Pharmacophore refinement guides the design of nanomolar-range botulinum neurotoxin serotype a light chain inhibitors

Article abstract of DOI:10.1021/ml100056v

Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins. The enzymes' zinc(II) metalloprotease, referred to as the light chain (LC) component, inhibits acetylcholine release into neuromuscular junctions, resulting in the disease botulism. Currently, no therapies counter BoNT poisoning postneuronal intoxication; however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity in the neuronal cytosol. Herein, we describe the pharmacophore-based design and chemical synthesis of potent [non-zinc(II) chelating] small molecule (nonpeptidic) inhibitors (SMNPIs) of the BoNT serotype A LC (the most toxic of the BoNT serotype LCs). Specifically, the three-dimensional superimpositions of 2-[4-(4-amidinephenoxy) phenyl]indole-6-amidine-based SMNPI regioisomers [K_i = 0.600 μM (±0.100 μM)], with a novel lead bis-[3-amide-5-(imidazolino)phenyl] terephthalamide (BAIPT)-based SMNPI [K_i = 8.52 μM (±0.53 μM)], resulted in a refined four-zone pharmacophore. The refined model guided the design of BAIPT-based SMNPIs possessing K_i values = 0.572 (±0.041 μM) and 0.900 μM (±0.078 μM).

Full text of DOI:10.1021/ml100056v

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Pharmacophore Refinement Guides the Design of Nanomolar-
Range Botulinum Neurotoxin Serotype A Light Chain Inhibitors
Jonathan E. Nuss,†,# Yuxiang Dong,‡,# Laura M. Wanner,† Gordon Ruthel,† Peter Wipf,§
Rick Gussio, Jonathan L. Vennerstrom,‡ Sina Bavari,*,† and James C. Burnett*,^
†United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, ‡Department of
Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, §Department
of Chemistry and Combinatorial Chemistry Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, Developmental
Therapeutics Program, National Cancer Institute at Frederick, Frederick, Maryland 21702, and ^Target Structure-Based Drug
Discovery Group, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702
ABSTRACT Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins.
The enzymes' zinc(II) metalloprotease, referred to as the light chain (LC) component,
inhibits acetylcholine release into neuromuscular junctions, resulting in the disease
botulism. Currently, no therapies counter BoNT poisoning postneuronal intoxication;
however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity
in the neuronal cytosol. Herein, we describe the pharmacophore-based design and
chemical synthesis of potent [non-zinc(II) chelating] small molecule (nonpeptidic)
inhibitors (SMNPIs) of the BoNT serotype A LC (the most toxic of the BoNT serotype
LCs). Specifically, the three-dimensional superimpositions of 2-[4-(4-amidinephenoxy)-
phenyl]indole-6-amidine-based SMNPI regioisomers [Ki = 0.600 μM ((0.100 μM)],
with a novel lead bis-[3-amide-5-(imidazolino)phenyl]terephthalamide (BAIPT)-based
SMNPI [Ki = 8.52 μM ((0.53 μM)], resulted in a refined four-zone pharmacophore. The
refined model guided the design of BAIPT-based SMNPIs possessing Ki values = 0.572
((0.041 μM) and 0.900 μM ((0.078 μM).
