A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A

Article abstract of DOI:10.1016/j.bmcl.2016.11.019

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and

Full text of DOI:10.1016/j.bmcl.2016.11.019

Accepted Manuscript
A matrix-focused structure-activity and binding site flexibility study of quino-
linol inhibitors of botulinum neurotoxin serotype A
William A. Harrell Jr., Rebecca C. Vieira, Susan M. Ensel, Vicki Montgomery,
Rebecca Guernieri, Vanessa S. Eccard, Yvette Campbell, Virginia Roxas-
Duncan, John H. Cardellina II, Robert P. Webb, Leonard A. Smith
PII:
S0960-894X(16)31158-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.019
BMCL 24414
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 August 2016
7 November 2016
8 November 2016
Please cite this article as: Harrell, W.A. Jr., Vieira, R.C., Ensel, S.M., Montgomery, V., Guernieri, R., Eccard, V.S.,
Campbell, Y., Roxas-Duncan, V., Cardellina, J.H. II, Webb, R.P., Smith, L.A., A matrix-focused structure-activity
and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.019
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
A matrix-focused structure-activity and binding site flexibility study of
quinolinol inhibitors of botulinum neurotoxin serotype A
William A. Harrell, Jr.a,1†, Rebecca C. Vieira a,2‡, Susan M. Ensel b, Vicki Montgomery a,
ω
Rebecca Guernieri a, Vanessa S. Eccard a,§, Yvette Campbell a, Virginia Roxas-Duncan a, ,
γ
John H. Cardellina II a, , Robert P. Webb a,*, and Leonard A. Smith c
a Division of Molecular and Translational Sciences, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, 21702, United States
b Department of Chemistry and Physics, Hood College, Frederick, Maryland, 21701, United States
c Senior Research Scientist, Medical Countermeasures Technology, Office of the Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases,
Frederick, Maryland, 21702, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of
enantiomers of one of the more active leads indicated considerable flexibility in the binding site.
We designed a limited study to investigate this flexibility and probe structure-activity
relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC
inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and
four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-
based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ≤ 1 µM and 11
effective at ≤ 2µM in an ex vivo assay.
Keywords:
Botulinum Neurotoxin Serotype A
BoNT/A Inhibitor
Betti Reaction Products
Structure-Activity Relationship
Binding Site Flexibility
2009 Elsevier Ltd. All rights reserved.
———
Abbreviations: BoNTs, botulinum neurotoxins; BoNT/A, botulinum neurotoxin serotype A; HC, heavy chain; LC, light chain; SNARE, soluble N-
ethylmaleimide-sensitive factor attachment protein receptor; SNAP-25, 25 kDa synaptosomal-associated protein; BIG-IV, Botulism Immune Globulin
Intravenous (Human); SAR, structure-activity relationship; IC50, 50% inhibitory concentration; MPNHDA, Mouse Phrenic Nerve Hemidiaphragm Assay
* Corresponding author: Tel. 301-619-6686.
E-mail address: robert.p.webb6.civ@mail.mil.
†
Present address: Department of Chemistry, Stevenson University, Owings Mills, Maryland, 21117, United States.
‡
Present address: Afton Chemical Corporation, Richmond, Virginia, 23219, United States.
§
Present address: Leidos Biomedical Research, Inc., Frederick, Maryland, 21701, United States.
ωPresent address: Biosurety Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, 21702, United States.
γ Present address: ReevesGroup, Virginia Beach, VA, 23451, United States.

