Probing the S2′ subsite of the anthrax toxin lethal factor using novel N-alkylated hydroxamates

Article abstract of DOI:10.1021/acs.jmedchem.5b01446

The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet av

Full text of DOI:10.1021/acs.jmedchem.5b01446

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Article
Probing the S2# Subsite of the Anthrax Toxin Lethal
Factor Using Novel N-Alkylated Hydroxamates
Elbek K. Kurbanov, Ting-Lan Chiu, Jonathan Solberg, Subhashree
Francis, Kimberly M Maize, Jenna Fernandez, Rodney L. Johnson, Jon E.
Hawkinson, Michael A. Walters, Barry C. Finzel, and Elizabeth Ambrose Amin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01446 • Publication Date (Web): 22 Oct 2015
Downloaded from http://pubs.acs.org on October 26, 2015
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Probing the S2′ Subsite of the Anthrax Toxin Lethal Factor Using Novel NꢀAlkylated
Hydroxamates
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Elbek K. Kurbanov†, TingꢀLan Chiu†, Jonathan Solberg‡, Subhashree Francis‡,
Kimberly M. Maize†, Jenna Fernandez†, Rodney L. Johnson†, Jon E. Hawkinson‡,
Michael A. Walters‡, Barry C. Finzel†, Elizabeth Ambrose Amin†§*
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†Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55414; ‡Institute for Therapeutics
Discovery and Development, University of Minnesota, Minneapolis, MN 55414; §Minnesota Supercomputing
Institute for Advanced Computational Research, University of Minnesota, Minneapolis, MN 55455, USA
ABSTRACT
The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is
chiefly responsible for anthraxꢀrelated cell death. Although many studies have been conducted
toward the design of smallꢀmolecule LF inhibitors, no LF inhibitor is yet available as a
therapeutic agent. Inhibitors with considerable chemical diversity have been developed and
investigated; however, the LF S2′ subsite has not yet been systematically explored as a potential
target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and
structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogs
specifically designed to extend into, and probe chemical preferences of, this S2′ subsite. We
discovered that this region accommodates a wide variety of chemical functionalities, and that a
broad selection of ligand structural modifications directed to this area can be incorporated
without significant deleterious alterations in biological activity. We also identified key residues
in this subsite that can potentially be targeted to improve inhibitor binding.
*To whom correspondence should be addressed: eamin@umn.edu
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INTRODUCTION
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The rodꢀshaped bacterium Bacillus anthracis, the causative agent of anthrax, was
disseminated in spore form via the US Postal Service in 2001, resulting in five American deaths.1
Anthrax continues to pose a significant threat to the public as a biological weapon, and effective
postexposure treatments remain elusive. The Centers for Disease Control (CDC) categorizes B.
anthracis as a Tier 1 Select Agent, a pathogen that poses great risk to national security,
economy, and critical infrastructure; this characterization is also due to the fact that the
extremely hardy anthrax spores can be weaponized to cause high mortality rates in inhalational
form.2 Notably, the former Soviet Union and Iraq are rumored to have built aerosolization
facilities for the largeꢀscale production of B. anthracis.3,4 The 1979 anthrax outbreak in
Sverdlovsk, in the former Soviet Union, has been attributed to the inhalation of anthrax spores
accidentally released at a military microbiological facility in that city.5
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The lethality of anthrax is largely due to its tripartite exotoxin, comprising lethal factor
(LF), a calmodulinꢀactivated adenylate cyclase, edema factor (EF), and protective antigen (PA).6
LF, a 89ꢀkDa zinc metalloprotease, is primarily responsible for anthrax pathogenesis, and binds
PA to form the anthrax lethal toxin which subsequently enters host target cells.7 Once in the
cytoplasm, LF cleaves mitogenꢀactivated protein kinase kinases (MAPKKs) MEK1, MEK2,
MKK3, MKK4, MKK6, and MKK7.8 This leads to the inhibition of MAPK phosphorylation,
thereby interfering with cellular immune defense mechanisms.7–12 In later stages of the disease,
the toxin invades endothelial cells, causing disruption of endothelial barriers and leakage of
vasculature, leading to hypovolemia, circulatory shock, and eventual host death.13–15 Antibiotics
such as fluoroquinolones are effective against B. anthracis, but they must be administered early
in the disease cycle due to rapid exotoxin secretion. Unfortunately, diagnosis of anthrax in its
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early stages poses major challenges, as symptoms of infection are often nonspecific.16 In later
stages of the disease course, antibiotics may yet clear the bacilli, but high levels of toxin can
remain systemically and cause fatal residual toxemia. The current arsenal of available antibiotics,
of course, has no effect on the LF toxin itself.
