Anthrax lethal factor protease inhibitors: Synthesis, SAR, and structure-based 3D QSAR studies

Article abstract of DOI:10.1021/jm050892j

We have recently identified a series of compounds that efficiently inhibit anthrax lethal factor (LF) metallo-protease. Here we present further structure-activity relationship and CoMFA (comparative molecular field analysis) studies on newly derived inhibitors. The obtained 3D QSAR model was subsequently compared with the X-ray structure of the complex between LF and a representative compound. Our studies form the basis for the rational design of additional compounds with improved activity and selectivity.

Full text of DOI:10.1021/jm050892j

J. Med. Chem. 2006, 49, 27-30
27
Anthrax Lethal Factor Protease Inhibitors:
Synthesis, SAR, and Structure-Based 3D QSAR
Studies
Sherida L. Johnson,† Dawoon Jung,† Martino Forino,‡
Ya Chen, Arnold Satterthwait, Dmitry V. Rozanov,
Alex Y. Strongin, and Maurizio Pellecchia*
Cancer Research Center and Infectious and Inflammatory Disease
Center, Burnham Institute for Medical Research, 10901 North
Torrey Pines Road, La Jolla, California 92037
Figure 1. Detail of the X-ray structure of compound 1 in complex
with LF (PDB_ID 1ZXV). Side chains of Zn2+-coordinating amino
acids are displayed.
ReceiVed September 9, 2005
Abstract: We have recently identified a series of compounds that
efficiently inhibit anthrax lethal factor (LF) metallo-protease. Here we
present further structure-activity relationship and CoMFA (comparative
molecular field analysis) studies on newly derived inhibitors. The
obtained 3D QSAR model was subsequently compared with the X-ray
structure of the complex between LF and a representative compound.
Our studies form the basis for the rational design of additional
compounds with improved activity and selectivity.
multiple substitutions on the phenyl ring significantly increases
the inhibitory activity.12 Furthermore, details of the 3D structure
of the complex between LF and a representative compound, 1
(BI-MFM3), revealed that the rhodanine ring is capable of
interacting with the Zn2+ metal ion via the thiazolidinedione
sulfur atom (Figure 1).12
Anthrax is an infectious disease caused by the bacterium
Bacillus anthracis.1 This rod-shaped bacterium infects humans
through the respiratory system, skin, or digestive tract. Depend-
ent upon the entry route into the human body, anthrax can be
highly lethal. Although cutaneous anthrax is rarely lethal,
inhalation anthrax is dangerous and usually fatal.2 Upon
inhalation, the anthrax spores adhere to the alveolar macrophages
and germinate. Bacteria migrate to the lymph node, in which
they rapidly multiply and excrete a tripartite exotoxin comprised
In this work, we report on further synthesis and SAR studies
in which we explored the relative importance of various
chemical substructures of 1 in inhibiting the protease activity
of LF. In this respect, exploration of substituting the rhodanine
ring with thiazolidinedione, thiobarbituric acid, creatinine, and
creatinine acetic acid was investigated. In addition, we synthe-
sized a set of analogues in which we varied the nature of the
phenyl and furan rings, as well (Tables 1 and 2). The synthesis
of each compound was achieved in part as described in our
previous work11 by preparing the appropriate aldehyde deriva-
tives and by using a final condensation step using the Knoev-
enagel reaction.13 The latter was carried out either under reflux
in acetic acid or by using microwave-assisted conditions.14-16
The compounds were obtained with average yields ranging from
80% to 96%. The details of the experimental conditions are
reported as Supporting Information. Once the compounds were
synthesized and characterized, we then performed an enzymatic
assay to evaluate the inhibitory activity of the resulting
compounds against LF. A fluorescence peptide cleavage assay
(100 µL) was performed in a 96 well plate. Each reaction
consisted of MAPKKide (4 µM) and LF (50 nM) (Lists
Biological Laboratories) in 20 mM Hepes, pH 7.4, and the small-
molecule inhibitor. Kinetics of the peptide cleavage was
examined for 30 min by using a fluorescent plate reader at
excitation and emission wavelengths of 485 and 590 nm,
respectively, and IC50 values were obtained by dose response
measurements. For a number of compounds, Lineweaver-Burk
analysis was also carried out to verify that the compounds are
competitive against the substrate.12
of protective antigen (PA, 83 kDa), lethal factor (LF, Zn2+
-
metalloproteinase, 90 kDa), and calmodulin-activated edema
factor adenylate cyclase (EF, 89 kDa).3,4 The combined actions
of these proteins constitute the anthrax toxins (AT), which
induce cell death. Unless properly and promptly treated,
inhalation anthrax will lead to the death of the host organism.5
Initially, PA binds to an AT receptor on the host cell surface,
where it is cleaved by a furin-like protease to produce a 20
kDa N-terminal fragment (PA20) and a 63 kDa C-terminal
fragment (PA63).6 PA63, which remains bound to the membrane,
oligomerizes into a heptameric prepore capable of binding LF
and EF.7 Upon binding of LF and EF, the complex undergoes
receptor-mediated endocytosis, and the PA63 conformational
change allows the two enzymatic moieties LF and EF to
translocate into the cell cytosol. Once in the cytosol, LF is then
able to cleave several members of the MAPKK family near the
N-terminus.8-10 This cleavage prevents interaction with and
phosphorylation of downstream MAPK, thereby inhibiting one
or more signaling pathways through a mechanism not yet
understood.11
With the long term goal of developing novel potential
treatments for anthrax disease, we previously identified several
small molecule inhibitors that inhibit anthrax LF protease
activity with IC50’s in the submicromolar range.12 Cell-based
and peptide cleavage assays were subsequently used to confirm
the potency of the iterate leads. The most potent compounds
were subsequently tested in mice models of the disease showing
a protection against Bacillus anthracis spores, when used in
combination with the antibiotic ciproflaxin.12 Initial structure-
activity relationship (SAR) data suggested that the presence of
From the data reported in Tables 1 and 2, it appears clear
that substitutions of the rhodanine ring give the most dramatic
effects with a severe loss of activity when the ring is substituted
with a creatinine or creatinine acetic acid moiety. However,
substitution with a thiobarbituric acid ring is allowed. The furan
ring can also be substituted with thiophene or a thiazole ring
without a dramatic effect on the inhibitory affinity of the
resulting compounds, while a variety of substitutions on the
phenyl ring are very well tolerated.
To obtain further insights on the mechanism of action of our
compounds, we have recently obtained the X-ray high-resolution
structure for LF in complex with a representative compound,
112 (Figure 1). The data reported in Tables 1 and 2 and the
X-ray structure of the complex between compound 1 and LF
provided a platform that should enable us to identify the
* To whom correspondence should be addressed. Tel: (858) 646-3159.
Fax: (858) 713-9925. E-mail: mpellecchia@burnham.org.
† These authors contributed equally to this work.
‡ Present address: Dipartimento di Chimica delle Sostanze Naturali,
Facolta` di Farmacia, Universita` degli Studi “Federico II” di Napoli, Italy.
10.1021/jm050892j CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/01/2005