Figure 1. Serving as structural inspiration for this study, compounds 1, 215
and 316 all showed low μM BoNT/A inhibitory activity in previous enzymatic
and ex vivo studies.
Figure 1. Serving as structural inspiration for this study, compounds 1, 215
and 316 all showed low μM BoNT/A inhibitory activity in previous enzymatic
and ex vivo studies.
Botulinum neurotoxins (BoNTs), produced by the anaerobic
bacterium Clostridium botulinum, are the direct cause of
botulism, which is characterized by flaccid muscle paralysis
resulting from blockage of acetylcholine release from nerve cells.
BoNTs occur as seven immunologically distinct serotypes (A-G)
and are some of the most potent toxins found in nature. Recently,
a new purported serotype designated as type H has been reported,
but this requires experimental confirmation.1,2 Botulinum
neurotoxin serotype A (BoNT/A) is the most common, with a
lethal dose (LD50) of less than 1 ng/kg in humans.3 The high level
of toxicity associated with BoNT/A, coupled with its apparent
ease of production makes it a dangerous potential biowarfare
agent.4
entered the nerve cell, antibodies are ineffective. Prophylactic
medical countermeasures against botulism do exist with
vaccines,14 but the increasing number of medical disorders being
treated with therapeutic BoNT (e.g., Botox) makes vaccination of
the general population inadvisable. Therefore, small-molecule
therapeutics effective in the neuronal cell are attractive targets for
the post-exposure treatment of botulism.
In our original disclosure of BoNT inhibitory activity of the 7-
substituted 8-hydroxyquinolines, we tested 55 analogs and found
that over half of them, including 1 and 2, exhibited >50%
inhibition of BoNT/A at doses <50 µM (Figure 1).15 Those
compounds contained a wide array of substituent groups attached
at C-7, as did the less active or inactive analogs, providing no
obvious or definitive structure-activity relationship (SAR)
information. Early on, we hypothesized that the binding site of
BoNT/A was very accommodating or flexible,16 a rather widely
occurring phenomenon that has recently been reviewed.17 When
we subsequently separated the enantiomers of 3, one of our more
potent compounds, and found them to have essentially equivalent
inhibitory activity,16 that hypothesis became more solidly
supported. Recently, Caglič et al. examined 188 8-
hydroxyquinoline compounds and found similar levels of activity
and reached similar conclusions about the binding site.18
Synthesized as 150 kDa protoxins, BoNTs are proteolytically
cleaved to form the active holotoxin comprised of a 100 kDa
heavy chain (HC) and a 50 kDa light chain (LC) linked via a
disulfide bond.5,6 The HC is responsible for the binding of the
toxin to the surface of cholinergic nerve cells, as well as
translocation of the LC into the cytosol of the nerve cell.7-10 The
LC is a zinc metalloprotease that cleaves various portions of
SNARE (soluble N-ethylmaleimide-sensitive factor attachment
protein receptor) protein complex. In BoNT/A, the LC cleaves
SNAP-25 (25 kDa synaptosomal-associated protein), preventing
acetylcholine release from the synaptic terminals causing flaccid
muscle paralysis.11,12
After our original observations about the activity of
compounds in this class and the flexibility or plasticity of the
binding site, we embarked on a detailed study of a limited,
focused matrix of analogs in an effort to determine the relevance
of substitutions on the 8-hydroxyquinoline ring and compositions
of the aldehyde and amine components of the Betti reation19,20
used to construct these compounds. We report here the results of
this study and its implications for further development of this
class of BoNT/A inhibitors.
Current therapies for BoNT intoxication, which include
supportive care, primarily mechanical ventilation, and the
administration of antitoxins, are impractical in the event of
widespread biological attack. Botulism Immune Globulin
Intravenous (Human) (BIG-IV), an FDA-approved drug from the
California Department of Public Health, is available to treat
infant botulism caused by neurotoxin serotypes A and B. An
FDA-approved heptavalent equine antitoxin (HBAT) is available
for treatment of botulism in adults. Antitoxins of equine origin,
however, carry the risk of serum-sickness and are only effective
while toxin is in the circulatory system.13 Once the toxin has
The Betti reaction is a one pot, Mannich-type reaction that
involves the condensation of an amine, aldehyde, and phenol. We
utilized this chemistry to develop a 3 x 3 x 4 matrix of potential
Figure 2. A 3 x 3 x 4 matrix of BoNT/A inhibitors using three substituted 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes.