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Other treatment options include antibodyꢀbased therapeutics, which exhibit their own set
of limitations. Antibodyꢀbased treatments belong to a wellꢀestablished drug class that has a fairly
high success rate for clinical approval, and are usually well tolerated by humans, but key caveats
exist: they are very expensive due to the high cost of manufacturing and the often large doses
required; also, rare but serious adverse effects have been reported. Antibodies can display
significant pharmacokinetic liabilities, limited tissue accessibility, and impaired interactions with
the immune system.17,18 Consistent with antibodyꢀbased target restriction to those on the surface
or exterior of host cells, all antiꢀanthrax antibodies developed to date target the protective antigen
in order to interfere with LF translocation into host cells. One of the first fully human
monoclonal antibodies developed was MDXꢀ1303, which has now been incorporated into the
CDC Strategic National Stockpile (SNS). However, postꢀexposure studies performed on nonꢀ
human primates (NHPs) showed that MDXꢀ1303 could only achieve 70% efficacy.19 In 2012,
the Food and Drug Administration (FDA) approved raxibacumab in combination with antibiotics
for the treatment of inhalational anthrax. Raxibacumab is a recombinant human immunoglobulin
G1λ monoclonal antibody that prevents binding of PA to cell surfaces. Despite achieving FDA
approval, efficacy studies performed on Cynomolgus macaques and New Zealand white (NZW)
rabbits showed that raxibacumab is inferior to currently approved antimicrobials.20,21
More recently, the FDA approved Anthrax Immune Globulin Intravenous (Human), a
purified human immune globulin G in combination with antibiotics to treat patients with
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inhalational anthrax. Its mechanism of action is similar to that of raxibacumab. Efficacy studies
performed with Cynomolgus macaques and NZW rabbits demonstrated that even in combination
with antimicrobials, complete protection of animals from B. anthracis by Anthrax Immune
Globulin Intravenous (Human) remains challenging.22 Overall, for postꢀexposure anthrax
treatment, there is still a key unmet need for novel therapeutics that reliably and effectively
protect against the direct effects of the anthrax toxin itself. Given the critical role of LF in
anthrax pathogenesis and lethality, the discovery of strategies to inhibit this enzyme is still the
most promising approach to combat postꢀexposure anthrax.23,24
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The lethal factor consists of four domains: the Nꢀterminal domain (I), a large central
domain (II), a small helical domain (III), and the Cꢀterminal catalytic domain (IV).25 The Nꢀ
terminal domain (I) (residues 1ꢀ263), binds to PA and is responsible for enabling LF
translocation into host cells. The functions of domains II (residues 264ꢀ297 and 385ꢀ550) and III
(residues 303ꢀ382) are not fully understood; however, it is known that domain III plays an
important role in terms of LF selectivity for MAPKKs. The Cꢀterminal domain IV (residues 552ꢀ
776) contains the LF active site, which features a catalytic Zn2+ coordinated to three active site
residues: His686, His690, and Glu735. Residues His686 and His690 form part of the signature
HEXXH consensus motif that is characteristic of most matrix metalloproteinases (MMPs) as
well as of other related Znꢀbinding enzymes.25,26
Numerous studies have been conducted toward the design of small molecule LF
inhibitors.27–36 The first reported LF inhibitors were small peptide sequences, designed as mimics
of the natural MAPKK substrate, chemically linked to hydroxamic acid zincꢀbinding groups
(ZBGs).37–39 However, their development as therapeutic agents has been limited because of the
pharmacokinetic and toxicological liabilities of hydroxamates. Recent research has therefore
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focused on the development of nonꢀhydroxamateꢀbased LF inhibitors.24,28–32,40–51 Reported
inhibitors have included cationic polyamines,30 aminoglycosides,41 pyrazolones,31 polyphenols,47
tetracyclins,42 αꢀdefensins,48 quinolines,40 rhodanines,43 and catechols24 with most of these
compounds exhibiting lowꢀ to midꢀmicromolar activity against LF.
