, MD 21010ꢀ5400.
Abstract
Botulinum neurotoxin A (BoNT/A) is one of the most deadly toxins, and is the etiological agent of the
potentially fatal condition, botulism. Herein, we investigated 8ꢀhydroxyquinoline (quinolinꢀ8ꢀol) as a potential
inhibitor scaffold for preventing the deadly neurochemical effects of the toxin. Quinolinols are known chelators
that can disrupt the BoNT/A metalloprotease zincꢀcontaining active site, thus impeding its proteolysis of the
endogenous protein substrate, synaptosomalꢀassociated protein 25 (SNAPꢀ25). Using this information, the
structureꢀactivity relationship (SAR) of the quinolinolꢀ5ꢀsulfonamide scaffold was explored through preparation
of a crude sulfonamide library, and evaluating the library in a BoNT/A LC enzymatic assay. Potency
optimization of the sulfonamide hit compounds was undertaken as informed by docking studies, granting a lead
compound with a submicromolar K . These quinolinol analogues demonstrated inhibitory activity in a cellꢀbased
i
model for SNAPꢀ25 cleavage and an ex vivo assay for BoNT/Aꢀmediated muscle paralysis.
Introduction
Clostridium botulinum is a Gramꢀpositive, anaerobic bacterium that naturally biosynthesizes one of the most
1
toxic known substances to mammals, botulinum neurotoxin (BoNT). Although <1 µg of aeresolized toxin is
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lethal to an adult human, BoNT also possesses a variety of important medical uses: of the seven different
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7ꢀ
toxin serotypes (AꢀG), serotypes A and B are routinely used to treat facial wrinkles, migraines and dystonias.
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Unintentional human exposure to BoNT most commonly takes the form of foodborne botulism in adults. The
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majority of botulism cases in the U.S. were caused by BoNT/A, which is also the serotype that induces the
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most severe and persistent symptoms. Indeed, in April of 2015 over 25 people in Ohio were stricken with
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foodborne BoNT/A, resulting in one patient’s death and eleven patients requiring mechanical ventilation. The
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botulism outbreak was the largest in 40 years in the United States, and was caused by improper home canning
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techniques that failed to kill toxinꢀproducing C. botulinum spores.
Furthermore, BoNT is considered a bioterrorism threat due to its extreme potency; dissemination of BoNT into
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the air or into a food supply could be devastating to a human population.
The currently approved
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treatment for botulism is a heptavalent equinine antitoxin that is only effective within a limited timeframe.
Thus, immunotherapeutic approaches can neutralize the BoNT holotoxin only until the neurotoxin’s heavy
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chain initiates cellular endocytosis. However, following toxin entry into cells, antibodies can no longer
prevent the zincꢀmetalloprotease light chain (LC) of BoNT/A from cleaving its 25 kDa substrate, synaptosomalꢀ
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associated protein 25 (SNAPꢀ25). Proteolysis of the SNAPꢀ25 protein disrupts fusion of acetylcholineꢀ
containing synaptic vesicles with the axon terminal membrane. As a result, muscle fibers no longer receive
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signals from motor neurons, thus causing muscle paralysis and autonomic dysfunction.
Over the past 20 years, a variety of BoNT/A inhibitors have been developed, which target LC endosomal
18ꢀ22
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translocation
or the LC protease directly.
Zincꢀchelating molecules have been shown to be privileged
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inhibitor scaffolds against zinc metalloproteases e.g. matrix metalloproteinases (MMPs);
for this reason,
23ꢀ25
hydroxamates have been intensely studied as BoNT/A inhibitors.
Despite the numerous disclosures of active
site BoNT/A LC inhibitors, rational design of subꢀmicromolar inhibitors remains challenging. This is due in
part to the highly flexible nature of the enzyme, contributing to an inducedꢀfit mechanism that obfuscates
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prediction of inhibitor binding poses.
An alternative to the hydroxamate, 8ꢀhydroxyquinoline (quinolinꢀ8ꢀ
2
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ol), has also been found to be a promising BoNT/A LC inhibitor scaffold e.g. compound 15 (NSC84094).
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The quinolinol core structure is present in the FDAꢀapproved antibiotic chloroxine as well as in the
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experimental drug PBT2, which has been tested in clinical trials as a therapy for Alzheimer’s disease.
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distinct advantage that quinolinols possess over more traditional zincꢀchelating hydroxamates is increased
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lipophilicity that enhances their ability to penetrate cell membranes, which in effect would increase a
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molecule’s ability to inhibit the intracellular BoNT/A LC.
Previously, the structureꢀactivity relationship
(SAR) of the 7ꢀposition on the quinolinol ring was explored extensively via screening of a Betti reaction library
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(
Figure 1). We hypothesized that functionalization at alternative positions on the quinolinol ring could also be
fruitful for the discovery of new and possibly more potent BoNT/A inhibitors.
In our current endeavor, we exploited synthetically facile methods for installing 5ꢀposition sulfonamides as well
as 2ꢀposition amino groups to rapidly identify BoNT/A inhibitor quinolinols (Figure 1). A FRETꢀbased assay
for BoNT/A LC substrate cleavage was used for initial inhibitor assessment of our molecules. The most potent
leads were tested in a cell assay and ex vivo mouse hemidiaphragm assay to determine the therapeutic potential
for these molecules to ameliorate the effects of BoNT/A.
Figure 1. Synthetic methods for generating quinolinol libraries
Results and Discussion
Crude Library Screening
Our primary objective for inhibitor discovery was to identify compounds as efficiently as possible; therefore we
sought a simple, yet, highꢀyielding synthetic methodology to generate molecules without the requirement of
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purification. Given this criterion, we opted to create a sulfonamide library via the reaction of quinolinolꢀ5ꢀ
sulfonyl chloride with a variety of amine fragments (Figure 2A). A total of 63 crude reaction mixtures (Table
S1) at a concentration of ~30 µM were screened directly against BoNT/A LC in a FRETꢀbased assay
(SNAPtide assay). The majority of the mixtures showed mild inhibition of the LC, and excitingly, 1 in 8 of
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these crude mixtures reduced enzymatic activity to less than 15% (Figure 2B). The eight initial hits were
rescreened at a threeꢀfold lower concentration to identify the two most potent compounds (sulfonamides 1 and
3), which were derived from piperidine and 3ꢀchloroaniline, respectively. It should be noted that the presence of
the quinolinꢀ8ꢀol moiety did not guarantee BoNT/A LC inhibition as some of the compounds showed little to no
activity; and furthermore, quinolinꢀ8ꢀol alone does not inhibit the LC. Thus, design and selection of quinolinol
aryl ring substituents was essential for achieving potent quinolinolꢀbased inhibitors.
Figure 2. Screening of crude quinolinol-5-sulfonamide library against BoNT/A LC reveals potent
inhibitors
A
OH
OH
OH
OH
BoNT/A LC
Screening
N
N
Amine
Library
N
N
O S O
NH
O
S O
N
O
R
S O
O S O
Cl
Cl
Most
Potent
Hits
N
R
Crude Sulfonamide
Library
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(A) Scheme showing generation of sulfonamide library and structures of hit compounds. (B) Histogram
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showing distribution of sulfonamide inhibitory activity against BoNT/A LC (relative to DMSO as a control) in
the SNAPtide assay. Bin width = ±10% enzyme activity. Compounds were tested at ~30 µM and the
compounds identified in the red colored bin were rescreened at ~10 µM to verify 1 and 3 as hits.
Inhibitor Validation and Preliminary SAR
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Through the discovery of 1 and 3 as hit compounds, we were able to demonstrate that the sulfonamide 5ꢀ
position contributed to BoNT/A LC inhibition. In order to refine inhibitor potency, a series of quinolinol
sulfonamides grounded upon 1 and 3 were designed, synthesized and tested (Figure 3). When examined in pure
form these compounds indeed possessed low µM potency. Analogues of 3 were pursued over 1 because of
greater inhibitor potency and availability of a rich source of starting material anilines. Unsubstituted Nꢀphenyl
sulfonamide 2 showed reduced inhibition, demonstrating the necessity of the 3ꢀchloro substituent. Other
functional groups and positions on the phenyl ring were explored in the crude sulfonamide library, but the 3ꢀ
chloro showed superior inhibitory activity (Table S1). Addition of an Nꢀmethyl substituent increased BoNT/A
inhibition as seen with 4 and 5. Lastly, conformational restriction of the 3ꢀchlorophenyl substituent was
explored by introducing 4ꢀ and 6ꢀindoline for mimicry of two possible locked rotational conformers; 4ꢀ
chloroindoline (7) displayed markedly better BoNT/A LC inhibition over 6ꢀchloroindoline (6) (Figure 3).
