Mechanistic differences between in vitro assays for hydrazone-based small molecule inhibitors of anthrax lethal factor

Article abstract of DOI:10.1016/j.bioorg.2006.07.004

A systematically generated series of hydrazones were analyzed as potential inhibitors of anthrax lethal factor. The hydrazones were screened using one UV-based and two fluorescence-based in vitro assays. The study identified several inhibitors with IC₅₀ values in the micromolar range, and importantly, significant differences in the types of inhibition were observed with the different assays.

Full text of DOI:10.1016/j.bioorg.2006.07.004

Bioorganic Chemistry 35 (2007) 50–58
www.elsevier.com/locate/bioorg
Mechanistic differences between in vitro assays
for hydrazone-based small molecule inhibitors
of anthrax lethal factor
*
M. Leslie Hanna, Theodore M. Tarasow, Julie Perkins
Chemistry and Materials Science Directorate, Lawrence Livermore National Laboratory,
7000 East Avenue, Livermore, CA 94551, USA
Received 27 June 2006
Available online 1 September 2006
Abstract
A systematically generated series of hydrazones were analyzed as potential inhibitors of anthrax
lethal factor. The hydrazones were screened using one UV-based and two fluorescence-based in vitro
assays. The study identified several inhibitors with IC50 values in the micromolar range, and impor-
tantly, significant differences in the types of inhibition were observed with the different assays.
Ó 2006 Elsevier Inc. All rights reserved.
Keywords: Anthrax; Lethal factor; Inhibitor; Hydrazone
1. Introduction
Inhalation of Bacillus anthracis spores is often fatal if not appropriately treated in a
timely fashion. Inhaled spores in the lung alveoli are phagocytosed by alveolar macro-
phages and transported to the lymph nodes, where the spores germinate and multiply.
The bacteria release a toxin that kills host macrophages, disabling the host immune system
and thereby allowing the bacteria to escape the lymphonode defense barrier to reach the
blood system causing bacteraemia and toxaemia, which rapidly kills the host [1,2].
*
Corresponding author. Fax: +1 925 422 3570.
E-mail address: perkins14@llnl.gov (J. Perkins).
0045-2068/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.07.004

M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
51
Antibiotic treatment can be effective following low dose exposure. However, there is no
effective method to treat an established infection, as the toxin continues to damage the
host even after the bacteria may have been killed by antibiotic treatment. The release of
B. anthracis spores as an act of terrorism necessitates the search for effective treatments
to inhibit the action of the toxin, which could be used alone or in combination with anti-
biotics to treat high dose exposure or established infections.
Anthrax toxin consists of three proteins: protective antigen (PA) [3,4], edema factor
(EF) [5] and lethal factor (LF). PA is responsible for binding to cell surface receptors
where it is proteolitically cleaved to produce a 20 kDa fragment and a 63 kDa fragment
[6,7]. Following cleavage, the 63 kDa fragment heptamarises to form a prepore that, after
binding either LF or EF, is transported into the cell by receptor-mediated endocytosis [8].
EF is a calmodulin-dependant adenylyl cyclase that can elevate intracellular cAMP to
pathological levels [5,9]. LF is a zinc-dependant metalloprotease that cleaves mitogen-ac-
tivated protein kinase kinases (MAPKK) at their amino termini [10–12]. Cleavage disrupts
the ability of these kinases to interact with and phosphorylate downstream substrates,
which leads finally to macrophage lysis via a mechanism not entirely understood to
date.[13] As outlined previously, the inhibition of LF proteolytic activity is a promising
method for treating exposure to B. anthracis spores [14] and inhibitors may also act as
the molecular recognition unit in an anthrax toxin detection assay.
Assays based on UV, fluorescence and mass spectrometry detection of cleavage prod-
ucts are commonly used for the analysis of proteolytic activity and the discovery of pro-
tease inhibitors. Several groups have reported the use of in vitro protease assays to identify
LF inhibitors and in some cases, effective inhibitors were further analyzed in cell-based
assays [15–24]. In an elegant study to demonstrate the use of self-assembled monolayer
mass spectrometry screening for protease inhibitors, Mrksich and coworkers [25] identified
a hydrazone-based small molecule as an effective non-competitive inhibitor of LF.
In this study, the hydrazone bond was used as a foundation to generate a series of
libraries to investigate the structure-activity relationships of hydrazone-based inhibitors
and LF using a UV-based in vitro assay. The most effective hydrazone-based LF inhibitors
were further evaluated using two fluorescence-based in vitro assays and the mechanism of
the inhibitors were compared over all experiments. Inhibition mechanism differences were
observed between the assay that most accurately reflects the native peptide bond hydroly-
sis and the two assays that utilize unnatural peptide bond cleavage to release optical
reporter groups. The differences demonstrate that caution should be taken when interpret-
ing inhibition data based upon unnatural protease substrates and helps to identify the site
of inhibition of the hydrazone-based LF inhibitors.
2. Materials and methods
2.1. Hydrazone formation
Component aldehydes and hydrazides were purchased from Sigma, Aldrich, Avacodo,
Alfa or TCI and used as received. Concentrated solutions of the aldehydes and hydrazides
were prepared in DMSO, and in most cases, were diluted to 20 mM. Some molecules were
restricted in amount available or solubility and these were prepared at lower concentra-
tions. The aldehyde and hydrazide solutions were mixed in 1:1 ratios (except with bis-hy-
drazide h, where a 2:1 ratio of aldehyde: hydrazide was used) and agitated in the dark