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The LF active site consists of three prominent subsites: a strongly hydrophobic and
sterically constrained S1′ subsite, a mostly hydrophobic but less sterically restricted, openꢀended
S1ꢀS2 region that forms a solventꢀexposed groove, and the less well characterized, flexible S2′
area (Figures 1a and 1b). The S1′ and S1ꢀS2 subsites have been previously explored and well
characterized in the literature.27,33 Our pharmacophore map assembly UM152 (Figure 2a)
reported earlier includes features representing interactions involving all three subsites of the LF
catalytic binding region, but due to the general dearth of inhibitors targeting the S2’ subsite, our
assembly only partly explores that area. Importantly, UM1 only incorporated features interacting
with Lys656 at the entrance of the S2′ subsite, and did not include any interactions located
beyond this entry region (Figure 2b). Therefore, although our pharmacophore hypothesis defined
part of the S2′ region, it remained to be established whether residues deeper within the S2′
subsite were capable of influencing LF inhibitory activities.
In order to more thoroughly explore this subꢀregion, we synthesized and experimentally
evaluated a series of novel LF inhibitors based on existing scaffolds, in order to explore deeper
regions of the S2′ subsite, and thereby identify residues in that area, if any, that play critical roles
in LF inhibition. Upon examining the published Xꢀray structure of the Merck sulfonamide
hydroxamate compound 13 (MKꢀ702/LF1ꢀB) (Figure 3) coꢀcrystallized with LF (PDB ID:
1YQY),27,50 it was noted that the S2′ subsite could be reached by means of substitutions
extending from the sulfonamide nitrogen. We therefore designed and synthesized a series of 13
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and related (compound 3r (MKꢀ31)27) derivatives functionalized at that nitrogen, and thereby
uncovered key design principles and pinpointed previously unidentified ligandꢀreceptor
interactions in the S2′ subsite. It is important to note that our goal in the work reported here was
not to design a new series of potential therapeutics per se, nor necessarily to increase compound
potency over the parent compound in this particular series. Our objective, rather, was to
construct a series of probe compounds based on a known LF inhibitor scaffold with well
characterized binding modes, in order to explore practical boundaries of this subsite and identify
preferred contact points for favorable intermolecular interactions. Such information could then
be applied to the design and optimization of novel LF inhibitors based on nonꢀhydroxamate or
other types of scaffolds.
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RESULTS
Structural Biology, Modeling, and Synthesis
Compound 3r (Table 1), an analog of 13 with the tetrahydropyranyl group replaced by a
methyl moiety, was selected as the scaffold for initial analog enumeration due to synthetic
tractability and functionalizability at the sulfonamide nitrogen. In order to confirm previously
reported inhibition by this compound and to affirm that its binding mode could be accurately
predicted, 3r was reꢀsynthesized and a crystal structure in complex with the Nꢀterminally
truncated LF enzyme was determined at 2.65 Å resolution by Xꢀray crystallography. Diffraction
statistics are summarized in Supporting Information. We found that this compound binds as
predicted from analysis of the 13 complex structure: the hydroxamate coordinates to the Zn2+
cation coꢀfactor, the fluoromethylbenzyl group directs into the S1' pocket between Leu677,
His686 and Tyr728, and the sulfonamide oxygen engages in Hꢀbonds with backbone amides of
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Lys656 and Gly657. Also as in the complex with 13, the sulfonamide NH makes a Hꢀbond with
the OH of Tyr728, and is in contact with an aspartic acid (Asp328) reaching across the inhibitor
binding groove from domain III (Figure 4a).
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To confirm that analogs of 3r alkylated at the sulfonamide nitrogen continue to inhibit
LF, compound 3a was prepared and characterized (Table 1). This analog, with only a methyl
group attached to the sulfonamide N, is expectedly less potent (LF IC50 = 37 µM) than the parent
compound, but crystallographic analysis confirms that its binding mode is similar to that of 3r
(Figure 4b). This loss in potency is attributable to the disruption of important hydrogen bonds
involving the sidechain OH of Tyr728, as well as to the nonꢀbonded strain introduced into the
system upon 3a binding. Specifically, removal of a hydrogen bond donor at the ligand
sulfonamide eliminates a key hydrogen bond to Tyr728, and the hydroxyl moiety moves
approximately 0.8 Å in response to the increased steric bulk of the methyl group. In the novel
analog, residue Tyr728 does preserve a hydrogen bond with the hydroxamate carbonyl, but an
additional hydrogen bond to Asp328 found in the 3r and 13 complexes is eliminated. This loss of
this Hꢀbond does not appear to be sufficient to induce domain III movement, as the complex with
3a remains in the tight conformation exhibited by Xꢀray structures of the 13 and 3r complexes
(see below). A sulfonamide methyl or methylene can fit within this severely constrained
environment, whereas bulkier branched substituents such as the isopropyl of 3q cannot fit, and
consequently retain no activity (Table 1).