Figure 3. Synthesis and activity of quinolin-8-ol-5-sulfonamides
OH
OH
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. HSO Cl
N
3
N
2. Amine,
THF,
Pyridine
1
-7
O S O
N
13-69%
R
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Amine Used to Prepare
Corresponding Sulfonamide
SNAPtide
IC (µM)
Compound
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Piperidine
Aniline
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4.28±0.26
3ꢀChloroaniline
NꢀMethylaniline
NꢀMethylꢀ3ꢀChloroaniline
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SAR Optimization Through Docking Studies
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In performing SAR optimization on 3, we arrived at 7 with improved BoNT/A LC inhibition. To achieve further
inhibition of the protease, we recruited the insight of computational docking to elucidate positions and
functional groups on the quinolinol ring that could garner increased potency. Briefly, lowest energy conformers
39ꢀ41
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of 5 were generated using OMEGA
and were docked with FRED
into the active site which has
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previously been cocrystalized with hydroxamate inhibitors. Because of its zincꢀchelating properties,
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quinolinols, similar to hydroxamates, presumably bind to the BoNT/LC active site zinc.
Docking of 5 into
the active site revealed a possible hydrogen bond between the sulfonamide oxygen and a backbone NH of
Phe163 in addition to the expected bidentate quinolinol chelation of the zinc. Hydrophobic interactions and/or
piꢀpi stacking were noted between the chlorophenyl group and Phe369/194. The orientation of compound 5
within the active site suggested that additional interactions with BoNT/A LC could be achieved by
functionalizing position 2 or 3 (Figure 4). Upon further docking studies, the 2ꢀhydrazino group (12) was found
to produce favorable fitness scores and appeared to form a hydrogen bond with Glu262. Possible hydrogen
bonds were also noted with Glu164 and Gln162, although hydrogen bond lengths were greater than normal.
Figure 1. Docking of compounds 5 and 12 into the BoNT/A LC active site
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(
A) Docking pose of compound 5 shown in tan colored carbons. (B) Docking pose of compound 5 with a 2ꢀ
hydrazino group added (analogous to compound 12) shown in light blue colored carbons. Protein receptor
carbons are shown in grey with a transparent surface. Relevant residues are labelled and distances between
compoundꢀprotein metal and possible hydrogen bond interactions are drawn with yellow lines. Receptor crystal
structure PDB ID: 4HEV.
In light of this promising docking result, we synthesized a series of 2ꢀaminoquinolinols via an S Ar reaction
N
with the precursor 2ꢀchloro compound 8 (Figure 5). Poor inhibitory activity of compound 8 was noted with an
IC >30 µM, revealing stringency for the 2ꢀposition chemotype. Moreover, quinolinols with 2ꢀcarboxy or 2ꢀ
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methylamino substituents were inactive, even as the corresponding amides in both cases. In contrast, addition of
the 2ꢀamino group in compound 9 presented a similar IC to the unsubstituted compound 7, although 2ꢀalkyl
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amines 10 and 11 showed drastically reduced inhibition, possibly due to repulsion between the lipophilic alkyl
groups and polar enzyme amino acid side chains. As we had hoped, a 2.4ꢀfold increase in potency was observed
when adding the hydrazino group (12) versus the unsubstituted compound 7 (Figure 5). Further modification of
the hydrazine with a methyl (13) or acetyl (14) group slightly decreased inhibitory activity, possibly by
interfering with the hydrogen bonding observed in the docking pose (Figure 4). Docking scores showed some
correlation with IC values, and three of the most potent compounds (5, 9 and 13) gave the highest scores
5
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(Table S9). Since the quinolinol scaffold is known to inhibit MMPs, our most potent compounds were tested
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against MMPꢀ1 and ꢀ2, revealing inhibitory activity; however, significant selectivity for BoNT/A LC was
achieved through addition of 2ꢀhydrazino groups (Table S10).
Figure 2. Synthesis and activity of quinolinol sulfonamides containing a 2-amino substituent
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NH
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NHAc 5.39±0.15
Quinolinol Sulfonamide Inhibition Kinetics
In considering 12 to be our optimized lead compound, we investigated inhibition kinetics of 12 in the SNAPtide
assay along with 7. Surprisingly, a LineweaverꢀBurke plot of the kinetic data suggested an uncompetitive
inhibition mechanism for both compounds; with increasing concentrations of inhibitor, both K and V
m
max
decreased (Figure 6A, 6C). This implies that 7 and 12 bind to the enzymeꢀsubstrate (ES) complex, which
increases BoNT/A LC affinity for the substrate while inhibiting protease activity. A global fit of this data with
an uncompetitive inhibition model revealed a fairly potent, submicromolar K of 895 nM for 12 (Figure 6D)
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while 7 was less potent as expected (Figure 6B). Active site inhibitors of BoNT/A LC such as hydroxamates
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typically demonstrate competitive inhibition, thus the observation of uncompetitive inhibition is unexpected.
A notable example of an uncompetitive inhibitor is the natural product brefeldin A; the nature of its inhibition
45
mechanism is attributed to its potent effects on disrupting protein transport from the endoplasmic reticulum. It
is difficult to interpret the biological implications of the uncompetitive mechanism of 7 and 12, but it could play
a role in its activity against BoNT/A (Figure 8). Because the compounds presumably bind only to the ES
complex, it may exclusively target an active enzyme conformation, possibly lending to their efficacy in
advanced BoNT/A models (Figure 7, 8). On the other hand, quinolinol uncompetitive inhibition may not occur
within BoNT/Aꢀintoxicated cells in the presence of the natural substrate SNAPꢀ25 and could be a kinetic
anomaly from the synthetic FRET substrate used in the in vitro assay. Overall, the kinetic results neither support
nor refute the predicted binding mode in Figure 4 because uncompetitive inhibitors can bind to either active or
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Figure 3. Inhibition kinetics of compounds 7 and 12 against BoNT/A LC
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A) LineweaverꢀBurke plot of compound 7 kinetic data. Paralell lines suggest an uncompetitive inhibition
mechanism as both K and Vmax are decreasing with increasing [I]. (B) Global fit of compound 7 kinetic data
using an uncompetitive inhibition model for K determination. (C) LineweaverꢀBurke plot of compound 12
kinetic data. (D) Global fit of compound 12 kinetic data (uncompetitive model). All points represent means ±
SEMs. Data were obtained in duplicate in the SNAPtide FRET assay using the flP6 substrate.
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Evaluation of Quinolinol-5-Sulfonamides in Advanced BoNT/A Assays
Given the promising inhibitory activities of our quinolinols against BoNT/A LC in the enzymatic assay, we
investigated these compounds in more advanced biological models for BoNT/A intoxication, which included a
cell assay and an ex vivo mouse muscle assay. For the cell assay, we employed a specific neuronꢀlike cell line
(BoCell) that expresses a recombinant fullꢀlength SNAPꢀ25 containing cyan fluorescent protein (CFP) and
yellow fluorescent protein (YFP) at the Nꢀ and Cꢀtermini, respectively. The fusion protein acts as a BoNT/A
biosensor for realꢀtime monitoring of SNAPꢀ25 cleavage by the LC protease within live cells resulting in a
47
decreased YFP/CFP ratio. Treatment of these cells with 7, 9 and 13 caused a reduction in BoNT/Aꢀmediated
proteolysis of the SNAPꢀ25 reporter, as indicated by statistically significant YFP/CFP ratio increases in the
presence of the compounds (Figure 7). Compound 13 was the most potent and even showed efficacy at a twoꢀ
fold lower concentration (Table S11). As a cautionary note, 7 and 13 decreased the YFP/CFP ratio without
toxin (Table S11), which could be explained by modest cytotoxicity in XTT and MTT assays (Figure S3, S4);
however, this did not obscure their observed efficacy, since +toxin values are normalized to their respective –
2
9
toxin controls (Figure 7). Compounds 12, 14 and previously reported quinolinol 15 were not active. Although
not particularly surprising for 14, since it showed less inhibition in the SNAPtide assay, lack of cellꢀbased
activity for 12 was unexpected. We cannot fully account for the inactivityo of 12, but the presence of an
unmodified hydrazino group may result in a reduced cellꢀmembrane permeability or intracellular modification
compared to the active, methylated hydrazide 13. Fortunately, three of our compounds showed noticeably better
28, 29
potency versus a benchmark BoNT/A LC inhibitor 15,
until now (Figure 7).
which had never been examined in the BoCell assay,
Figure 4. Quinolinols inhibit BoNT/A LC-mediated cleavage of SNAP-25 in live cells
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BoCells expressing a recombinant SNAPꢀ25 linked to CFP and YFP at the Nꢀand Cꢀtermini, respectively, were
exposed to 50 pM BoNT/A (Tx) for 1 h followed by the listed compounds at 25 µM or DMSO vehicle for 20 h.