28 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Table 1. Inhibitory Activity and Training Set Data for QSARa
Letters
a ND (not determined) indicates compounds not included in the analysis.
chemical determinants for the activity of the compounds. Details
of the three-dimensional structure of the complex between LF
and 1 revealed that the rhodanine ring is able to interact with
the Zn2+ metal ion via the thiazolidine sulfur atom. It is
reasonable to predict that even small changes in this position
may largely affect activity. This is observed with closely related
compounds in which the rhodanine ring is substituted with a
thiazolidinedione ring (for example, 17 (BI-11D8) and 28 (BI-
11D9), Tables 1 and 2). Likewise, the activity of thiobarbiturate
derivatives could be attributed to the presence of the sulfur atom
that could presumably interact similarly with the metal ion.
Finally, in such a scenario, substitution of the rhodanine ring
tolerance of substitutions at this position (Tables 1 and 2).
Therefore, analysis of the X-ray structure of 1 in complex with
LF provides a qualitative interpretation of the structure-activity
relationship data reported in Tables 1 and 2. These studies should
enable us to design additional compounds with possibly
improved affinity, selectivity, and drug-likeness.
In this respect, having in hand the X-ray structure of a
representative compound gives us the possibility to establish
an alignment rule for the superposition of the diverse set of
derivatives to carry out a CoMFA (comparative molecular field
analysis) study.17 It has been shown18,19 that this combined
experimental and statistical approach is more robust then using
simple in silico docking strategies that are hindered by the lack
of suitable force fields and scoring functions especially when
the binding site contains metal ions.20 Docking simulations of
our novel inhibitors into the LF binding pocket were performed
using GOLD 2.221 and by using the GOLD fitness function.21
All torsion angles in each compound were allowed to rotate
freely, but the distance between the LF metal ion and the sulfur
atom in each inhibitor was constrained (2.5-3.0 Å). The starting
coordinates of the binding sites were taken from the X-ray
crystal structure from our previous work (PDB_ID 1ZXV). The
preparation and calculation of molecular coordinates of all
molecules and CoMFA studies were carried out using SYBYL7.0
(TRIPOS, St. Louis).22 The docked conformations of 17
compounds were used as a training set for the CoMFA study
with a creatinine moiety is predicted to abolish the Zn2+
-
chelating ability of the compounds, with concomitant loss of
activity, as indeed observed (Table 1). The carboxylic group of
1 is pointing toward a hydrophilic region of the protein close
to its surface (Figure 1), which explains the variability of the
substitutions allowed at this position and the increased affinity
of the compounds with a small charged group (Tables 1 and
2). In addition, hydrophobic interactions between the phenyl
ring and hydrophobic side chains of LF were also observed.
However, electron density of the benzene ring is less evident
in the X-ray structure of 112 indicating a possible conformational
mobility around the carbon-carbon bond of the para-substituted
benzene ring and the larger available space around this portion
of the ligand. These observations correlate with the higher