BoNT/A inhibitors for this SAR study. These 36 compounds
were synthesized using the three substituted 8-
hydroxyquinolines, three heteroaromatic amines, and four
demonstrating starting IC50 values at or below 4.6 μM. Upon first
glance, it seems no single compound stands out and no
discernible pattern exists. However, interesting general trends
across each group are apparent upon taking the average of all
IC50 values for each starting material type (Table 1, for an
inclusive list of biological data for each of the 36 individual
compounds, see Table 2).
substituted benzaldehydes shown in Figure 2.
Small-molecule BoNT/A inhibitors 1 and 2 share the same
general structural scaffold, differing only in the placement of the
nitrogen in the pyridyl ring and a fluorine substituent. One goal
of this study was to determine the importance of these structural
features in regard to BoNT/A inhibitory activity. In choosing the
starting material for the amine portion of the inhibitors for this
SAR study, we selected 2- and 3-pyridyl amines as we previously
observed that both components gave rise to potent BoNT/A
inhibitors. We also chose to use 2-pyrimidyl amine as we
envisioned that an extra nitrogen on this ring might provide
additional contacts in the active site and improve solubility and
formulation characteristics. As for the aldehyde portion, we
wanted to probe the effects of fluorine substitutions on the
aromatic ring. In this case, benzaldehyde was chosen as a starting
A
review of the data for the three different 8-
hydroxyquinoline starting materials indicated that the 5-chloro
substituted analogs show the highest activity against BoNT/A
(average IC50 of 1.9 μM), a two-fold increase in activity relative to
the unsubstituted 8-hydroxyquinoline and the 2-methyl version,
which both have average IC50 values of 3.9 μM. Upon
examination of the BoNT/A inhibitory activity with respect to the
three heteroaromatic amines used in this study, the two pyridine
reagents provided most of the active compounds. The 2-amino
derivatives have an average IC50 of 1.8 μM, the most active of the
group. This is in stark contrast to the 2-aminopyrimidine
derivatives which have an average IC50 of 4.6 μM across the
group, approximately 2.5x less potent than the 2-aminopyridine
containing compounds.
material, along with 2-fluoro,
difluorobenzaldehydes.
4-fluoro and 2,4-
In addition to studying the effects of these structural
differences on BoNT/A inhibitory activity, we also evaluated
changes to the core 8-hydroxyquinoline structure. In our two
previous studies, we discovered that compounds 1 and 3,
differing in a hydrogen vs. a chlorine attached at C-5 on the 8-
hydroxyquinoline moiety, both inhibit BoNT/A with an IC50 of
1.5 μM. Additionally, compounds containing a methyl group at
the C-2 position showed promising activity in preliminary
(unpublished) studies. With this in mind, we chose 8-
hydroxyquinoline as well as the 5-chloro and 2-methyl
substituted versions as the last three components in this 3 x 3 x 4
matrix SAR study.
When looking at the aldehyde group, the results are not as
straightforward, with the 4-fluoro derivatives showing slightly
higher inhibitory activity when compared to the rest of the group.
Inhibitors employing benzaldehyde, 2-fluorobenzaldehyde, and
4-fluorobenzaldehyde all have average IC50 values ranging from
2.3 to 2.9 μM, roughly twice as potent the 2,4-
difluorobenzaldehyde derivatives.
After finding interesting inhibitory trends across these
structural groups in our in vitro BoNT/A LC protease assay, we
wanted to see if these general trends held true in biological
systems at the tissue level. We tested our 3 x 3 x 4 matrix of
small-molecule inhibitors in an ex vivo mouse phrenic nerve
hemidiaphragm assay (MPNHDA).15 Inhibitors were tested at
final concentrations of 20 and 2 μM, with the exception of
compounds 1-3, which were previously tested at 10 and 2 μM.15,17
Compounds listed as not effective did not cause a delay in time to
50% loss of twitch tension at a concentration of 20 μM.
To determine IC50 values for the 36 BoNT/A inhibitors, an
HPLC-based enzymatic assay was implemented using a full-
length recombinant BoNT/A LC in the presence a 17-mer SNAP-
25 peptide substrate. This substrate consists of residues 187 to
203 of SNAP-25 and catalytically cleaves the BoNT/A LC
between residues Q197 and R198.21 All 36 compounds were very
effective in inhibiting cleavage of the BoNT/A LC, and had IC50
values ranging from 0.6 to 12.7 μM, with all but four compounds
Trends similar to those observed in the in vitro assay emerged
across each group in the ex vivo assay. When considering the 8-
hydroxyquinoline, the 5-chloro derivative appears to be the most
potent (Table 2; for ease of comparison, this table sorted by
amine and aldehyde groups can be found in Tables S1 and S2,
respectively, in the supporting information). This group has the
largest number of compounds active at 2 μM and has only one
compound that was not effective at 20 μM in the MPNHDA. The
unsubstituted 8-hydroxyquinoline and the 2-methyl analogs show
similar activity to one another, with three ineffective compounds
in each group. These two groups have five compounds combined
that are active at 2 μM, the same number as the 5-chloro group
alone.
Table 1. Average IC50 values for starting material
components
Inhibitor Moiety
Starting Material
Average
IC50 (μM)
8-hydroxyquinoline
8-hydroxyquinoline
2-methyl-8-hydroxyquinoline
5-chloro-8-hydroxyquinoline
2-aminopyridine
3.9
3.9
1.9
1.8
3.3
4.6
2.9
2.8
2.3
4.9
amine
When observing the data across the amine substituents, there
is a stark contrast between different starting materials (Table S1).
Inhibitors containing either 2- or 3-pyridyl groups are all active
in this assay. Interestingly, all of the compounds that are not
effective at delaying onset of paralysis at 20 μM can be found in
the group incorporating the pyrimidine moiety. Data from both
the MPNHDA and the BoNT/A protease assay indicate that
having two nitrogens in this ring is clearly detrimental to
BoNT/A inhibitory activity.
3-aminopyridine
2-aminopyrimidine
aldehyde
benzaldehyde
2-fluorobenzaldehyde
4-fluorobenzaldehyde
2,4-difluorobenzaldehyde
Inhibitors synthesized using 4-fluorobenzaldehyde seem to be
the most active in the MPNHDA in the aldehyde group, with all
4-fluoro derivatives showing activity at either 2 or 20 μM (Table