YFP and CFP fluorescence was then measured and expressed as the YFP/CFP ratio. A decrease in this ratio is
indicative of an increase in SNAPꢀ25 cleavage. –Tx controls were included for each compound and +Tx
fluorescence ratios were normalized to these controls to produce mean percentage values and SDs over twelve
replicates as represented in the figure. *p<0.001 by oneꢀway ANOVA with Dunnett’s postꢀhoc test vs. +Tx
DMSO control (left bar).
Lead structures 7, 9 and 12-14 were further evaluated in a goldꢀstandard ex vivo assay for BoNT/A intoxication
that consists of electrophysiological monitoring of mouse hemidiaphragm muscles as they succumb to the
paralytic effects of the toxin. The assay has been used previously to evaluate a variety of inhibitors including
2
9, 48
1
5.
Results from our experiments demonstrate that all quinolinols tested, to some degree, extended halfꢀ
paralysis time (HPT) of BoNT/Aꢀintoxicated mouse hemidiaphragms (Figure 8, Table S12) with 9 and 12
being the most potent (see Figure S1 for representative time course of muscle twitch tension). Statistically
significant increases in HPT for 9 and 12 were observed compared to 15, which was the weakest at ameliorating
BoNT/A paralysis (Figure 8). Although 12 was inactive in the cell assay, its marked activity in the ex vivo
assay could be attributed to a beneficial, offꢀtarget effect in imparing BoNT/A LC endosomal translocation
19, 20
because previous studies have demonstrated that quinolines possess this mechanism of action.
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Demonstration of efficacy in the hemidiaphragm assay is crucial for establishing therapeutic relevance of
quinolinols for mitigating BoNT/A. Although the cellꢀbased assay is important for assessing inhibition of
BoNT/A LC proteolysis of SNAPꢀ25, the ex vivo assay gives a more complete picture of BoNT/A intoxication
through measuring diaphragm muscle contraction signaled by the phrenic nerve. The fact that our quinolinols
show activity in both assays indicates their potential use for the symptomatic relief of botulism.
Figure 8. Quinolinols mitigate BoNT/A paralysis of mouse hemidiaphragms
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Pairs of mouse hemidiaphragms (n = 4ꢀ7) were treated with the indicated compounds at 20 µM in DMSO or
DMSO vehicle for 1 h and then exposed to 5 pM BoNT/A. The time required to achieve 50% twitch tension
was noted (halfꢀparalysis time, HPT), and the % increases in HPT for inhibitorꢀtreated hemidiaphragms versus
vehicleꢀtreated hemidiaphragms are reported as means and SEMs. The difference between vehicle and
compound treated groups was statistically significant (*p<0.05, **p<0.01, ***p<0.001) by a paired tꢀtest.
Significant differences between compounds were noted by a oneꢀway ANOVA, F5,26=5.237, p=0.0019; ^p<0.01
versus 15 (left bar) by Dunnett’s postꢀhoc test.
Conclusion
The inhibitory activity of a series of quinolinol derivatives against BoNT/A LC was explored by screening a
crude sulfonamide library, utilizing an LC enzymatic assay for readout. Screening the library revealed a clear
SAR centered upon the quinolinol 5ꢀposition in which Nꢀaryl sulfonamides were the most inhibitory (Figure 2).
Investigation of these initial hits allowed for potency optimization, which gravitated to a conformationallyꢀ
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constrained molecule 7 (Figure 3). An in silico docking survey of potential poses of 7 inside the BoNT/A LC
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active site revealed that 2ꢀposition substituents could provide additional binding interaction with the enzyme
(
Figure 4). Indeed, functionalization of the ring with a 2ꢀhydazino group (compound 12) increased potency
Figure 5). A kinetic evaluation of 12 revealed a submicromolar K with an interesting uncompetitive inhibition
(
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mechanism (Figure 6). The most active quinolinols caused BoNT/A LC inhibition in both a cellꢀbased assay
Figure 7), and an ex vivo mouse hemidiaphragm assay (Figure 8). Overall, compounds 7, 9 and 12ꢀ14 showed
(
greater efficacy in these assays than a previously reported reference compound 15, demonstrating an improved
inhibitor design of the new quinolinols. The physiochemical properties of these quinolinols may require further
optimization for in vivo studies, but our current results provide an excellent starting point for development of a
potential smallꢀmolecule BoNT/A therapeutic.
Experimental
Crude Sulfonamide Library Screening. In each well of a polypropylene 96ꢀwell plate, 75 µmol (3 eq) of each
amine was added followed by 100 µL of 1:1 DMSO/1,4ꢀdioxane. Quinolinꢀ8ꢀolꢀ5ꢀsulfonyl chloride (6 mg, 25
µmol) in 100 µL 1,4ꢀdioxane was then added to each well. The 63 reaction mixtures in the plate were shaken
for 18 h. Each crude product mixture was diluted 100ꢀfold into DMSO to make an estimated 2.5 mM stock
solution, which was tested against the BoNT/A LC in the SNAPtide assay at ~30 µM for all compounds and
~10 µM for the hits. For each row on the plate, a DMSO only control well was included as a 100% enzyme
activity reference, and inhibitor treated wells were normalized to the control. See Table S1 for identity of
amines used for library preparation.
SNAPtide Assay. In a 96ꢀwell, halfꢀarea opaque plate, 50X solution of compound in DMSO (1 µL) was
preincubated for 20 min with 24.5 µL of 56 nM recombinant 425aa BoNT/A LC in 40 mM HEPES + 0.1%
Triton Xꢀ100, pH 7.4 buffer. In the same buffer, 24.5 µL of 6 µM SNAPtide substrate (#521 from List Labs)
was added to the enzyme+compound solution and the plate was read for 25 min at 490 nm (ex)/523 nm
(
em)/495 nm (cutoff). The linear slopes of the assay readout in RFU/s were used to determine V . Resulting V
0 0
values, obtained in duplicate at at least four different inhibitor dilutions, were normalized to the control V0
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ꢂꢁꢁ
(DMSOꢀtreated well) and curves were fit in GraphPad Prism with the equation ꢀ =
ꢁ
ꢄꢅꢆꢇꢄꢈꢉ ꢌꢍꢅꢆꢇꢄꢎꢈꢏꢌꢌ∗ꢐꢑꢒꢒꢓꢒꢔꢕꢖꢌ
ꢂꢃꢂꢁ
ꢊꢋ
to calculate each IC . For kinetic experiments, a different SNAPtide substrate with a reduced K was used
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m
(flP6, #523 from List Labs). Kinetic data were fit in GraphPad Prism with the uncompetitive inhibition model
with a hillslope of 5 to determine Ki.
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Docking. The Openeye software suite was used for docking under the use of an academic license. The BoNT/A
LC receptor files were generated from the active sites of PDB structures 3QIY, 4HEV, 3QIZ, 3QJ0, 2IMB and
49
2
IMA using the Make Receptor program. The lowest energy conformers of a set of known quinolinols were
3
9ꢀ41
generated with OMEGA
and were docked into the receptors using FRED with the Chemgauss3 scoring
4
2ꢀ44
25
function.
Docking into the 4HEV receptor gave the most reasonable compound poses with the most metal
interaction; therefore 4HEV was selected as the receptor for docking experiments (see Tables S2-9 for docking
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scores). Figures were generated using the VIDA GUI.
Materials for Cell Culture. BoCell assay mediumꢀ2 (BAMꢀ2) and BAMꢀ2 supplement were purchased from
the BioSentinel Inc., Madison, WI. Glutamax (100X), Bꢀ27 (50X), 1X phosphate buffer saline (PBS) and fetal
bovine serum (FBS) were purchased from Life Technologies Corp., Carlsbad, CA. Eagle’s Minimum Essential
Medium (EMEM) with glutamine and penicillinꢀstreptomycin mixture (100X) were purchased from the
American Type Culture Collection (ATCC), Manassas, VA. BoNT/A was purchased from Metabiologics Inc.,
Madison, WI. PolyꢀDꢀlysine coated 96ꢀwell (black/clear) plates were purchased from the Corning Inc., Corning,
NY.
