Discovery and structure-activity relationship analysis of Staphylococcus aureus sortase A inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.067

Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using high-throughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure-activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC₅₀ values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.

Full text of DOI:10.1016/j.bmc.2009.08.067

Bioorganic & Medicinal Chemistry 17 (2009) 7174–7185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure–activity relationship analysis of Staphylococcus aureus
sortase A inhibitors
Nuttee Suree a,b, Sung Wook Yi a, William Thieu a, Melanie Marohn a, Robert Damoiseaux c, Albert Chan a,b
,
a,b,d,
Michael E. Jung a, , Robert T. Clubb
*
*
a Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1570, United States
b Molecular Biology Institute, University of California, Los Angeles, CA 90095-1570, United States
c Molecular Screening Shared Resource, University of California, Los Angeles, CA 90095-1570, United States
d UCLA-Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095-1570, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing
need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-
infective agents as this enzyme attaches virulence factors to the cell wall. Using high-throughput screen-
ing, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure–
activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione
analogs that inhibit SrtA with IC50 values in the sub-micromolar range. Many of these molecules also
inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase
inhibitors.
Received 27 May 2009
Revised 22 August 2009
Accepted 29 August 2009
Available online 6 September 2009
Keywords:
Antibiotic
Staphylococcus aureus
Sortase
Ó 2009 Elsevier Ltd. All rights reserved.
SrtA
Inhibitor
1. Introduction
reaction may be powerful anti-infective agents as srtAꢀ strains of
S. aureus fail to display many virulence factors and exhibit reduced
The rise of community- and hospital-acquired methicillin resis-
tant Staphylococcus aureus (MRSA) is a major health problem that
has created a pressing need for new antibiotics.1 More than
90,000 Americans acquire potentially deadly MRSA infections each
year, which annually are estimated to kill more people than AIDS
in the United States.2 Proteins displayed on the surface of S. aureus
play key roles in the infection process as they promote bacterial
adhesion to host cells and tissue, acquire essential nutrients, and
circumvent the immune response.3 Most surface proteins in S. aur-
eus are attached to the cell wall by the sortase A (SrtA) enzyme.4–8
SrtA is located on the extracellular surface and catalyzes a
transpeptidation reaction that joins an LPXTG sorting signal within
the surface protein precursor to the cell wall precursor molecule
virulence.14–24 SrtA inhibitors may also be useful in treating infec-
tions caused by other Gram-positive pathogens, since many also
use related enzymes to attach virulence factors to the cell wall
and to assemble pili that promote bacterial adhesion.25,26 Sortases
can be classified into five distinct families based on their primary
sequence.27 Enzymes most closely related to the S. aureus SrtA pro-
tein appear to be the best candidates for inhibitor development as
their elimination in other bacterial pathogens attenuates virulence
(e.g., Listeria monocytogenes, Streptococcus pyogenes, and Strepto-
coccus pneumoniae (reviewed in Refs. 28,29)). Finally, SrtA is not re-
quired for the growth of S. aureus in cell cultures. Therefore, anti-
infective agents that work by inhibiting SrtA could have a distinct
advantage over conventional antibiotics as they may be less likely
to induce selective pressure that leads to drug resistance.7,30
A number of different strategies have been employed to search
for sortase inhibitors (reviewed in Refs. 28,29,31). These include
screening natural products32–38 and small compound libraries,39
as well as synthesizing rationally designed peptidomimetics and
small molecules.40–44 Recently, mechanism-based aryl (b-ami-
no)ethyl ketone (AAEK) inhibitors have been reported.39 AAEK
molecules are specifically activated by sortase via a b-elimination
reaction that generates an olefin intermediate that covalently
modifies the active site cysteine thiol group.39 However, these
lipid-II [undecaprenyl-pyrophosphate-MurNAc(-
L-Ala-D-iGln-L-Lys
(NH2–Gly5)- -Ala-
D
D
-Ala)-b1–4-GlcNAc)].7–10 The lipid-II linked
protein product is then incorporated into the cell wall by the trans-
glycosylation and transpeptidation reactions of cell wall synthe-
sis.11–13 Small molecules that inhibit the SrtA transpeptidation
* Corresponding authors. Tel.: +1 310 825 7954; fax: +1 310 206 3722 (M.E.J.);
tel.: +1 310 206 2334; fax: +1 310 206 4779 (R.T.C.).
E-mail addresses: jung@chem.ucla.edu (M.E. Jung), rclubb@mbi.ucla.edu
(R.T. Clubb).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.067