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M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
overnight in the presence of 0.1% formic acid. The resultant hydrazones were subsequently
screened against LF following dilution in DMSO to the appropriate concentration. The
hydrazone formation was confirmed with numerous aldehyde/hydrazide combinations
in d6-DMSO using Nuclear Magnetic Resonance spectroscopy.
2.2. Control experiments
A representative number of aldehydes and hydrazides were screened in a similar man-
ner to the hydrazones to confirm that hydrazone formation was required for inhibition.
No inhibition was observed with the representative component aldehydes and hydrazides.
2.3. p-Nitroaniline (p-NA) release UV assay [25]
The substrate, Ac-NleKKKKVLP-pNA, was synthesized as described previously [25].
0.05–0.2 lg of LF was pre-incubated for 5 min with 0–10 lM inhibitor in 25 mM HEPES,
pH 7.0 containing 10 mM NaCl, 5 mM MgCl2, 50 lM ZnCl2, 50 lM CaCl2 and 0.5%
DMSO at room temperature. Aliquots (97.5 lL) were added to duplicate wells of a 96 well
plate containing 2.5 lL of 10 mM substrate, Ac-NleKKKKVLP-pNA (final concentration
0.25 mM), in the same buffer. The release of p-NA was monitored at 385 nm at 1–2 min
intervals for 20 min (using a Bio-Tek Synergy HT plate reader) and quantified using a
p-NA standard.
2.4. Coumarin release fluorescent assay
AcGYbARRRRRRRRVLR-7-AMC was commercially synthesized as described previ-
ously [22]. Assays were carried out as described previously, [22], except using HEPES buff-
er (same as in MAPKKide experiments) in place of sodium phosphate for continuity in the
experimental procedures. 100 lM inhibitor was used in each experiment to achieve >50%
inhibition. The probe has an excitation wavelength of 360 nm and emission wavelength of
460 nm. Significant interference from the hydrazone inhibitors in the fluorescence of the
released coumarin was observed. This background was subtracted by running a non-enzy-
matic control experiment for each hydrazone analyzed.
2.5. MAPKKide assay
These assays were carried out as described by Pellecchia and coworkers [23] using
100 lM inhibitor to achieve >50% inhibition. MAPKKide peptide substrate (DAB-
CYL/FITC) has an excitation wavelength of 485 nm and an emission wavelength of
590 nm, was obtained from List Biological Laboratories and used as received.
3. Results and discussion
The structure of the hydrazone LF inhibitor identified by Mrksich and coworkers [25] is
shown in Fig. 1. To carry out a structure–activity study to identify more potent hydrazone
inhibitors and map the non-covalent interactions between enzyme and inhibitor, hydraz-
ones formed from all possible combinations of a broad range of component aldehydes and
hydrazides were screened using a p-NA release, UV-based assay. The structures of the