BoCell Culture. BoCells are a division arrested genetically modified neuronꢀlike cellꢀline that expresses fullꢀ
length SNAPꢀ25 with CFP and YFP at the Nꢀ and Cꢀterminal residues of SNAPꢀ25, respectively. A frozen vial
6
of BoCells (>2.5 x 10 cells per vial) was thawed at 37 ºC for ~2 min with occasional shaking. Cells were
transferred from the cryogenic vial to a 15 mL conical tube containing 9 mL of preꢀwarmed EMEM, mixed
gently with a 10 mL pipette and centrifuged at 1000 rpm for 6 min. The supernatant was removed and cells
were reꢀsuspended in EMEM (10.5 mL) with 10% FBS, and 1X penicillinꢀstreptomycin. A 100 µL suspension
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of cells was dispensed into each well of a 96ꢀwell (black–clear) polyꢀDꢀlysine coated plate. Cells were grown in
a 37 ºC humidified cell culture incubator in an atmosphere of 95% air/5% CO2.
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BoCell Assay Method. After 24 h, EMEM was removed and replaced by complete BoCell assay mediumꢀ2
(CBAMꢀ2), a proprietary formulation that increases sensitivity of cells to BoNT. CBAMꢀ2 was prepared by
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adding glutamax, Bꢀ27 and BAMꢀ2 supplement to basic BAMꢀ2 media according to the manufacturer’s
instructions. For evaluating inhibitors, cells were exposed to BoNT/A (50 pM) for 1 h. BoNT/Aꢀcontaining
media was then aspirated and cells were washed once with PBS. The test compounds were dissolved in CBAMꢀ
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at 25 µM and added to cells (quadruplicate wells performed three times) for 20 h. At the end of this
incubation period, CBAMꢀ2 media containing test compound was removed and replaced with PBS after an
intermediate PBS wash. Fluorescence was measured using a plate reader (Bioteck Synergy H4), and cleavage
was quantified by the YFP/CFP emission ratio at 526/470 nm in response to excitation at 500 and 434 nm,
respectively. Emissions were recorded sequentially for each fluorescent protein. The principle of the assay is
that YFP (and its associated 9ꢀmer SNAPꢀ25 fragment) dissociates following cleavage by BoNT/A LC and
subsequently undergoes rapid degradation by the cell. In contrast, CFP (and associated 187ꢀmer truncated
SNAPꢀ25) remains membrane bound and is retained by the cell. Thus reduction of the YFP emission signals
cleavage of SNAPꢀ25, while CFP emission allows data to be normalized for variations cell density. Statstical
analysis was performed in GraphPad Prism version 6.
Hemidiaphragm Assay. Experiments were performed in vitro on hemidiaphragm muscles dissected from male
CDꢀ1 mice (20ꢀ25 g). Mice were housed in AAALAC International accredited facilities with food and water
available ad libitum. To obtain muscle preparations, the animals were euthanized by an overdose of isoflurane
and decapitated. Hemidiaphragms with attached phrenic nerves were dissected, mounted in temperatureꢀ
o
controlled tissue baths and immersed in KrebsꢀRinger bicarbonate solution at 37 C (SigmaꢀAldrich, St. Louis,
MO). The solution was bubbled with a gas mixture of 95% O /5% CO and had a pH of 7.3. The phrenic nerve
2
2
was stimulated with 0.2ꢀmsec supramaximal (6 V) pulses at 0.033 Hz using a Grass S88 stimulator (AstroꢀMed,
Inc., West Warwick, RI). Muscle twitches were measured with Grass FT03 isometric force displacement
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transducers (AstroꢀMed), digitized and analyzed offꢀline with pClamp software (Molecular Devices, Sunnyvale,
CA, USA). Resting tensions were maintained at 1.0 g to obtain optimal nerveꢀevoked tensions. Stock solutions
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of test compounds were dissolved in DMSO at 10 mM and stored at ꢀ20 C until use. The stock solution was
added slowly (30 sec) to the bath to prevent precipitation. 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 and the Animal Welfare Act of 1966 (P.L. 89ꢀ544), as amended.
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Compounds were studied at a final inhibitor concentration of 20 µM. Hemidiaphragm preparations were
generally pretreated with test compounds for ≥ 1h prior to addition of 5 pM BoNT/A (Metabiologics, Madison,
WI). Control muscles were pretreated for 1 h with 0.2% DMSO prior to challenge with 5 pM BoNT/A. At the
concentration tested, some compounds produced a partial inhibition of twitch tension (9, 12, 13), which was of
similar magnitude and time course for the different inhibitors. Inhibition had an onset time of 3ꢀ5 min, and
resulted in a reduction in tension of 40.4 ± 1.5% before reaching steadyꢀstate at 45.9 ± 5.1 min. To minimize the
effect of muscle to muscle variation, one hemidiaphragm was pretreated with test compound and the
contralateral diaphragm muscle from the same mouse received an equivalent volume of DMSO. Halfꢀparalysis
time (HPT) was determined as the time required for the twitch tension to fall to 50% of its value just prior to
BoNT/A addition. Toxin was added from a 100 nM stock solution which was first diluted to 5 nM (1:20 µL in
KrebsꢀRinger bicarbonate solution) just prior to adding to a 20ꢀml muscle bath. Data were expressed as the
percent increase in HPT in the test muscle relative to the HPT in the DMSOꢀtreated muscle. For each
compound, four to seven replicates were run, and statstical analysis as described in each figure caption was
performed in GraphPad Prism version 6.
General Chemistry. All starting materials and reagents were purchased from CombiꢀBlocks or SigmaꢀAldrich.
Organic solvents were anhydrous or distilled prior to use. NMR spectra were recorded on a Bruker 600 MHz
instrument and peaks are reported in ppm, using the solvent peak as a chemical shift reference. High resolution
mass analysis was performed on an Agilent ESIꢀTOFꢀMS system. Analysis of purity was performed by
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integrating compound peaks on a 254 nm UV trace recorded on a Agilent LCMS with an Agilent Zorbax
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300SBꢀC8 4.6 X 50 mm 5 µm column, flow rate 0.5 mL/min, solvent A: H O+0.1% COOH, solvent B:
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MeCN+0.1% COOH, gradient 0ꢀ4 min: 5ꢀ95% B, 4ꢀ7 min: 95% B. All compounds with determined IC50
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values (1ꢀ15) were ≥95% pure. Compound 15 was synthesized as previously described.
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General Procedure for Sulfonamide Synthesis. (Adapted from previously reported synthetic methods)
To
a cooled flask (0 °C) containing 8ꢀhydroxyquinoline (850 mg, 5.9 mmol) was added 2.5 mL chlorosulfonic
acid. The mixture was stirred for 2 h, allowing to warm to rt and poured into 200 mL iced brine. An extraction
was performed with 100 mL 1:1 THF/EtOAc, which was dried over MgSO and evaporated to yield the crude
4
8
ꢀhydroxyquinolineꢀ5ꢀsulfonyl chloride (278 mg, 19%). The sulfonyl chloride (22 mg, 0.09 mmol) was
dissolved in 2 mL THF and 2 eq pyridine (0.18 mmol) and 1 eq aniline or amine (0.09 mmol) was added. The
solution was stirred for 18 h, diluted with 10 mL EtOAc and washed once with 1 N HCl followed by saturated
sodium bicarbonate solution. The organic layer was collected, dried and evaporated to yield the pure
sulfonamide.
General Procedure for S Ar Reaction to 2-Amino-8-hydroxyquinoline-5-sulfonamides. In a sealed vial, 2ꢀ
N
chloroꢀquinolinol compound 8 (10 mg, 26 µmol) was dissolved in 0.5 mL THF and 50 eq of the desired amine
or hydrazide. The mixture was stirred at 85 °C for 22 h. Alternatively, the same reaction could be performed
under 150 W microwave radiation at 150 °C for 22 min in 0.25 mL dioxane. The volatiles were removed under
high vacuum to afford the crude product which was dissolved in 1 mL 1:1 EtOAc/THF and washed with water
and saturated sodium bicarbonate solution. The organic layer was dried and evaporated to afford the pure 2ꢀ
amino or hydrazino product as a solid.