S2). Both the benzaldehyde and 2-fluoro groups have three
compounds active at 2 μM, as well as two each that do not delay
onset of BoNT/A induced paralysis at 20 μM. However, if we
remove the pyrimidine containing compounds that are clearly far
less active in this assay, the benzaldehyde, 2-fluoro, and 4-fluoro
groups all show similar activity, with three compounds each that
cause a delay in time to 50% loss of twitch tension at a
concentration of 2 μM. The 2,4-difluoro analogs are the least
Interestingly, compounds containing an extra nitrogen in this ring
(2-aminopyrimidine) were the least active in this series. All
compounds that were not effective in the MPNHDA came from
this group. The active site seemed to accommodate fluorine
substitutions at the R3 or R4 positions, but substitution at both
sites proved less active. While the flexibility of the BoNT/A
active site does seem to accommodate added steric bulk at the R1
position, the addition of a methyl group here provides no
Compound
1
R1
R2
R3
R4
R5
IC50 (μM)
MPNHDAb (μM)
H
H
H
H
2-pyridyl
1.5
10c
active group, with only one compound active at 2 μM and three
ineffective compounds. These results are very consistent with
those from the in vitro BoNT/A LC protease assay.
discernable difference in activity from that of the unsubstituted 8-
hydroxyquinoline. However, the 5-chloro substituted compounds
were the most active, with 31 and 35 (IC50 = 0.6 μM) being the
most potent quinolinol-based BoNT/A inhibitors discovered to
date. Our current efforts are directed toward further medicinal
chemistry exploration of the aldehyde and amine components to
determine whether additional improvements can be made in the
BoNT/A inhibitory activity profiles.
Overall, this study has produced some of the most effective
quinolinol-based BoNT/A inhibitors to date, with seven
compounds displaying submicromolar IC50 values. Some of our
original observations about the activity of compounds in this
series were confirmed and some additional support for our
hypothesis about the flexibility of the binding site was developed,
with all compounds in this study displaying IC50 values ≤ 12.7
μM. As expected, active compounds were found throughout,
although certain structural preferences emerged. Inhibitors
containing a 2-pyridyl ring were the most active across the group.
A recent report from our laboratory disclosed that two early
hits from our investigation of 8-hydroxyquinoline based
inhibitors also inhibit BoNT’s B, C, E, and F and, intriguingly,
display different profiles of inhibition.22 This work has further
expanded the potential for this class of compounds in the
Table 2. IC50 and MPNHDA BoNT/A inhibitory data sorted by 8-hydroxyquinoline componenta