M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
53
Fig. 1. Structure of LF hydrazone-based inhibitor identified by Mrksich and coworkers (1a in Table 1).
aldehydes and hydrazides used in this initial screen are shown in Fig. 2 and include the
components of the hydrazone in Fig. 1—2-hydroxy-5-nitro-benzaldehyde and 3,4-dihy-
droxy-benzhydrazide.
Only hydrazones with a 2-hydroxy-5-nitro-benzaldehyde component showed any signif-
icant inhibitory effect. Therefore, a further series of hydrazones formed using aldehydes dif-
ferentially substituted only in the 2 and/or 5 positions were analyzed. These aldehydes are
listed in the supplementary material. All inhibitors identified in the second screen consistent-
ly had a 2-position substitution of the aldehyde component, and the substituent was often an
OH group, which may participate in coordination to the zinc ion, creating an inhibitory
effect. This second screen yielded more inhibitors and therefore, this larger library of 2-
substituted, and in some cases also 5-substituted, aldehydes were used as the basis to screen
a broader range of component hydrazides (shown in supplementary material).
The components of all the hydrazones that showed an inhibitory effect in the p-NA UV
assay (including the lead candidate hydrazone in Fig. 1) are shown in Table 1. Subsequent-
ly, these lead hydrazone inhibitors were further analyzed using a fluorescence-based assay
Fig. 2. Component (a) aldehydes and (b) hydrazides of hydrazone inhibitors examined during an initial broad
screen using the p-NA UV-based assay.

Table 1
LF inhibition
1–8; aldehydes and a–j; hydrazides used to construct hydrazone LF inhibitors. + ,p-NA assay (10 lM inhibitor); o, AcGYbARRRRRRRRVLR-7-AMC assay (100 lM
inhibitor); x, MAPKKide assay (100 lM inhibitor). Three symbols <50% activity remaining; two symbols 50–75% activity remaining; one symbol 75–80% activity
remaining. No symbols, no significant inhibitory effect observed. The inhibitory effects of the highlighted inhibitors 1a, 2h, 3h and 7h were assessed more thoroughly.

M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
55
previously described by Montecucco and coworkers [22]. In this case, many of the most
potent inhibitors contained component aldehyde 1 or aldehyde 8, and hydrazide a or
hydrazide h.
Finally, the hydrazones in Table 1 were further analyzed using the commercially avail-
able MAPKKide FRET-based assay. Similarly to the coumarin assay, the MAPKKide
assay identified strongest inhibitor candidates composed of aldehyde 1, hydrazide a
(although a little weaker) and hydrazide h. The inclusion of hydrazide h gave inhibitors
of a long, narrow, hydrophobic nature, and this is consistent with the observations of Pan-
chal et al., [21] where they observed their hydrophobic inhibitors lie over the catalytic zinc
binding site, blocking the activity of the protease. The MAPKKide assay uses a peptide
Table 2
Four MAPKK residues flanking the LF cleavage site [12,26]
Cleavage
P4
P3
P2
P1
P10
P20
P30
P40
MKK-1
MKK-2
MKK-3
MKK-4
MKK-4
MKK-6
MKK-7
MKK-7
K
P
K
K
S
P
P
H
P
T
L
L
L
A
L
L
L
P
P
I
A
I
L
F
I
L
L
Q
L
S
N
T
P
P
P
L
T
C
F
L
K
L
S
N
I
M
A
N
E
V
D
A
T
G
T
R
K
R
K
Q
G
A
T
M
Cleavage occurs between P and P0 residues.
Table 3
IC50 values and structures of hydrazone inhibitors highlighted in Table 1
Assay
IC50 (lM)
Hydrazone inhibitor
1a
2h
3h
7h
p-NA
Coumarin
MAPPKide
8
150
200 18
0.2
5
85
90
80
3
3
7
45
50
50
1
2
5
40
70
50
1
2
5
Data is an average of two repeats.