5-(piperidin-1-ylsulfonyl)quinolin-8-ol (1). The title compound was prepared from piperidine using the general
1
sulfonamide synthesis procedure as a yellow solid (17.9 mg, 69% yield). H NMR (600 MHz, Acetoneꢀd ) δ
6
9
.15 (d, J = 8.7 Hz, 1H), 8.95 (d, J = 2.7 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.77 (dd, J = 8.8, 4.0 Hz, 1H), 7.26
1
3
(
d, J = 8.2 Hz, 1H), 3.10 – 3.05 (m, 4H), 1.58 – 1.51 (m, 4H), 1.44 – 1.37 (m, 2H). C NMR (151 MHz,
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Acetoneꢀd ) δ 158.31, 149.71, 139.04, 135.20, 133.92, 126.46, 124.40, 123.68, 109.37, 47.09, 26.07, 24.13.
6
+
HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H N O S 293.0954; Found 293.0956.
1
4
17
2
3
8
-hydroxy-N-phenylquinoline-5-sulfonamide (2). The title compound was prepared from aniline using the
1
general sulfonamide synthesis procedure as a yellowꢀgreen solid (3.6 mg, 13% yield). H NMR (600 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Acetoneꢀd ) δ 9.25 (brs, 1H), 9.10 (dd, J = 8.7, 1.5 Hz, 1H), 8.92 (dd, J = 4.1, 1.5 Hz, 1H), 8.23 (d, J = 8.3 Hz,
6
13
1
H), 7.72 (dd, J = 8.7, 4.1 Hz, 1H), 7.20 – 7.12 (m, 3H), 7.15 – 7.06 (m, 2H), 6.98 (tt, J = 7.6, 1.3 Hz, 1H). C
NMR (151 MHz, Acetoneꢀd ) δ 158.47, 149.73, 138.99, 138.52, 134.42, 133.91, 129.89, 125.64, 125.50,
6
+
1
25.20, 124.41, 121.37, 109.16. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H N O S 301.0641; Found
1
5
13
2
3
3
01.0641.
N-(3-chlorophenyl)-8-hydroxyquinoline-5-sulfonamide (3) The title compound was prepared from 3ꢀ
chloroaniline using the general sulfonamide synthesis procedure as a yellowꢀgreen solid (7.8 mg, 26% yield).
1
H NMR (600 MHz, Acetoneꢀd ) δ 9.50 (s, 1H), 9.09 (dd, J = 8.7, 1.5 Hz, 1H), 8.94 (dd, J = 4.2, 1.5 Hz, 1H),
6
8.29 (d, J = 8.3 Hz, 1H), 7.76 (dd, J = 8.8, 4.2 Hz, 1H), 7.23 – 7.14 (m, 3H), 7.06 (ddd, J = 8.1, 2.2, 0.9 Hz,
13
1
1
H), 7.00 (ddd, J = 7.9, 2.1, 0.9 Hz, 1H). C NMR (151 MHz, Acetone) δ 158.79, 149.89, 140.08, 140.01,
39.05, 134.95, 134.20, 134.16, 131.48, 125.56, 124.86, 124.63, 120.45, 119.02, 109.25. HRMS (ESIꢀTOF)
+
m/z: [M + H] Calcd for C H ClN O S 335.0252; Found 335.0248.
15
12
2
3
8
-hydroxy-N-methyl-N-phenylquinoline-5-sulfonamide (4). The title compound was prepared from Nꢀ
methylaniline using the general sulfonamide synthesis procedure as a yellowꢀgreen solid (5.3 mg, 19% yield).
1
H NMR (600 MHz, CDCl ) δ 8.78 (m, 1H), 8.42 (d, J = 8.8 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.36 – 7.15 (m,
3
1
3
4
H), 7.12 – 6.95 (m, 3H), 3.23 (s, 3H). C NMR (151 MHz, CDCl ) δ 156.90, 148.37, 141.34, 137.81, 134.75,
3
+
133.54, 129.02, 127.56, 127.26, 125.70, 123.00, 122.34, 108.21, 38.13. HRMS (ESIꢀTOF) m/z: [M + H] Calcd
for C H N O S 315.0798; Found 315.0797.
1
6
15
2
3
N-(3-chlorophenyl)-8-hydroxy-N-methylquinoline-5-sulfonamide (5). The title compound was prepared from Nꢀ
methylꢀ3ꢀchloroaniline using the general sulfonamide synthesis procedure as a yellowꢀgreen solid (5.1 mg, 16%
1
yield). H NMR (600 MHz, CDCl ) δ 8.78 (dd, J = 4.2, 1.5 Hz, 1H), 8.42 (dd, J = 8.8, 1.4 Hz, 1H), 8.13 (d, J =
3
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.3 Hz, 1H), 7.33 (dd, J = 8.8, 4.2 Hz, 1H), 7.22 (ddd, J = 8.1, 2.0, 1.0 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.16
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
(t, J = 8.0 Hz, 1H), 7.03 (t, J = 2.0 Hz, 1H), 6.99 (ddd, J = 8.0, 2.1, 1.1 Hz, 1H), 3.15 (s, 3H). C NMR (151
MHz, CDCl ) δ 157.16, 148.57, 142.66, 137.88, 134.54, 134.50, 133.77, 129.95, 127.71, 127.32, 125.60,
3
+
1
25.45, 123.20, 121.96, 108.28, 38.07. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H ClN O S 349.0408;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16 14
2
3
Found 349.0407.
5
-((6-chloroindolin-1-yl)sulfonyl)quinolin-8-ol (6). The title compound was prepared from 6ꢀchloroindoline
1
using the general sulfonamide synthesis procedure as an offꢀwhite solid (6.2 mg, 19% yield). H NMR (600
MHz, CDCl ) δ 9.08 (d, J = 8.8 Hz, 1H), 8.90 – 8.86 (m, 1H), 8.31 (d, J = 8.3 Hz, 1H), 7.67 (s, 1H), 7.63 – 7.56
3
13
(
m, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.02 – 6.96 (m, 2H), 4.01 (t, J = 8.5 Hz, 2H), 2.86 (t, J = 8.5 Hz, 2H). C
NMR (151 MHz, CDCl ) δ 157.34, 148.71, 143.28, 137.99, 134.14, 133.43, 132.94, 130.25, 125.92, 125.48,
3
+
1
23.89, 123.74, 123.18, 115.64, 108.51, 50.64, 27.61. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for
C H ClN O S 361.0408; Found 361.0408.
17 14
2
3
5-((4-chloroindolin-1-yl)sulfonyl)quinolin-8-ol (7). The title compound was prepared from 4ꢀchloroindoline
1
(
0.53 mmol scale) using the general sulfonamide synthesis procedure as a yellow solid (130 mg, 68% yield). H
NMR (600 MHz, CDCl ) δ 9.02 (d, J = 8.7 Hz, 1H), 8.84 – 8.80 (m, 1H), 8.25 (d, J = 8.3 Hz, 1H), 7.53 (dd, J =
3
8
.7, 4.1 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.1 Hz,
13
1
H), 3.99 (t, J = 8.5 Hz, 2H), 2.90 (t, J = 8.4 Hz, 2H). C NMR (151 MHz, CDCl ) δ 157.42, 148.72, 143.41,
3
138.07, 134.10, 133.04, 131.08, 130.20, 129.25, 125.51, 123.83, 123.74, 123.22, 113.20, 108.46, 49.77, 27.49.
+
HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H ClN O S 361.0408; Found 361.0409.
1
7
14
2
3
2
-chloro-5-((4-chloroindolin-1-yl)sulfonyl)quinolin-8-ol (8). The title compound was prepared in the same
manner as compound 7 using 2ꢀchloroꢀ8ꢀhydroxyquinoline as a starting material (115 mg, 0.64 mmol), yielding
1
the product as a yellow solid with a bright pink impurity (97 mg, 38%). H NMR (600 MHz, CDCl ) δ 8.97 (d,
3
J = 9.0 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.44 (dd, J = 8.0, 0.7 Hz, 1H), 7.20 (d, J =
8
.4 Hz, 1H), 7.14 – 7.10 (m, 1H), 6.94 (dd, J = 8.2, 0.8 Hz, 1H), 3.97 (t, J = 8.5 Hz, 2H), 2.89 (t, J = 8.4 Hz,
13
2
H). C NMR (151 MHz, CDCl ) δ 156.24, 150.48, 143.12, 137.94, 136.97, 132.74, 131.14, 130.17, 129.26,
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
+
1
24.99, 124.13, 124.00, 123.67, 113.13, 110.17, 49.74, 27.43. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for
C H Cl N O S 395.0018; Found 395.0018.