KEYWORDS Botulinum neurotoxin, small molecule inhibitor, gas-phase pharma-
cophore, rational design, biothreat agent
otulinum neurotoxins (BoNTs) are the deadliest of
known microbial toxins;1 for example, it is estimated
that a lethal BoNT dose (LD50) for Homo sapiens is 1 ng
neuron-to-muscle signaling, resulting in the life-threatening
flaccid paralysis associated with botulism. The BoNTserotype
A, E, and C LCs cleave synaptosomal-associated protein of
25 kDa (SNAP-25),1 the BoNT serotype C LC also cleaves
syntaxin,1 and the BoNT serotype B, D, F, and G LCs cleave
vesicle-associated membrane protein.1
B
kg-1 body mass.1 As a result, these enzymes are classified as
category A, highest priority biothreat agents by the Centers
for Disease Control and Prevention.2
Secreted by strains of anaerobic bacillus Clostridium botu-
linum,1 there are seven known BoNT serotypes (designated
A-G). Serotypes A-C, E, and F cause human botulism.3,4
The mechanism of BoNT neuronal intoxication can be
summarized as follows:1 (1) Postproteolytic activation, the
enzyme consists of a disulfide-linked 100 kDa heavy chain
(HC) and a 50 kDa light chain (LC); (2) the HC binds to
neuronal cell surface receptors, triggering toxin internaliza-
tion via an intracellular vesicle; (3) the HC mediates LC
translocation out of the intracellular vesicle into the neuronal
cytosol; and (4) the LC [a zinc (Zn)(II) metalloprotease]
cleaves, depending on the serotype, one of three proteins
(see below) composing the soluble N-ethylmaleimide-sensi-
tive fusion protein attachment protein receptor (SNARE)
complex. The SNARE complex negotiates the release of
acetylcholine-containing synaptic vesicles into neuromuscular
junctions. The toxin-induced inhibition of acetylcholine re-
lease, via the proteolysis of SNARE proteins, terminates
Currently, there are no therapies available for the treat-
ment of BoNT LC-mediated paralysis postneuronal intoxica-
tion. This is pivotal, as the current treatments for BoNT
poisoning are limited to (1) the administration of antitoxin(s),1,5
which is not effective postneuronal intoxication6 and, there-
fore, would be of limited use following an act of bio-
terror (as it is likely that victims would seek medical attention
only after enervation), and (2) mechanical ventilation, which
is necessary once BoNT-induced paralysis compromises
thoracic muscle contraction.5 However, the latter form of
treatment would also be impractical following even a limited
act of bioterror employing a BoNT(s), as critical care re-
sources would likely be overwhelmed.7 Hence, there is
considerable interest in discovering and developing small
Received Date: March 18, 2010
Accepted Date: June 21, 2010
Published on Web Date: June 24, 2010
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molecules as therapeutics to inhibit BoNT LC proteolytic
activity postneuronal intoxication.1,8,9 Moreover, with res-
pect to the development of small molecule therapeutics, the
BoNT serotype A LC (BoNT/A LC) represents a top priority,
as it, versus other BoNT LCs known to cause botulism in
humans, possesses the longest duration of activity in the
neuronal cytosol.3,4 Thus, the hypothesis rationalizing a
small molecule-based therapeutic approach for the treat-
ment of BoNT/A LC intoxication is as follows: Small, druglike
molecules can penetrate into the neuronal cytosol10 and
inhibit the toxin's LC proteolytic activity postneuronal intoxi-
cation. In turn, this mechanism would protect SNAP-25 and,
hence, the function of the SNARE protein complex. Addi-
tionally, if stockpiled in dry, sunlight-free, temperature-con-
trolled locations, chemically stable small molecules would
remain viable for many years. In contrast, vaccines possess
comparatively shorter shelf-lives.