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M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
substrate containing a natural cleavage site for LF with natural amino acid residues flank-
ing the scissile bond, and consequently interacts with the entire LF active site [26]. Table 2
illustrates representative examples of amino acids located on either side of the LF cleavage
site of natural MAPKK substrates, where cleavage is between the P1 and P10 sites [12,26].
a
b
c
Fig. 3. Lineweaver–Burke plots of inhibitor 2h for each assay. (a) p-NA assay — 2h at 10 lM (b) Coumarin assay
— 2h at 100 lM (c) MAPKKide assay — 2h at 100 lM. Data is an average of two repeats.

M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
57
In contrast, the p-NA- and coumarin-based assays employ substrates that only contain
amino acid residues in the P sites. It is unclear how the unnatural optical tag interacts with
the active site, although given the nature of the substrate and the active site, the tag is
likely to at least partially inhabit the P10 site. Cleavage at the terminus of the peptide sub-
strate (P1 site) releases the unnatural UV-active or fluorescent molecule. Based on these
substrate differences, any active site inhibitor in the FRET assay would result in compet-
itive inhibition, whereas in the p-NA and coumarin assays, active site inhibitors could dis-
play competitive (binding in the P sites) or non-competitive (binding in the P0 sites)
inhibition depending on what portion of the LF active site they interact with.
Table 3 shows the IC50 and the hydrazone structures of the selection of the inhibitors
highlighted in Table 1. In this case, 0.5 lg of LF enzyme was used to obtain these values.
The inhibitors identified in this study do not show a significantly enhanced inhibitory effect
over the original lead candidate 1a (8 lM in this study). When the p-NA assay was repeat-
ed at lower enzyme concentration (0.17 lg), an IC50 of 2 lM for 1a was obtained, consis-
tent with previously reported results.[25] A representative example (inhibitor 2h) of
Lineweaver–Burke plots for the p-NA and coumarin assays (Fig. 3a and b respectively)
shows the inhibition mechanism is not distinct. The lines clearly intersect to the left of
the y-axis but not on the x-axis, suggesting a mixed mode of inhibition. However, the
inhibitors exhibit competitive inhibition with the MAPKKide assay as shown by a repre-
sentative example (inhibitor 2h) of a Lineweaver–Burke plot in Fig. 3c. These differences
suggest the hydrazone inhibitors are binding in the P0 sites since occupation of these sites
would directly compete for active site binding with the MAPKKide substrate, but not nec-
essarily the p-NA and coumarin substrates. The p-NA and coumarin leaving groups may
interact with the P0 sites to some extent, causing the mixed mode of inhibition to be
observed. For future work, these results indicate that more potent inhibitors may be dis-
covered by extending the hydrazone inhibitors to bind to the P sites.
These experiments highlight the quite different results that can be obtained when using
different in vitro assays to identify protease inhibitors. Importantly, biochemical investiga-
tions using different substrates can yield information on inhibition mechanisms and iden-
tify sites of inhibitor–enzyme interaction.
4. Conclusions
In summary, three in vitro assays were used to analyze a series of hydrazones for their
inhibitory effects on the proteolytic action of anthrax LF. A number of inhibitors were
identified, although not with significantly enhanced inhibitory effect over the initial lead
compound. However, notable differences in the types of inhibition observed using the dif-
ferent assays identified the likely binding site of the inhibitors, and these observations will
be taken into account in future protease inhibitor assay development.
Acknowledgments
This work was carried out under the auspices of the US Department of Energy by the
University of California, Lawrence Livermore National Laboratory under Contract W-
7405-Eng-48 with financial support from the Laboratory Directed Research and Develop-
ment fund. The authors thank Sandra L. McCutchen-Maloney and Todd H. Corzett
(LLNL) for access and assistance with the UV/fluorescence plate reader. The authors

58
M.L. Hanna et al. / Bioorganic Chemistry 35 (2007) 50–58
thank Erica L. Gjersing and Julie L. Herberg (LLNL) for assistance in acquiring NMR
spectra.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
doi:10.1016/j.bioorg.2006.07.004.
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