Preliminary modeling based upon the 3r complex structure led to the synthesis and
evaluation of a series of compounds with a benzyl moiety substituting for the methyl of 3a as
described earlier.53 While these compounds resulted in only marginal increases in potency over
3a, crystal structures with five of these analogs revealed key structural information, namely, the
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extent to which LF is capable of dynamic adaptation in conformation in response to ligand
binding. These structures confirmed that the benzyl substituent occupies the S2′ site, but also
illustrate that movement of domain III can dramatically expand S2′. A second conformation
(which we call “open”) was observed with some inhibitors that exposes crucial residues such as
Asp328, Lys380, and His654 to ligand binding, offering multiple potential sites for favorable
chargeꢀcharge and Hꢀbonding interactions between ligand and protein. However, specific
identification of particular ligand features that influence conformational state was not possible
from the study of these compounds; therefore, a larger empirical exploration of more diverse
analogs was initiated.
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An exhaustive docking and scoring validation analysis was first conducted that
established Glide 5.954–57 as the most suitable virtual screening package for the LF system from
among a series of commercially available docking algorithms. We found that Glide 5.9
performed superior to SurflexꢀDock,58,59 Glide,54–57 AutoDock,60 and MOE61 (see Supporting
Information for data and analysis), as assessed by RMSD values between experimental and
docked ligand configurations. To select the most appropriate receptor structures for docking, we
crossꢀdocked the five coꢀcrystallized ligands into each of the five protein structures published in
Ref. 53. Structures 4PKS and 4PKV were selected for further docking studies since these
complexes exhibited the lowest ligand RMSD values, 0.91 Å and 0.93 Å respectively, between
experimental and predicted bound configurations. Glide 5.9 was therefore chosen to carry out all
subsequent docking experiments.
To begin the evaluation of new 3r analogs, an in silico library was designed from three
SigmaꢀAldrich structure sets of aliphatic bromides, benzyl bromides and aliphatic iodides.
Compounds with reactive moieties such as Michael acceptors, aldehydes, and cyanides were
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excluded from the set. Interactive enumeration in Maestro was used to computationally generate
90 synthetically tractable analogs of 3r. All 90 structures were docked using the Glide XP
protocol in the Schrödinger Maestro Discovery Suite 9.4 (Schrödinger, Inc.) into the PDB
structure 4PKR (obtained by us), the best representative LF complex adopting the open
conformation. Docking and scoring revealed that incorporating polar functionalities such as
amino, amido, or carboxyl groups into the Nꢀalkylating substituent allows for favorable chargeꢀ
charge interactions with the side chains of Lys380 and Asp328, as well as Hꢀbonding
interactions with the backbone of His654 and Asp328. We then proceeded to design and
synthesize an extended library of 3r analogs incorporating polar functionalities based on our
docking results (Table 1).
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Complexes with inhibitors 5a (5D1T) and 5c (5D1U) were also characterized
crystallographically by our team. These inhibitors do induce the open conformation of LF which,
as reported by us previously, is characterized by an extended distance between domain III and
the catalytic center, and by a larger hinge angle.53 We believe this can be attributed to the bulk of
the Nꢀalkylation that cannot be accommodated within the smaller confines of S2′ in the tight LF
conformation. In this previous work,53 a metaꢀsubstituted benzylated 3r derivative was reported
(PDB ID 4PKU), wherein the benzyl was found to adopt a unique orientation within the S2′
subsite, possibly due to the substitution pattern. However, in complex with 5a, it is evident that
metaꢀsubstitution is not sufficient to cause this change; in this structure, the standard position of
the S2′ component is observed. Importantly, structure 5D1U represents the only successfully LFꢀ
crystallized compound (5c) with an extended aliphatic chain. Note that while the Nꢀsubstituent of
this compound is only ordered through the first three carbons (see Supporting Information), this
moiety clearly occupies the S2′ subsite.
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Based on previous studies,53 it was expected that the stabilization of domain III in the
tight position would be advantageous for potent inhibitors, as this domain configuration is
observed both when no ligand is bound and when the potent inhibitors 13 or 3r (PDB ID: 4WF6)
are coꢀcrystallized with LF. However, three LF Xꢀray coꢀcrystal structures that we have just
deposited ꢀꢀ with 3a (5D1S), 5a (5D1T), and 5c (5D1U) ꢀꢀ demonstrate that this is not always the
case. Structures 5D1T, a complex with compound 5a, and 5D1U, a complex with compound 5c,
present with domain III in the open position, yet the coꢀcrystallized ligands are among the more
potent analogs tested. While the primary amine functionality is not well resolved in either of
these structures, it is clear that these ligands induce the open rather than the tight configuration of
the protein. The coꢀcrystallized compound in 4PKT, on the other hand, induces the tight position
without enhancement of activity, as opposed to the coꢀcrystallized compound in 4PKR which
induces the open position.53 From these studies, we have concluded that inhibitors are not
necessarily required to induce the tight conformation of LF in order to achieve favorable
potency; rather, the position of domain III is more closely correlated with the size and
substitution pattern at the sulfonamide N on the ligand. The open position is observed when a
large substitution or nonꢀpara benzylation is introduced, and the tight position is manifested
when the substitution is small or when benzylation is pꢀsubstituted (as in 4PKT and 4PKV), as
the latter is too large and inflexible to be accommodated by the open position of domain III.