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 29
Table 2. Inhibitory Activity and Test Set Data for the 3D QSAR
Studies
Figure 2. Superimpositions of docked conformers used for CoMFA
studies. In panel A, the structures of the compounds for the training
set are displayed, the compound highlighted in green being compound
1 (whose coordinates are from the PDB_ID 1ZXV). Panel B shows
calculated versus observed pIC50 values against LF for the compounds
in the training set (q2 ) 0.51, r2 ) 0.98, no. of components ) 4, no.
of compounds ) 17). In panel C, the aligned structures for the
compounds in the test sets are displayed. Panel D shows predicted
versus observed pIC50 values against LF for the 10 compounds in the
test set.
(Table 1, Figure 2A), while the docked structures for 10
additional compounds were used as a test set (Table 2, Figure
2C). However, inhibitors with IC50 values equal to or greater
than 100 µM and purity lower than 75% (see Supporting
Information) were not included in the CoMFA. Partial charges
for the protein (LF) were assigned from the AMBER02 force
field,23 and atomic charges for the 27 inhibitors were calculated
using PM3 (MOPAC6.0).24 The inhibition constants were
expressed in pIC50 values (pIC50 ) -log[IC50]) and correlated
with the steric and electrostatic fields (CoMFA), as well as the
total molecular surface area (TMSA) of each compound. The
cross-validation with leave-one-out option and the SAMPLS
program,25 rather than column filtering, was carried out to obtain
the optimal number of components to be used in the final
analysis. After the optimal number of components (four) was
determined, a non-cross-validated analysis was performed
without column filtering. The q2 (cross-validated r2 of 0.51),
SPRESS (cross-validated standard error of prediction of 0.60),
r2 (non-cross-validated r2 of 0.98, Figure 2B), and F values
(145.94) were computed according to the definitions in SYBYL.
The relative contributions to this CoMFA model were 40.9%
for the steric field, 38.5% for electrostatic field, and 20.6% for
total molecular surface area (TMSA). To evaluate the predictive
ability of this model, we subsequently calculated the pIC50
values for the 10 compounds in the test set (Figure 2D, Table
2). As it can be seen in Figure 2D, the model exhibits a
remarkably good predictive ability (r2 ) 0.83). The comparison
Figure 3. Comparison of (A) hydrophobic and hydrophilic potential
molecular surface (MOLCAD)26 of the substrate binding site of LF in
complex with compound 8 with (B) CoMFA contour plots of steric
field contributions and comparison of (C) the electrostatic potential
molecular surfaces (MOLCAD) with (D) the CoMFA contour plots of
electrostatic field contributions. In panel A), the hydrophobic and
hydrophilic areas are displayed in brown and blue, respectively, while
green surfaces represent an intermediate hydrophobicity. In panel B,
green contours indicate the regions where the addition of bulky groups
may increase activity and yellow contours indicate the regions where
the addition of bulky groups may decrease activity. In panel C, positive
and negative areas are displayed in red and blue, respectively, while
cyan surfaces represent neutral areas. The color code follows the
definitions of MOLCAD.26 In panel D, blue contours indicate regions
where less electronegative groups may increase activity and red contours
indicate regions where more electronegative groups may increase
activity.
between the CoMFA contours and the docking site for the
compounds is reported in Figure 3, which displays our most
active compound, 8 (BI-11B3). To evaluate whether the results
are biased toward the selected training and test sets, we have

30 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
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Acknowledgment. This work was supported in part by NIH
Grant 1UO1 AI056385.
Supporting Information Available: Procedures for the syn-
thesis of the reported compounds and analytical data and Figures
S1 and S2 and Table S1 related to additional CoMFA and CoMSIA
studies. This material is available free of charge via the Internet at
http://pubs.acs.org.
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