17
13
2
2
3
2
-amino-5-((4-chloroindolin-1-yl)sulfonyl)quinolin-8-ol (9). To
a
stirred solution of 2ꢀaminoꢀ8ꢀ
hydroxyquinoline (250 mg, 1.56 mmol) and pyridine (1.2 eq, 151 µL) in 12 mL 1:1 DCM/THF was slowly
added trifluoroacetic anhydride (1.1 eq, 242 µL) and the mixture was stirred for 1 h. The volatiles were
evaporated to produce the crude trifluoroamideꢀprotected intermediate as a grey solid. The solid was suspended
in 1 mL chloroform and 1 mL chlorosulfonic acid was added. The mixture was stirred for 3 h and poured into
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 mL iced brine. An extraction was performed with 50 mL 1:1 THF/EtOAc, which was dried over MgSO4
and evaporated to yield the crude sulfonyl chloride. The sulfonyl chloride (143 mg, 0.40 mmol) was dissolved
in 5 mL THF and 1.5 eq pyridine (48 µL, 0.60 mmol) and 1.1 eq 4ꢀchloroindoline (68 mg) was added. The
solution was stirred for 18 h, diluted with 20 mL EtOAc and washed with 1 N HCl, saturated sodium
bicarbonate solution and brine. The organic layer was collected, dried and evaporated. The resulting solid was
dissolved in 2 mL 1:1 THF/MeOH and 2 mL saturated sodium carbonate solution and the mixture was stirred
for 1 h. The mixture was diluted with water and EtOAc and the organic layer was dried and evaporated to yield
1
the pure product as a tan solid (96 mg, 16%). H NMR (600 MHz, DMSOꢀd ) δ 8.47 (d, J = 9.4 Hz, 1H), 7.75
6
(
d, J = 8.3 Hz, 1H), 7.28 (dd, J = 8.2, 0.8 Hz, 1H), 7.19 (tt, J = 8.0, 0.8 Hz, 1H), 7.01 (dd, J = 8.1, 0.8 Hz, 1H),
6
.97 (d, J = 8.3 Hz, 1H), 6.91 (s, 0H), 6.89 (d, J = 9.4 Hz, 1H), 6.75 (s, 2H), 3.97 (t, J = 8.5 Hz, 2H), 2.89 (t, J =
1
3
8.4 Hz, 2H). C NMR (151 MHz, DMSOꢀd ) δ 156.99, 155.83, 143.29, 137.99, 133.63, 130.32, 129.85,
6
+
1
29.37, 126.66, 123.09, 121.48, 118.90, 114.52, 112.68, 109.24, 49.57, 26.81. HRMS (ESIꢀTOF) m/z: [M + H]
Calcd for C H ClN O S 376.0517; Found 376.0517.
1
7
15
3
3
5-((4-chloroindolin-1-yl)sulfonyl)-2-(methylamino)quinolin-8-ol (10). The title compound was prepared using
the general S Ar procedure using a 2 M methylamine in THF solution. The crude material was purified by silica
N
1
gel preparatory TLC with EtOAc as an eluent to yield the product as a light yellow solid (3.1 mg, 61%). H
NMR (600 MHz, CDCl ) δ 8.61 (d, J = 9.3 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.12 –
3
7
.08 (m, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 9.3 Hz, 1H), 3.96 (t, J = 8.5 Hz,
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H), 3.07 (d, J = 4.9 Hz, 3H), 2.89 (t, J = 8.5 Hz, 2H). C NMR (151 MHz, CDCl ) δ 156.09, 155.15, 143.74,
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
137.76, 134.56, 130.97, 130.26, 129.18, 127.40, 123.61, 123.07, 119.44, 114.26, 113.32, 108.65, 49.67, 29.85,
+
2
7.53. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H ClN O S 390.0674; Found 390.0674.
18
17
3
3
5
-((4-chloroindolin-1-yl)sulfonyl)-2-(ethylamino)quinolin-8-ol (11). The title compound was prepared using the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
general S Ar procedure using a 2 M ethylamine in THF solution yielding the product as a light yellow solid
N
1
(
2.8 mg, 55%). H NMR (600 MHz, CDCl ) δ 8.61 (d, J = 9.4 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.1
3
Hz, 1H), 7.12 – 7.08 (m, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 8.1, 0.8 Hz, 1H), 6.66 (d, J = 9.4 Hz, 1H),
1
3
3
.96 (t, J = 8.5 Hz, 2H), 3.55 – 3.49 (m, 2H), 2.90 (t, J = 8.5 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). C NMR (151
MHz, CDCl ) δ 155.32, 155.01, 143.74, 137.62, 134.61, 130.97, 130.25, 129.18, 127.36, 123.60, 123.06,
3
+
119.41, 113.30, 110.13, 108.62, 49.71, 36.53, 27.53, 14.70. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for
C H ClN O S 404.0830; Found 404.0830.
19
19
3
3
5
-((4-chloroindolin-1-yl)sulfonyl)-2-hydrazinylquinolin-8-ol (12). The title compound was prepared using the
general S Ar procedure (19 µmol scale) using hydrazine hydrate yielding the product as a brown solid (7.4 mg,
N
1
9
7
7
9%). (See Figure S2 for NMR spectra) H NMR (600 MHz, DMFꢀd ) δ 8.59 (d, J = 9.5 Hz, 1H), 8.03 (s, 1H),
7
.89 (d, J = 8.3 Hz, 1H), 7.43 (dd, J = 8.1, 0.8 Hz, 1H), 7.25 (tt, J = 8.1, 0.8 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H),
1
3
.06 – 7.03 (m, 1H), 7.04 (dd, J = 8.1, 0.8 Hz, 1H), 4.10 (t, J = 8.4 Hz, 2H), 2.99 (t, J = 8.4 Hz, 2H). C NMR
(
151 MHz, DMFꢀd ) δ 157.98, 156.69, 144.08, 138.39, 133.37, 130.93, 130.55, 129.63, 127.24, 123.43, 122.68,
7
+
119.90, 113.90, 113.23, 109.37, 50.09, 27.34. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H ClN O S
1
7
16
4
3
3
91.0603; Found 391.0304.
5
-((4-chloroindolin-1-yl)sulfonyl)-2-(2-methylhydrazinyl)quinolin-8-ol (13). The title compound was prepared
1
using the general S Ar procedure using methylhydrazine yielding the product as a brown solid (5 mg, 99%). H
N
NMR (600 MHz, Acetoneꢀd ) δ 8.74 (d, J = 9.6 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.44 (dd, J = 8.1, 0.8 Hz,
6
1
H), 7.19 (tt, J = 8.1, 0.9 Hz, 1H), 7.16 – 7.12 (m, 1H), 7.12 – 7.07 (m, 1H), 6.97 (dd, J = 8.1, 0.8 Hz, 1H), 4.06
1
3
(
t, J = 8.5 Hz, 2H), 3.37 (s, 3H), 2.94 (t, J = 8.5 Hz, 2H). C NMR (151 MHz, Acetoneꢀd ) δ 157.66, 156.64,
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
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5
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5
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5
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1
44.66, 134.89, 131.35, 131.21, 130.03, 128.70, 127.86, 123.95, 123.77, 120.59, 114.17, 113.84, 109.43, 50.50,
+
38.68, 27.86. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H ClN O S 405.0783; Found 405.0786.
1
8
18
4
3
N'-(5-((4-chloroindolin-1-yl)sulfonyl)-8-hydroxyquinolin-2-yl)acetohydrazide (14). Hydrazide 12 (25 mg, 0.064
mmol) was dissolved in 400 µL THF and 1.1 eq acetic anhydride (6.7 µL, 0.07 mmol) and pyridine (7.7 µL,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.096 mmol) were added. The mixture was stirred for 1 h and then diluted with 400 µL of MeOH, 400 µL
saturated sodium carbonate solution and 400 µL H O. After 30 min of stirring, 500 µL EtOAc was added and
2
the organic layer was washed with sat. sodium bicarbonate solution, collected, dried and evaporated to give the
product as a yellowꢀbrown solid (22.2 mg, 80%). The product could also be prepared using the general S Ar
N
1
procedure with acetohydrazide. H NMR (600 MHz, Acetoneꢀd ) δ 8.78 (d, J = 9.3 Hz, 1H), 7.97 (d, J = 8.3 Hz,
6
1H), 7.46 (d, J = 8.1 Hz, 1H), 7.20 (m, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.1
13
Hz, 1H), 4.05 (t, J = 8.4 Hz, 2H), 2.94 (t, J = 8.4 Hz, 2H), 2.07 (s, 3H). C NMR (151 MHz, Acetoneꢀd ) δ
6
1
68.80, 156.61, 156.31, 144.55, 138.00, 135.60, 131.33, 131.20, 129.96, 129.00, 123.96, 123.86, 121.31,
+
113.83, 113.36, 109.68, 50.46, 27.83, 26.12. HRMS (ESIꢀTOF) m/z: [M + Na] Calcd for C H ClN O SNa
19
17
4
4
4
55.0557; Found 455.0553.