Table 1. SMNPI Two-Dimensional Structures and Inhibition
Constants
Current X-ray structures of the BoNT/A LC fail to rationalize
the structure-activity relationships (SAR) for our competitive,
non-Zn(II) chelating, small molecule (nonpeptidic) inhibitors
(SMNPIs) of the BoNT/A LC.10-12 For this reason, we are
successfully employing a gas-phase pharmacophore-based
approach for SMNPI discovery and optimization.11 , 1 2 This
paradigm uses an efficient, iterative approach: SMNPIs are
continually integrated into the pharmacophore to both deve-
lop three-dimensional (3D) search queries to discover novel
SMNPI chemotypes (via the 3D database mining of small
molecule libraries) and guide the rational design of more
potent SMNPI derivatives. Following, structure-activity data
from both database-mined and rationally designed SMNPIs
are incorporated back into the model, resulting in further
pharmacophore refinement.10-13
Employing this strategy, we recently reported a three-zone
iteration of the pharmacophore for BoNT/A LC inhibition11 that
guided the design of 2-[4-(4-amidinephenoxy)phenyl]indole-6-
amidine (2,4,4-APPIA)-based SMNPI regioisomers 1a and 1b
(Ta ble 1 ).11 In vitro testing of 1a and 1b indicated that the regio-
isomers possess a Ki=0.600 μM ((0.100 μM). However, it is
important to note that the substituted bis-amidine moieties of
1a and 1b are not chemically stable during high-performance
liquid chromatography (HPLC) separation, which prohibits
regioisomer isolation. Additionally, the selective syntheses of
individual regioisomers 1a and 1b are also prohibitive; there-
fore, we are currently exploring alternative cationic moieties to
further advance the development and the optimization of this
chemotype. Furthermore, we have reported how the three-zone
pharmacophore was employed to generate a 3D search query
that identified novel bis-[3-amide-5-(imidazolino)phenyl]tere-
phthalamide (BAIPT)-based SMNPI 2(Table 1),12 a lead inhibitor
possessing a Ki=8.52 μM ((0.53 μM). The discovery of 2 was
particularly compelling because one of its bis-butylamide sub-
stituents is located outside of the three-zone iteration of the
pharmacophore,12 implying the possibility of a fourth zone of
SMNPI occupancy (Figure 1a). Importantly, a new pharmaco-
phore zone 4 could potentially be exploited to discover new
SMNPI chemotypes, as well as guide the synthetic optimization
of our known SMNPIs.
a Published Ki value (ref 11).
1a, 1b, and 2 were superimposed in gas phase. As shown
in Figure 2a, the SMNPIs overlaid with good chemical
complementarity. Of particular significance to pharmaco-
phore refinement, the 4-amino-7-chloroquinoline (4,7-ACQ)
components of 1a and 1b and the terminal methylenes of
the butylamide substituents of 2 can occupy proximal loca-
tions within empirically verified zone 311 and hypothesized
pharmacophore zone 4, respectively (Figure 2a) (with the
4,7-ACQ components providing greater zonal occupancy).
To initially examine the possibility of a four-zone pharma-
cophore, sterically feasible intramolecular conformations of
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Figure 1. SMNPI alignments in a previously reported three-zone
iteration of the pharmacophore for BoNT/A LC inhibition.11 ,12
Chlorine atoms are light green, nitrogen atoms are blue, and
oxygen atoms are red. Squares indicate planar pharmacophore
components, and dashed spheres indicate cationic pharmaco-
phore components. (a) SMNPI 2 (magenta carbons) as it was
previously predicted to fit within the three-zone iteration of the
pharmacophore.12 (b) SMNPI regioisomers 1a (green carbons) and
1b (cyan carbons) were previously predicted to fit the three-zone
pharmacophore such that their “core” 2,4,4-APPIA components
oriented in opposite directions within zones 1 and 2, and as a
result, their 4,7-ACQ substructures occupied only zone 3.11
Additionally, with respect to model refinement, the new
SMNPI superimpositions are in contrast to our previously
published three-zone pharmacophore,11 which predicted
that the 4,7-ACQ components of 1a and 1b occupy only
zone 3 of this model (Figure 1b).
Further supporting a four-zone pharmacophore model are
the superimpositions of the SMNPIs within the requirements
of reported zones 1 and 2 (Figure 2a).11 , 1 2 Specially, in zone 1
(Figure 2a), (1) the cationic amidines of 1a and 1b and the
cationic imidazoline of 2 are located in close proximity and
possess comparable directional orientations, (2) the hydrogen
bond-donating indole nitrogens of 1a and 1b superimpose
with a hydrogen bond-donating amide nitrogen of the “core”
terephthalamide of 2, (3) the phenyl components of the
indoles of 1a and 1b align closely with the zone 1-occupying
phenyl of 2, and (4) the central phenyls of 1a and 1b super-
impose with the terephthalamide phenyl of 2. It is notable that
in the refined four-zone pharmacophore (Figure 2a) versus the
three-zone iteration of the model (Figure 1), the central phenyl
rings of 1a, 1b, and 2 form the key basis for SMNPI alignment
and, therefore, are now included within the zone 1 planar com-
ponent (reference Figure 1 vs Figure 2a). In zone 2 (Figure 2a),
(1) the ether oxygens of 1a and 1b superimpose with, and are
oriented in the same direction as, a terephthalamide carbonyl
oxygen of 2, (2) the terminal phenyls of 1a and 1b are located
proximal to, and partially overlay with, the zone 2-occupying
phenyl of 2, and (3) the cationic amidines of 1a and 1b and the
cationic imidazole of 2 align in good order and with equivalent
directional orientations.