Interestingly, with a pꢀsubstituted benzylation, the SꢀNꢀCαꢀCβ torsion angle is decreased,
allowing domain III to once again occupy the tight configuration.
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Chemistry
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Synthetic modifications to the 3r sulfonamide were accomplished from advanced
intermediate 1, the synthesis of which was previously reported by our laboratory.53 Intermediate
1 was alkylated using various bromides and K2CO3 in DMF to give 2aꢀ2p in 52%ꢀ88% yield
(Scheme 1a). Intermediate 2q was preferentially alkylated under Mitsunobu conditions
employing 2ꢀpropanol, PPh3, and DIAD, which increased the yield to 54% (from 17% with the
SN2 methodology). Esters 1 and 2aꢀ2q were converted to their corresponding hydroxamic acids
3aꢀ3r using hydroxylamine hydrochloride and NaOMe in MeOH in 27% – 87% yield (Scheme
1b). 3b was further hydrogenated to 4a using 10% wt. Pd/C in 43% yield. Compounds 3iꢀ3o
were deprotected using 4 N HCl in 1,4ꢀdioxane or a solution of TFA/DCM (1:2) to their
corresponding hydrochloride or trifluoroacetate salts 5aꢀ5g (Scheme 2). Acetylated analogues
7aꢀ7f were synthesized by means of a threeꢀstep procedure from 2hꢀ2m (Scheme 3). Initially,
Bocꢀprotected intermediates 2hꢀ2m were deprotected using 4 N HCl in 1,4ꢀdioxane, and the
resulting amines were acetylated with Ac2O, TEA, and DMAP to give 6aꢀ6f in 33%ꢀ95% yield.
Finally, the penultimate esters were converted to their corresponding hydroxamic acids using
hydroxylamine hydrochloride and NaOMe in MeOH as described above. Methyl amide 9 was
synthesized in parallel fashion over three steps (Scheme 4). Initially, tBuꢀester 2o was
deprotected with TFA/DCM (1:3). The resulting carboxylic acid was coupled with methylamine
hydrochloride using EDCI, HOBt, and NMM to yield methyl ester 8 in 71% yield. The desired
hydroxamate was accomplished using the previously described procedure in 30% yield.
Compound 12 was synthesized according to Scheme 5. The mꢀnitrobenzyl substituent was first
hydrogenated to the mꢀamine using 10% wt. Pd/C in MeOH in 77% yield. The free amine was
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acetylated with AcCl to yield 11, which was subsequently converted to hydroxamic acid 12
under the previously described conditions.
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Scheme 1. Synthesis of Compounds 3aꢀ3r.
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Reagents and Conditions: (a) RꢀBr, K2CO3, DMF, rt, 2 days; for 2q: 2ꢀpropanol, PPh3, DIAD,
THF (b) NH2OH·HCl, NaOMe, MeOH, 0 °C to rt, 16 h.
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Scheme 2. Synthesis of Compounds 5aꢀ5g.
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Reagents and Conditions: (a) 4 N HCl in dioxane, rt, 1 h; or TFA/DCM (1:2), TES, 0 °C.
Scheme 3. Synthesis of Compounds 7aꢀ7f.
Reagents and Conditions: (a) 4 N HCl in dioxane, rt, 1 h; (b) Ac2O, TEA, DMAP, THF, 0 °C, 16
h; (c) NH2OH·HCl, NaOMe, MeOH, 0 °C to rt, 16 h.
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Scheme 4. Synthesis of Compound 9.
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Reagents and Conditions: (a) TFA/DCM (1:3), rt, 2.5 h; (b) CH3NH2·HCl, NMM, HOBt, EDC,
THF, rt, 16 h; (c) NH2OH·HCl, NaOMe, MeOH, 0 °C to rt, 16 h.
Scheme 5. Synthesis of Compound 12.