Corresponding Author
Kim D. Janda email: kdjanda@scripps.edu
*
The authors declare no conflict of interest. The views expressed in this article are those of the authors and do
not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government.
Acknowledgements
We thank the funding support from NIH grant R01AI119564 (KDJ), The Defense Threat Reduction Agency,
Joint Science and Technology Office, and Medical S&T Division, award CB4080 (MA). We also acknowledge
Alex Ducime for synthesis of some inactive quinolinols and Openeye software for use of their docking software
under an academic license. This is TSRI manuscript # 29418.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
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Screening library, docking scores and coordinates, selected NMR spectra, MMP assay results, cytotoxicity assy
results, representative hemidiaphragm timecourse data and raw data for cell and ex vivo assays.
Author Contributions
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
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P.T.B. designed, synthesized and tested compounds in the SNAPtide assay, performed docking and wrote the
manuscript. M.A. coordinated research at ICD, ran the hemidiaphragm experiments and edited the manuscript.
C.H.P assisted with the hemidiaphragm experiments including analysis of data. A.K.S. performed the BoCell
experiments. K.D.J. was the head research advisor, directed research activities and edited the manuscript.
Abbreviations
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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BoNT/A LC, botulinum neurotoxin serotype A light chain; SNAPꢀ25, synaptosomalꢀassociated protein; MMP,
matrix metalloproteinase; HPT, halfꢀparalysis time.
References
1
.
Sugiyama, H. Clostridium botulinum neurotoxin. Microbiol. Rev. 1980, 44, 419ꢀ448.
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. Botulinum toxin as a biological weapon: Medical and public health
management. J. Am. Med. Assoc. 2001, 285, 1059ꢀ1070.
3
.
Schantz, E. J.; Johnson, E. A. Properties and use of botulinum toxin and other microbial neurotoxins in
medicine. Microbiol. Rev. 1992, 56, 80ꢀ99.
4
.
.
Jankovic, J. Botulinum toxin in clinical practice. J. Neurol. Neurosurg. Psych. 2004, 75, 951ꢀ957.
5
Fagien, S. Botox for the treatment of dynamic and hyperkinetic facial lines and furrows: Adjunctive use
in facial aesthetic surgery. Plast. Reconstr. Surg. 1999, 103, 701ꢀ713.
Frampton, J. E. Onabotulinumtoxin A (BOTOX ®): A review of its use in the prophylaxis of headaches
in adults with chronic migraine. Drugs 2012, 72, 825ꢀ845.
7. Odergren, T.; Hjaltason, H.; Kaakkola, S.; Solders, G.; Hanko, J.; Fehling, C.; Marttila, R. J.; Lundh, H.;
6
.
Gedin, S.; Westergren, I.; Richardson, A.; Dott, C.; Cohen, H. A double blind, randomised, parallel group study
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 29
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to investigate the dose equivalence of Dysport (R) and Botox (R) in the treatment of cervical dystonia. J.
Neurol. Neurosurg. Psych. 1998, 64, 6ꢀ12.
8
.
Troung, D. D.; Rontal, M.; Rolnick, M.; Aronson, A. E.; Mistura, K. Doubleꢀblind controlled study of
botulinum toxin in adductor spasmodic dysphonia. Laryngoscope 1991, 101, 630ꢀ634.
Roggenkamper, P.; Jost, W. H.; Bihari, K.; Comes, G.; Grafe, S.; Team, N. T. B. S. Efficacy and safety
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
.
of a new botulinum toxin type A free of complexing proteins in the treatment of blepharospasm. J. Neural
Transm. 2006, 113, 303ꢀ312.
1
0.
Woodruff, B. A.; Griffin, P. M.; Mccroskey, L. M.; Smart, J. F.; Wainwright, R. B.; Bryant, R. G.;
Hutwagner, L. C.; Hatheway, C. L. Clinical and laboratory comparison of botulism from toxin typeꢀA, typeꢀB,
and typeꢀE in the United States, 1975ꢀ1988. J. Infect. Dis. 1992, 166, 1281ꢀ1286.
1
1.
McCarty, C. L.; Angelo, K.; Beer, K. D.; CibulskasꢀWhite, K.; Quinn, K.; de Fijter, S.; Bokanyi, R.;
Germain, E. S.; Baransi, K.; Barlow, K.; Shafer, G.; Hanna, L.; Spindler, K.; Walz, E.; DiOrio, M.; Jackson, B.
R.; Luquez, C.; Mahon, B. E.; Basler, C.; Curran, K.; Matanock, A.; Walsh, K.; Slifka, K. J.; Rao, A. K. Large
outbreak of botulism associated with a church potluck meal ꢀ Ohio, 2015. MMWR Morb. Mortal. Wkly. Rep.
2
015, 64, 802ꢀ803.
2. Wein, L. M.; Liu, Y. F. Analyzing a bioterror attack on the food supply: The case of botulinum toxin in
1
milk. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9984ꢀ9989.
13.
4.
Henderson, D. A. The looming threat of bioterrorism. Science 1999, 283, 1279ꢀ1282.
1
Tacket, C. O.; Shandera, W. X.; Mann, J. M.; Hargrett, N. T.; Blake, P. A. Equine antitoxin use and
other factors that predict outcome in typeꢀA foodborne botulism. Amer. J. Med. 1984, 76, 794ꢀ798.
15. Simpson, L. I. The binary toxin produced by Clostridium botulinum enters cells by receptorꢀmediated
endocytosis to exert its pharmacologic effects. J. Pharmacol. Exp. Ther. 1989, 251, 1223ꢀ1228.
6. Blasi, J.; Chapman, E. R.; Link, E.; Binz, T.; Yamasaki, S.; De Camilli, P.; Sudhof, T. C.; Niemann, H.;
1
Jahn, R. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAPꢀ25. Nature 1993, 365, 160ꢀ163.
ACS Paragon Plus Environment

Page 25 of 29
Journal of Medicinal Chemistry
1
2
1
7.
Schiavo, G.; Rossetto, O.; Tonello, F.; Montecucco, C. Intracellular targets and metalloprotease activity
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of tetanus and botulism neurotoxins. Curr. Top. Microbiol. Immunol. 1995, 195, 257ꢀ274.
8. Seki, H.; Xue, S.; Pellett, S.; Šilhár, P.; Johnson, E. A.; Janda, K. D. Cellular protection of SNAPꢀ25
1
against botulinum neurotoxin/A: Inhibition of thioredoxin reductase through a suicide substrate mechanism. J.
Am. Chem. Soc. 2016, 138, 5568ꢀ5575.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9.
necessary for antagonist activity against botulinum neurotoxin. Toxicon 1997, 35, 1439ꢀ1451.
0. Deshpande, S. S.; Sheridan, R. E.; Adler, M. Efficacy of certain quinolines as pharmacological
antagonists in botulinum neurotoxin poisoning. Toxicon 1997, 35, 433ꢀ445.
21. Kiris, E.; Nuss, J. E.; Stanford, S. M.; Wanner, L. M.; Cazares, L.; Maestre, M. F.; Du, H. T.; Gomba,
Sheridan, R. E.; Deshpande, S. S.; Nicholson, J. D.; Adler, M. Structural features of aminoquinolines
2
G. Y.; Burnett, J. C.; Gussio, R.; Bottini, N.; Panchal, R. G.; Kane, C. D.; Tessarollo, L.; Bavari, S. Phosphatase
inhibitors function as novel, broad spectrum botulinum neurotoxin antagonists in mouse and human embryonic
stem cellꢀderived motor neuronꢀbased assays. Plos One 2015, 10.
2
2.
Kiris, E.; Burnett, J. C.; Nuss, J. E.; Wanner, L. M.; Peyser, B. D.; Du, H. T.; Gomba, G. Y.; Kota, K.
P.; Panchal, R. G.; Gussio, R.; Kane, C. D.; Tessarollo, L.; Bavari, S. Src family kinase inhibitors antagonize
the toxicity of multiple serotypes of botulinum neurotoxin in human embryonic stem cellꢀderived motor
neurons. Neurotox. Res. 2015, 27, 384ꢀ398.
23.
inhibitor using in situ lead identification chemistry. Org. Lett. 2006, 8, 1729ꢀ1732.