Figure 2. Three-dimensional alignments of SMNPIs 1a, 1b, and 2 in
the context of the refined four-zone pharmacophore for BoNT/A LC
inhibition, and rational designs based on the new model. (a) All colors
and shape definitions are as indicated in Figure 1. (b) Carbon atoms
are yellow, orange, and burgundy for designs 3, 4, and 5, respectively;
all other atom colors are as indicated in Figure 1.
(Figure 2a), formed the basis for the design of derivatives
to improve the inhibitory potency of the BAIPT-based
SMNPI chemotype. Specifically, the four-zone pharmaco-
phore indicated that tethering 4,7-ACQ substructures
onto the terminal amide amines of the BAIPT chemotype
“core” with alkyl linkers (see derivatives 3-5, Figure 2b)
would result in SMNPIs with improved inhibitory poten-
cies. Additionally, as shown in Figure 2b, 3-5 were de-
signed to provide SAR exploring the lengths of the alkyl
chains [i.e., ethyls (3), propyls (4), and butyls (5)] tether-
ing the 4,7-ACQ zones 3- and 4-occupying components
The 3D chemical complementarity observed for 1a, 1b,
and 2, when aligned in the four-zone pharmacophore
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Scheme 1. Synthesis of BAIPT-Based SMNPIs 3-5a
of Zn(II) concentrations equaling 5, 10, 25, and 50 μM did not
effect inhibitory efficacies (Supporting Information, Table S1),
thereby indicating that the SMNPIs do not chelate the Zn(II) of
the enzyme's catalytic engine. The in vitro results obtained for
3-5 (Ta bl e 1) confirm the presence of pharmacophore zone
4. Moreover, the increased inhibitory potencies of3-5, versus
2, demonstrate that, with each refinement of the gas-phase
pharmacophore, the model's predictive resolution is im-
proved and that it can be used to design more potent SMNPIs.
On the basis of the SAR provided by 3-5, the inhibitory
potency of 4 implies that the optimal length of the alkyl tether
connecting SMNPI components is a propyl chain. By contrast,
the inhibitory potency of 3indicates that shorter ethyl tethers do
not allow the SMNPI's 4,7-ACQ moieties to achieve optimal
zones 3 and 4 occupancy, while the inhibitory potency of 5
indicates that longer butyl tethers overextend optimal 4,7-ACQ
binding in zones 3 and 4. To provide SAR that will further guide
SMNPI optimization (in vitro), future BAIPT-based designs will
not only explore the effects of modified tethers (e.g., linkers
with increased rigidity and the inclusion of heteroatoms) on
BoNT/A LC inhibition, but will also incorporate a diversity of
zone 3- and zone 4-occupying components (e.g., indoles,
phenyls, pyridines, etc.). Furthermore, future studies will also
be conducted to examine BAIPT-based SMNPI SNAP-25 protec-
tion and toxicity in neurons.
a Reagents and conditions: (a) Ammonium formate, 10% Pd-C, H2O,
90 ꢀC, 21 h, and then HCOOH. (b) DMAP, DMA, roomtemperature, 48h. (c)
N1-(7-chloroquinolin-4-yl)-ethane-1,2-diamine (to provide 10), N1-(7-chlo-
roquinolin-4-yl)-propane-1,3-diamine (to provide 11), N1-(7-chloroquino-
lin-4-yl)-butane-1,4-diamine (to provide 12), HOBt, EDCl, DMA, room
temperature, 24 h. (d) Ethylene diamine, NaSH, DMA, 120 ꢀC, 2 h. (e)
Methanesulfonic acid, CH3CN, 70 ꢀC, 0.5 h.