4
H
H
H
H
H
H
F
H
H
H
H
H
F
3-pyridyl
2-pyrimidyl
2-pyridyl
6.2
2.7
1.2
2.4
1.9
4.1
4.6
1.7
3.6
4.4
12.7
0.7
1.4
4.6
1.4
0.7
12.0
1.6
0.8
4.2
2.7
12.3
4.3
1.5
3.5
4.5
1.4
0.7
4.0
0.6
2.3
0.7
0.7
0.6
2.2
20
NEd
20
10c
NEd
2
5
6
H
H
2
H
H
F
3-pyridyl
7
H
H
F
2-pyrimidyl
2-pyridyl
8
H
H
H
H
H
F
9
H
H
F
3-pyridyl
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3
H
H
F
2-pyrimidyl
2-pyridyl
20
20
20
NEd
2
H
H
F
H
H
F
F
3-pyridyl
H
H
F
F
2-pyrimidyl
2-pyridyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
F
H
H
H
H
H
H
F
H
3-pyridyl
20
NEd
2
H
2-pyrimidyl
2-pyridyl
H
H
F
3-pyridyl
20
NEd
20
20
20
20
2
H
F
2-pyrimidyl
2-pyridyl
H
H
H
H
F
H
F
3-pyridyl
H
F
2-pyrimidyl
2-pyridyl
H
F
H
F
F
3-pyridyl
H
F
F
2-pyrimidyl
2-pyridyl
NEd
2c
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
F
H
H
H
H
H
H
F
26
27
28
29
30
31
32
33
34
35
36
H
3-pyridyl
2
H
2-pyrimidyl
2-pyridyl
2
H
20
2
H
F
3-pyridyl
H
F
2-pyrimidyl
2-pyridyl
2
H
H
H
H
F
20
2
H
F
3-pyridyl
H
F
2-pyrimidyl
2-pyridyl
20
20
20
NEd
H
F
H
F
F
3-pyridyl
H
F
F
2-pyrimidyl
a Assays conducted as described in ref. 15 and the Experimental Procedures section.
b Mouse phrenic nerve hemidiaphragm assay. Compounds were tested at 20, 2, and 0.5 μM and data represent lowest effective concentration.
c Inhibitors 1-3 were also tested at 10 μM.
d Not Effective at concentration of 20 μM.
Acknowledgments
This work was supported by the Defense Threat Reduction
Agency, Joint Science and Technology Office for Chemical-
Biological Defense. We are grateful to E. Brueggemann for mass
spectrometry support and Y.F. Lam at UMD for NMR support.
treatment of BoNT intoxication. There has been interest in this
class of inhibitor in other laboratories as well,18 including a
detailed computer modeling study by Gómez-Jeria et al.23 that
expanded our earlier examination into the binding of enantiomers
of 3.16

Supplementary Material
Supplementary data associated with this article, including
tables of assay data sorted by starting material component,
experimental protocols, and characterization data, can be found,
in the online version, at http://
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National Research Council, 2011. The authors declare no competing financial
interest.

Graphical Abstract
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A matrix-focused structure-activity and
binding site flexibility study of quinolinol
inhibitors of botulinum neurotoxin serotype
A
Leave this area blank for abstract info.
William A. Harrell, Jr., Rebecca C. Vieira, Susan M. Ensel, Vicki Montgomery, Rebecca Guernieri, Vanessa S.
Eccard, Yvette Campbell, Virginia Roxas-Duncan, John H. Cardellina II, Robert P. Webb, and Leonard A.
Smith