4. Silvaggi, N. R.; Boldt, G. E.; Hixon, M. S.; Kennedy, J. P.; Tzipori, S.; Janda, K. D.; Allen, K. N.
Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Identification of a potent botulinum neurotoxin a protease
2
Structures of Clostridium botulinum neurotoxin serotype A light chain complexed with smallꢀmolecule
inhibitors highlight activeꢀsite flexibility. Chem. Biol. 2007, 14, 533ꢀ542.
2
5.
Silhar, P.; Silvaggi, N. R.; Pellett, S.; Capkova, K.; Johnson, E. A.; Allen, K. N.; Janda, K. D.
Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: Synthesis, crystallography,
modeling, kinetic and cellular based studies. Bioorg. Med. Chem 2013, 21, 1344ꢀ1348.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 29
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
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3
3
3
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
6.
Kumaran, D.; Rawat, R.; Ludivico, M. L.; Ahmed, S. A.; Swaminathan, S. Structureꢀ and substrateꢀ
based inhibitor design for Clostridium botulinum neurotoxin serotype A. J. Biol. Chem. 2008, 283, 18883ꢀ
1
2
8891.
7.
Silhar, P.; Capkova, K.; Salzameda, N. T.; Barbieri, J. T.; Hixon, M. S.; Janda, K. D. Botulinum
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
neurotoxin A protease: Discovery of natural product exosite inhibitors. J. Am. Chem. Soc. 2010, 132, 2868.
8. Caglic, D.; Krutein, M. C.; Bompiani, K. M.; Barlow, D. J.; Benoni, G.; Pelletier, J. C.; Reitz, A. B.;
2
Lairson, L. L.; Houseknecht, K. L.; Smith, G. R.; Dickerson, T. J. Identification of clinically viable quinolinol
inhibitors of botulinum neurotoxin A light chain. J. Med. Chem. 2014, 57, 669ꢀ676.
2
9.
RoxasꢀDuncan, V.; Enyedy, I.; Montgomery, V. A.; Eccard, V. S.; Carrington, M. A.; Lai, H. G.; Gul,
N.; Yang, D. C. H.; Smith, L. A. Identification and biochemical characterization of smallꢀmolecule inhibitors of
Clostridium botulinum neurotoxin serotype A. Antimicrob. Agents Chemother. 2009, 53, 3478ꢀ3486.
3
0.
Lai, H. G.; Feng, M. H.; RoxasꢀDuncan, V.; Dakshanamurthy, S.; Smith, L. A.; Yang, D. C. H.
Quinolinol and peptide inhibitors of zinc protease in botulinum neurotoxin A: Effects of zinc ion and peptides
on inhibition. Arch. Biochem. Biophys. 2009, 491, 75ꢀ84.
3
1.
Videnovic, M.; Opsenica, D. M.; Burnett, J. C.; Gomba, L.; Nuss, J. E.; Selakovic, Z.; Konstantinovic,
J.; Krstic, M.; Segan, S.; Zlatovic, M.; Sciotti, R. J.; Bavari, S.; Solaja, B. A. Second generation steroidal 4ꢀ
aminoquinolines are potent, dualꢀtarget inhibitors of the botulinum neurotoxin serotype A metalloprotease and
P. falciparum malaria. J. Med. Chem. 2014, 57, 4134ꢀ4153.
3
2.
metalloproteinases. J. Am. Chem. Soc. 2006, 128, 3156ꢀ3157.
33. Puerta, D. T.; Lewis, J. A.; Cohen, S. M. New beginnings for matrix metalloproteinase inhibitors:
Identification of highꢀaffinity zincꢀbinding groups. J. Am. Chem. Soc. 2004, 126, 8388ꢀ8389.
4. Jacobsen, J. A.; Fullagar, J. L.; Miller, M. T.; Cohen, S. M. Identifying chelators for metalloprotein
Jacobsen, F. E.; Lewis, J. A.; Cohen, S. M. A new role for old ligands: Discerning chelators for zinc
3
inhibitors using a fragmentꢀbased approach. J. Med. Chem. 2011, 54, 591ꢀ602.
ACS Paragon Plus Environment

Page 27 of 29
Journal of Medicinal Chemistry
1
2
3
5.
Zuniga, J. E.; Schmidt, J. J.; Fenn, T.; Burnett, J. C.; Arac, D.; Gussio, R.; Stafford, R. G.; Badie, S. S.;
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Bavari, S.; Brunger, A. T. A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very
different conformation than SNAPꢀ25 substrate. Structure 2008, 16, 1588ꢀ1597.
3
6.
Silhar, P.; Lardy, M. A.; Hixon, M. S.; Shoemaker, C. B.; Barbieri, J. T.; Struss, A. K.; Lively, J. M.;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Javor, S.; Janda, K. D. Cꢀterminus of botulinum A protease has profound and unanticipated kinetic
consequences upon the catalytic cleft. ACS Med. Chem. Lett. 2013, 4, 283ꢀ287.
37.
Part 5: The antibacterial action of 8ꢀhydroxyquinoline (oxine). Br. J. Exp. Pathol. 1950, 31, 425ꢀ441.
8. Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.; Jones, W. D.; McLean, C. A.; Barnham, K.
Rubbo, S. D.; Albert, A.; Gibson, M. I. The influence of chemical constitution on antibacterial activity.
3
J.; Volitakis, I.; Fraser, F. W.; Kim, Y. S.; Huang, X. D.; Goldstein, L. E.; Moir, R. D.; Lim, J. T.; Beyreuther,
K.; Zheng, H.; Tanzi, R. E.; Masters, C. L.; Bush, A. I. Treatment with a copperꢀzinc chelator markedly and
rapidly inhibits betaꢀamyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001, 30, 665ꢀ676.
39.
0.
OMEGA version 2.4.6. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com
Hawkins, P. C. D.; Nicholls, A. Conformer Generation with OMEGA: Learning from the data set and
4
the analysis of failures. J. Chem. Inf. Model. 2012, 52, 2919ꢀ2936.
1. Hawkins, P. C. D.; Skillman, A. G.; Warren, G. L.; Ellingson, B. A.; Stahl, M. T. Conformer generation
4
with OMEGA: Algorithm and validation using high quality structures from the Protein Databank and
Cambridge Structural Database. J. Chem. Inf. Model. 2010, 50, 572ꢀ584.
4
2.
3.
FRED version 2.2.5. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com
4
McGann, M. FRED pose prediction and virtual screening accuracy. J. Chem. Inf. Model. 2011, 51, 578ꢀ
596.
4.
4
McGann, M. FRED and HYBRID docking performance on standardized datasets. J. Comput. Aided
Mol. Des. 2012, 26, 897ꢀ906.
4
5.
Chardin, P.; McCormick, F. Brefeldin A: The advantage of being uncompetitive. Cell 1999, 97, 153ꢀ
1
55.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 29
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7
8
9
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
6.
inhibitor of human placental alkaline phosphatase. Biochem. J. 1971, 124, 509ꢀ516.
7. Min, D.; Tepp, W. H.; Johnson, E. A.; Chapman, E. R. Using fluorescent sensors to detect botulinum
neurotoxin activity in vitro and in living cells. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14701ꢀ14706.
8. Adler, M.; Dinterman, R. E.; Wannemacher, R. W. Protection by the heavy metal chelator N,N,N',N'ꢀ
Lin, C. W.; Sie, H. G.; Fishman, W. H. Lꢀtryptophan. A nonꢀallosteric organꢀspecific uncompetitive
4
0
1
2
3
4
5
6
7
8
9
0
1
2
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3
4
5
6
7
8
9
0
4
tetrakis (2ꢀpyridylmethyl)ethylenediamine (TPEN) against the lethal action of botulinum neurotoxin A and B.
Toxicon 1997, 35, 1089ꢀ1100.
4
9.
0.
Make Receptor version 3.0.1. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com
VIDA version 4.3.0.4. OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com
Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. Derivatives of 8ꢀhydroxyꢀ2ꢀmethylquinoline are
5
51.
powerful prototypes for zinc sensors in biological systems. J. Am. Chem. Soc. 2001, 123, 5160ꢀ5161.
2. Ariyasu, S.; Sawa, A.; Morita, A.; Hanaya, K.; Hoshi, M.; Takahashi, I.; Wang, B.; Aoki, S. Design and
5
synthesis of 8ꢀhydroxyquinolineꢀbased radioprotective agents. Bioorg. Med. Chem 2014, 22, 3891ꢀ3905.
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