Reagents and Conditions: (a) 10 wt. % Pd/C, MeOH, rt, 4h; (b) AcCl, TEA, DCM, 0 °C to rt, 16
h; (c) NH2OH·HCl, NaOMe, MeOH, 0 °C to rt, 16 h.
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In vitro Assessment of LF inhibition
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Two assays were used to measure the IC50 values of the synthesized compounds: a
quenched fluorescence resonance energy transfer (FRET) assay and a mobility shift assay
(MSA). Details are provided below. The FRET assay measures LF enzymatic activity using an
internally quenched peptide substrate derived from MAPKK. Briefly, LF cleavage separates the
Dnp quencher from the oABz fluorescent tag, leading to an increase in fluorescence intensity. In
order to address concerns about fluorescence interference, we employed a secondary, orthogonal
MSA assay, which uses a microfluidic chip to measure the conversion of fluorescent substrate to
fluorescent product. The terminated reaction mixture is introduced through a capillary sipper
onto the chip, where substrate and product are separated by electrophoresis and detected via
laserꢀinduced fluorescence.
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The MSA IC50 values of all synthesized analogs ranged from 1.1 ꢁM to 47 ꢁM, which are
consistently less favorable than the 0.5 ꢁM IC50 value demonstrated by the parent 3r (Table 1).
(Note that compounds 3b, 3i, 3k, 3l, 3m, 3n, and 3p are not included in Table 1 because FRET
and MSA triplicate data points unfortunately could not be obtained due to compound
degradation).
DISCUSSION
From previously published crystallographic data53 and the newer activity and structural
data reported here, we can now ascertain that the S2′ subsite of LF is able to accommodate many
diverse Nꢀalkylated analogs of 3r. In our previous as well as in our more recent structures, the
ligands align well and domain III is consistently observed to adopt the tight position.
Interestingly, another analog (3q) that incorporates an isopropyl modification is inactive, likely
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due to unavoidable steric clashes with Tyr728. Docking studies correctly predicted that 3q would
prove to be the least active of the 28 synthesized compounds listed in Table 1. In subsequent
data, we observed that this steric clash with Tyr728 could be relieved by substituting the
isopropyl group with an nꢀpropyl moiety, resulting in inhibitory activity of 6.4 ꢁM in the MSA.
The most active analogs from our new series incorporated primary amines into the Nꢀ
alkylated substituents. Our docking results shed further light on this, indicating that the improved
activity of primary amineꢀcontaining compounds may be due to additional interactions with the
3α1ꢀ3α2 loop in domain III that separates the S2′ subsite from the S4′, specifically involving
electrostatic interactions with the Asp328 sidechain, and/or Hꢀbond donating interactions with
the backbones of Asp325, Ser326 or Ser327. A notable exception is found in 5f, which when
docked into both crystal structures, adopts a completely different orientation in the active site
and was not predicted to engage in interactions with any of those four residues. We note that
analog 5f featured the shortest Nꢀalkyl substituent (a threeꢀcarbon propyl linker), a moiety that is
likely too short to reach that particular set of residues.
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Moreover, the amineꢀcontaining analogs 5aꢀ5f proved to be more active than their Bocꢀ
protected or acetylated counterparts 3h, 3j, and 7aꢀ7f. Docking studies revealed that upon Bocꢀ
protection or acetylation, analogs lose Hꢀbond donating interactions with the backbone of
Asp325, Ser236, and Ser327, and also electrostatic interactions with Asp328. However,
carboxylic acidꢀcontaining compound 5g exhibited a different pattern. The butyl protected
analog 3o and the amideꢀcontaining analog 9 were both more active against LF than 5g, which is
not surprising given that the carboxylic acid in 5g is unable to donate a hydrogen bond to the
3α1ꢀ3α2 loop. Also, hydrophobic compounds 3cꢀ3g do not engage in crucial proteinꢀligand
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electrostatic or Hꢀbond donating interactions with that loop, and are similarly less active than
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amines 5aꢀ5e.