In summary, our gas-phase pharmacophore for BoNT/A LC
inhibition is constantly enabling the rational design of more
potent SMNPIs. In the current study, a refined four-zone
iteration of the model was generated via the 3D super-
imposition of two different SMNPI chemotypes. To empiri-
cally validate the refined model, three target compounds
based on the BAIPTchemotype “core” were synthesized and
examined in vitro. Of the three designs, all are more potent
than parent compound 2, while 4 is the most potent non-
Zn(II)-chelating, nonhydroxamic acid-containing, SMNPI of
the BoNT/A LC reported to date. Moreover, comparison of the
inhibitory potencies of 1a, 1b, and 4 indicates that a propyl
tether may be optimal for accessing pharmacophore zones 3
and 4 with an aromatic moiety. In general, the refined
pharmacophore model forms the basis for increasing the
activities of our non-Zn(II)-chelating SMNPI chemotypes.
Future four-zone pharmacophore-based designs will focus
on developing SAR to provide strategies for optimizing the
chemical and steric compositions of SMNPI components
populating zones 3 and 4 (e.g., the 4,7-ACQ components of
1a, 1b, and 3-5), as well as the flexibility/rigidity and
composition of the tethers linking zones 1 and 4 and zones
2 and 3 (e.g., the propyl linkers of 4).
to the terminal amide amines of the BAIPT chemotype
“core”.
Scheme 1 displays the general synthetic procedure used to
prepare 3-5. Generation of the target compounds began with
repeated attempts to reduce 3-cyano-5-nitrobenzoic acid (6) to
corresponding aniline 7 via catalytic hydrogenation; however,
substantial quantities of incomplete reduction products were
constantly observed. To circumvent this obstacle, hydrogen
transfer, rather than catalytic hydrogenation, was used to suc-
cessfully generate 7 in 77% yield after acidification with formic
acid and crystallization from water. Following, 7 and 8 were
coupled using a modified procedure reported by Sellarajah
et al.14 to afford key intermediate 9 in 92% yield after quench-
ing with water and crystallization from acetonitrile. Next, amide
bond formation between 9 and the corresponding aminoquino-
lines15 was performed at room temperature using HOBt and
EDCl in DMA solvent to provide dinitrile intermediates 10-12,
respectively. The transformation of 10-12 into corresponding
imidazoline targets 3-5 was achieved via reaction with ethyle-
nediamine and NaSH in DMA solvent at 120 ꢀC, employing a
modified procedure of Sun et al.16 Finally, the salts of 3-5 were
obtained by stirring with methanesulfonic acid in acetonitrile at
70 ꢀC. Detailed synthetic procedures and compound characteri-
zations are provided as Supporting Information.
The Ki values for 3-5 (Table 1) were determined using a
well-documented HPLC-based assay.17-22 The four-zone
pharmacophore-based designs were all more potent than 2
[Ki=8.52 μM ((0.53 μM)]. Specifically, 3-5 possess Ki values
of 2.12 ((0.26 μM), 0.572 ((0.041 μM), and 0.900 μM
((0.078 μM), respectively (Ta bl e 1). Furthermore, plots of
the inhibition kinetics data for 3-5 indicate that all are com-
petitive inhibitors (Supporting Information, Figures S1-S3),
and additional in vitro analyses of the SMNPIs in the presence
SUPPORTING INFORMATION AVAILABLE Descriptions
of molecular modeling techniques employed for pharmaco-
phore refinement, general chemistry methods, experimental
details for the syntheses and characterization of 3-5, 7, and
9-12, and inhibition assay conditions and Ki value determination.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondemce should
be addressed. (S.B.) Tel: 301-619-4246. Fax: 301-619-2348.