N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
7175
compounds only inhibit SrtA with an IC50 of ꢁ5–50
l
M.39 Other re-
fluorescent substrate analog (o-aminobenzoyl (Abz)-LPETG-diami-
nopropionic acid-dinitrophenyl-NH2 (Dap(Dnp)). The assay was
miniaturized to enable its use in high-throughput screening
(HTS). A typical progress curve is shown in Figure 1A. The calcu-
lated Z0 score (a statistical measure of the assay’s robustness) is
0.75, which indicates that the assay can be effectively used for
screening.45 The DiverSet library (ChemBridge Corp.) was screened
for inhibitors of SrtA (see Section 4). Two criteria were used to cal-
culate the inhibition percentage (% inhibition) of each compound in
ported compounds also need to be optimized further to be thera-
peutically useful as they either have limited potency, undesirable
physicochemical features (e.g., high molecular weights) or inacti-
vate the enzyme slowly.28,29,39
To identify potent inhibitors of SrtA we performed high-
throughput screening (HTS) of a ꢁ30,000 compound library, which
led to the identification of three promising small molecule inhibi-
tors that can potentially be developed into anti-infective agents. A
structure–activity relationship (SAR) analysis revealed several
pyridazinone and pyrazolethione analogs that inhibit SrtA with
IC50 values in the sub-micromolar. These compounds are more po-
tent than any previously described natural or synthetic inhibitor,
and thus are excellent molecules for further development.
the library: (1) the initial velocity (mi) of product formation calcu-
lated from reaction progress curves, and (2) an end-point determi-
nation of product formation obtained by measuring the total
product fluorescence five hours after initiating the reaction. Com-
pounds in the library were first ranked by their end-point readings.
This revealed a Gaussian distribution (Fig. 1B), such that molecules
that exhibit >55% enzyme inhibition can be considered as hits with
a 99.7% confidence limit (their % inhibition value is at least three
standard deviation units above the mean).46 A total of 288 com-
pounds met this criterion. The number of potential inhibitors
was then further reduced by selecting only those molecules for
which >80% inhibition was observed in the end-point analysis, as
2. Results
2.1. High-throughput screening identifies several SrtA
inhibitors
In order to screen for small molecule inhibitors of SrtA we mod-
ified a fluorescence resonance energy transfer (FRET) assay that
monitors the SrtA-catalyzed hydrolysis of an internally quenched
well as statistically significant inhibition when their mi values were
considered (the mi value was less than or equal to 0 based on a 10
min progress curve). This reduced the total number of compounds
Figure 1. (A) FRET assay for measuring SrtA enzymatic activity. Three progress curves are overlaid and correspond to inhibitors with different potencies. (B) Histogram
showing the distribution of 30,000 compounds in the ChemBridge library as a function of% inhibition of SrtA determined by an end-point analysis during the high-throughput
screening campaign. (C) Venn diagram showing how the initial velocity (
mi) and end-point analyses were used to identify 44 inhibitors of S. aureus SrtA. Lead compounds 1–3
were selected from these inhibitors and have the best physicochemical and inhibitory properties. The number of compounds in each population is shown in parentheses.
O
OH
HS
O
H
N
N
Br
N
N
N
N
Cl
S
NO2
S
O
S
NO2
1 (IC50 = 3.7 μM)
2 (IC50 = 4.5 μM)
3 (IC50 = 5.2 μM)
Figure 2. Structures of the SrtA inhibitors identified by high-throughput screening. The IC50 value against S. aureus SrtA of each compound is indicated.