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CONCLUSION
The S2′ binding site of the anthrax toxin LF is a large, dynamic solvent exposed channel
that changes configuration in response to various ligands, such as our sulfonamide hydroxamate
3r analogs alkylated at the sulfonamide moiety, binding via the movement of domain III. We
synthesized a series of these analogs to take advantage of the residues that were exposed due to
this conformational change, and to probe binding preferences at this subsite. Biological activity
and Xꢀray data indicate that the decrease in inhibitory activity exhibited by these new analogs
with respect to the parent compound likely results from the elimination of a key ligandꢀreceptor
Hꢀbond to Tyr728. We did, however, find that biological activity can partly be recovered by
installing amineꢀcontaining substituents on the Nꢀsulfonamide that are capable of interacting
with Asp325, Ser326 and Ser327. Possible steric clashes with Tyr728 decrease potency of
compounds dramatically; this should be taken into account during optimization of inhibitors with
groups that are oriented near this residue. Also, inhibitor design targeting the S2′ subsite can
result in novel scaffolds with a wide variety of substituents that do not induce a significant loss
in potency, for example, 1.1 ꢁM for 5c compared to 0.5 ꢁM for 3r. One could therefore take
advantage of this accommodating S2′ subsite to modify other scaffolds to improve druglike and
related properties.
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EXPERIMENTAL SECTION
Biochemical Evaluation
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Synthesized compounds were evaluated utilizing an in vitro fluorescence resonance
energy transfer (FRET) and mobility shift (MSA) assays to determine LF inhibition as follows:
Quenched FRET Lethal Factor Protease Assay. The quenched fluorescence resonance
energy transfer (FRET) assay measures LF enzymatic activity using an internally quenched
peptide substrate derived from MAPKK. LF cleavage separates the Dnp quencher from the oABz
fluorescent tag, leading to an increase in fluorescence intensity. Endpoint assays were conducted
following modified published procedures.30,50 In this assay, 10 µL of 100 nM anthrax toxin lethal
factor prepared by us as described previously51 (final concentration 50 nM) in 2 X assay buffer
(40 mM HEPES containing 0.02 % Triton Xꢀ100, pH 8.0) was added to 384ꢀwell assay plates
(Corning #3677) using a MultiDrop (ThermoꢀFisher). The plate was preꢀincubated at 37 °C for
15 min and the reaction was initiated by the addition of 10 µL of 14 µM oAbz/Dnpꢀsubstrate
(developed inꢀhouse at the Biomedical Genomics Center, University of Minnesota) in water
(final substrate concentration 7 µM). The reaction was allowed to continue for 5 minutes at 37
°C, and then terminated with addition of 5 µL 50 mM EDTA (final concentration 10 mM).
Fluorescence intensity was measured on a SpectraMax M2e microplate reader with excitation
and emission wavelengths of 320 nm and 420 nm, respectively. For IC50 determinations, test
compounds were dissolved in DMSO at 10 mM and varying volumes were added to the assay
plate using a Labcyte Echo® 550 acoustic dispenser prior to LF addition to achieve 8
concentrations in duplicate (final DMSO concentration 1 %). IC50 values were determined as a
percent of control wells containing no inhibitor (following subtraction of background wells
lacking enzyme) from at least 3 independent experiments using GraphPad Prism software. IC50
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values were determined for the positive control compounds 13 and 14 (GM6001)39 which were
included on every plate.
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Mobility Shift Protease Assay. The offꢀchip mobilityꢀshift assay uses a microfluidic chip
to measure the conversion of fluorescent substrate to fluorescent product using a Caliper LC3000
(PerkinElmer). The terminated reaction mixture is introduced through a capillary sipper onto the
chip where substrate and product are separated by electrophoresis and detected via laserꢀinduced
fluorescence. As in the FRET assay, 10 µL of 100 nM anthrax toxin lethal factor prepared by us
as described previously51 (final concentration 50 nM) in 2X assay buffer (40 mM HEPES
containing 0.02 % Triton Xꢀ100, pH 8.0) was added to 384ꢀwell assay plates (Corning #3677)
using a MultiDrop (ThermoꢀFisher). The plate was preꢀincubated at 37 °C for 15 min and the
reaction was initiated by the addition of 10 µL of 8 µM FITCꢀsubstrate (Celtek Peptides, #RKꢀ
10ꢀ4) in water (final substrate concentration 4 µM). The peptide sequence of the substrate is
identical to the FRET assay substrate. The reaction was allowed to continue for 10 minutes at 37
°C, and then terminated with addition of 4 µL 0.5 mM phenanthroline/32.5 µM EDTA solution
(final concentration of 0.1 mM phenanthroline /6.5 µM EDTA). Samples were then analyzed via
the LabChip 3000 software. For IC50 determinations, test compounds were dissolved in DMSO
at 10 mM and varying volumes were added to the assay plate using a Labcyte Echo® 550
acoustic dispenser prior to LF addition to achieve 8 concentrations in duplicate (final DMSO
concentration 1%). IC50 values were determined as a percent of control wells containing no
inhibitor (following subtraction of background wells lacking enzyme) from at least 3
independent experiments using GraphPad Prism software. As before, IC50 values were
determined for the positive control compounds 13 and 14 included on every plate.