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E-mail: sina.bavari@us.army.mil. (J.C.B.) Tel: 804-225-0527.
Fax: 804-828-8556. E-mail: burnettjames@mail.nih.gov.
(10) Burnett, J. C.; Ruthel, G.; Stegmann, C. M.; Panchal, R. G.;
Nguyen, T. L.; Hermone, A. R.; Stafford, R. G.; Lane, D. J.;
Kenny, T. A.; McGrath, C. F.; Wipf, P.; Stahl, A. M.; Schmidt,
J. J.; Gussio, R.; Brunger, A. T.; Bavari, S. Inhibition of
Metalloprotease Botulinum Serotype A: From a Pseudo-Pep-
tide Binding Mode to a Small Molecule that is Active in
Primary Neurons. J. Biol. Chem. 2007, 282, 5004–5014.
(11) Burnett, J. C.; Wang, C.; Nuss, J. E.; Nguyen, T. L.; Hermone,
A. R.; Schmidt, J. J.; Gussio, R.; Wipf, P.; Bavari, S. Pharma-
cophore-Guided Lead Optimization: the Rational Design of a
Non-Zinc Coordinating, Sub-Micromolar Inhibitor of the
Botulinum Neurotoxin Serotype a Metalloprotease. Bioorg.
Med. Chem. Lett. 2009, 19, 5811–5813.
(12) Hermone, A. R.; Burnett, J. C.; Nuss, J. E.; Tressler, L. E.; Nguyen,
T. L.; Solaja, B. A.; Vennerstrom, J. L.; Schmidt, J. J.; Wipf, P.; Bavari,
S.; Gussio, R. Three-Dimensional Database Mining Identifies a
Unique Chemotype that Unites Structurally Diverse Botulinum
Neurotoxin Serotype A Inhibitors in a Three-Zone Pharmacophore.
ChemMedChem 2008, 3, 1905–1912.
(13) Burnett, J. C.; Opsenica, D.; Sriraghavan, K.; Panchal, R. G.;
Ruthel, G.; Hermone, A. R.; Nguyen, T. L.; Kenny, T. A.; Lane,
D. J.; McGrath, C. F.; Schmidt, J. J.; Vennerstrom, J. L.; Gussio,
R.; Solaja, B. A.; Bavari, S. A Refined Pharmacophore Iden-
tifies Potent 4-Amino-7-Chloroquinoline-Based Inhibitors
of the Botulinum Neurotoxin Serotype A Metalloprotease.
J. Med. Chem. 2007, 50, 2127–2136.
(14) Sellarajah, S.; Lekishvili, T.; Bowring, C.; Thompsett, A. R.;
Rudyk, H.; Birkett, C. R.; Brown, D. R.; Gilbert, I. H. Synthesis
of Analogues of Congo Red and Evaluation of Their Anti-Prion
Activity. J. Med. Chem. 2004, 47, 5515–5534.
Author Contributions: # The authors contributed equally to this
work.
Funding Sources: The presented research was supported by
Defense Threat Reduction Agency project 3.10084_09_RD_B and
also Agreement Y3CM 100505 (MRMC and NCI, National Institutes
of Health) for J.C.B. Furthermore, for J.C.B., in accordance with
SAIC-Frederick, Inc., contractual requirements, this project has
been funded in whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health, under Contract
HHSN261200800001E.
Notes: The authors report no conflicts of interest in this work. The
content of this publication does not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government or the U.S. Army.
ABBREVIATIONS 4,7-ACQ, 4-amino-7-chloroquinoline; 2,4,
4-APPIA, 2-[4-(4-amidinephenoxy)phenyl]indole-6-amidine;
BAIPT, bis-[3-amide-5-(imidazolino)phenyl]terephthalamide;
BoNT/A LC, botulinum neurotoxin serotype A light chain;
SMNPI, small molecule (nonpeptidic) inhibitor.
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