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Importantly, a high correlation between the quenched FRET and mobility shift assays
was observed (see Supporting Information) indicating that both assays provide reliable inhibitory
potency data for these compounds. On average, the compounds were 3.9ꢀfold more potent in the
mobility shift assay relative to the quenched FRET assay. This apparent potency difference is
likely due to a 1.75ꢀfold higher substrate concentration in the FRET relative to the mobility shift
assay, and a higher substrate Km in the mobility shift assay relative to that in the FRET assay.
Although the amino acid sequence is identical in the substrates used in both assays, the Nꢀ
terminal and Cꢀterminal modifications are different, which may affect LF cleavage efficiency.
Unfortunately, it is not technically feasible to determine the substrate Km in the mobility shift
assay because signal linearity is lost at high concentrations of substrate. The higher substrate
concentration in the FRET assay coupled with a higher Km in the mobility shift assay would
decrease the apparent potency of these competitive inhibitors in the FRET assay.
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Structural Biology
Lethal factor protein (residues 265ꢀ776, A266S) was prepared and small molecule ligands
were coꢀcrystallized as reported by us.53 Diffraction data for structure 4WF6 (3r) were collected
at 100 K using a Saturn 944+ detector and a Rigaku Miromaxꢀ007FHM source inꢀhouse (at the
University of Minnesota). The data were processed using HKL2000.62 Diffraction data for
structures 5D1S (3a), 5D1T (5a), and 5D1U (5c) were collected at 100 K on beamline 17ꢀIDꢀB
(IMCAꢀCAT) using a Dectris PILATUS 6M pixelꢀarray detector at the Advanced Photon
Source, at Argonne National Laboratories in Argonne, IL. The data were processed using
autoPROC.63 The structures were solved using molecular replacement with atomic coordinates
from either 1YQY or 4PKR using Phaser64 in the CCP4 suite.65 Structural refinement was done
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using both Refmac566 and Phenix,67,68 while the Coot modeling software69 was used for
visualization and model building. Ligand omit maps are provided in Supporting Information.
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ANCILLARY INFORMATION
Supporting Information
Details of chemical synthesis, compound characterization data, docking and scoring
algorithm selection, Xꢀray crystal structure selection for docking and scoring, correlation data
between the quenched FRET and mobility shift assays, Xꢀray crystallographic omit maps, and a
table of SMILES strings (in CSV format) and IC50 values for each compound reported here.
Accession Codes
Coordinates of 4WF6, 5D1S, 5D1T, and 5D1U have been deposited in the Protein Data
Bank.
Corresponding Author
Elizabeth Ambrose Amin; Eꢀmail: eamin@umn.edu. Telephone: 612ꢀ626ꢀ2387. Fax: 612ꢀ626ꢀ
6346
Notes
The authors declare no competing financial interest.
Acknowledgments
This work was funded partly by NIH/NIAID R01 AI083234, the University of Minnesota
Office of the Vice President for Research (GrantꢀinꢀAid of Research, Artistry, and Scholarship),
and the University of Minnesota College of Pharmacy Grants Award Program, all to E.A.A.
Additional support was provided by the University of Minnesota Doctoral Dissertation
Fellowship, and the University of Minnesota College of Pharmacy Bighley Fellowship, both
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awarded to E.K.K., and the American Foundation for Pharmaceutical Education PreꢀDoctoral
Fellowship, awarded to K.M.M. The authors gratefully acknowledge the Minnesota
Supercomputing Institute for Advanced Computational Research (MSI), particularly Ms. Nancy
Rowe, for valuable assistance. The authors would also like to thank Teresa de la MoraꢀRey for
initial structural work. Use of the IMCAꢀCAT beamline 17ꢀID at the Advanced Photon Source
was supported by the companies of the Industrial Macromolecular Crystallography Association
through a contract with HauptmanꢀWoodward Medical Research Institute.
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Abbreviations Used
LF, Anthax Toxin Lethal Factor; CDC, Centers for Disease Control; FDA, Food and Drug
Administration; EF, Anthrax Toxin Edema Factor; PA, Anthrax Toxin Protective Antigen;
MAPKKs, mitogenꢀactivated protein kinase kinases; SNS, Strategic National Stockpile; NHPs,
nonꢀhuman primates; NZW, New Zealand white; MMPs, matrix metalloproteinases; ZBGs, zincꢀ
binding groups; MSA, mobility shift assay; Dnp, 2,4ꢀdinitrophenyl; oꢀAbz, oꢀaminobenzoic acid.
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