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N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
to 44 (Fig. 1C). Their inhibitory activity was then confirmed by
manually repeating the FRET assay and they were ranked based
on their % inhibition as determined by the end-point analysis. From
this set, 10 compounds were selected for further study because
they had the highest inhibitory activity and because they had
physicochemical properties similar to known drugs.47–52 For these
inhibitors, the concentration that is required to reduce the activity
of SrtA by 50% (IC50) was determined using well established meth-
ods.34,46,53 The most potent SrtA inhibitors from this group are
shown in Figure 2 (compounds 1–3) and were chosen for further
study.
purchased closely related analogs of the lead compounds from
the ChemBridge Corp. and determined their IC50 values against S.
aureus SrtA. The analogs were identified through search of the
company’s database and share 75–95% similarity (based on the
chemical functionality and scaffolding as determined by the com-
pany’s similarity search engine) with one of the three lead com-
pounds. A total of 7, 9, and 21 analogs of lead compounds 1, 2,
and 3 were purchased and tested, respectively. This work enabled
regions of the chemical scaffold required for inhibition to be coar-
sely defined. Analogs of the rhodanine 1 and pyridazinone 2 were
then synthesized to make more subtle changes to discover mole-
cules with even higher potency or better physicochemical proper-
ties. Eight analogs of 1 (compounds 1-8 to 1-13) and a total of 41
analogs of 2 were produced and tested (compounds 2-10 to 2-
50). Tables 1–3 show the structures of all of the compounds that
were tested and their IC50 numbers. To gain insights into their
selectivity, for several of the compounds we also measured their
IC50 values against the Bacillus anthracis sortase enzyme (BaSrtA).
A discussion of this data is presented below.
2.2. Analysis of the reversibility of inhibition of SrtA
For the three lead molecules, the reversibility of enzyme inhibi-
tion was determined by measuring the enzymatic activity of each
enzyme–inhibitor complex immediately after it was rapidly di-
luted.46 In this study SrtA was first incubated with saturating con-
centrations of each compound (inhibitor concentrations 10-fold
higher than the IC50 value). The SrtA–inhibitor complexes were
then rapidly diluted and the enzyme activity immediately mea-
sured (data not shown). Inhibition by compound 1 is rapidly
reversible as 84% of the enzyme activity is recovered after dilution.
Compounds 2 and 3 also reversibly inhibit the enzyme, but more
slowly; 50% and 58% activity is regained immediately after dilu-
tion, respectively. Mass spectrometry was also employed to con-
firm that the molecules form a reversible complex with the
enzyme (described in Section 4). In this study, the mass spectrum
of each saturated SrtA–inhibitor complex was recorded 1, 48, or
96 h after forming the complex. Mass spectra of these enzyme–
inhibitor complexes showed no difference from the negative con-
trol (SrtA alone), suggesting that the inhibitors do not stably mod-
ify the enzyme (data not shown). Detailed studies on inhibitory
reversibility of the lead compounds and their derivatives are also
being conducted in our laboratory and will be reported elsewhere.
2.3.1. Synthesis and SAR of the rhodanine compounds (series 1)
Two scaffolds of the rhodanine compounds were examined by
SAR (Table 1). Compounds with scaffold A were purchased from
ChemBridge Corp. (1 to 1-8), while compounds with scaffold B
were synthesized in our laboratory (1-8 to 1-13). The synthesis
of these compounds followed literature precedence, namely reac-
tion of the N-alkyl isothiocyanate with methyl thioglycolate gave
the 3-alkyl-4-oxothiazolidine-2-thiones. Condensation of these
with the 5-arylfurfuraldehydes gave the compounds 1-8 to 1-13
in good yields.54,55 In scaffold A, replacing the 2,4-dimethyl groups
on the R2 position reduces the potency three–fivefold (compound 1
vs 1-1, 1-2, 1-3, 1-7). On the other hand, relocating the 2-OH group
on the R3 position reduces the potency by 10-fold (compound 1 vs
1-4). These data suggest that these functional groups play a critical
role in enzyme binding, presumably through hydrophobic interac-
tion via the 2,4-dimethyl groups on the R2 position, and hydrogen
bonding via the 2-OH group at the R3 position. The SAR results for
compounds with scaffold B are in general agreement with this
interpretation. Although these molecules retain the central rhoda-
nine nucleus, they differ in the R1 group and replace the R3 group
2.3. Structure–activity relationship (SAR) analysis
An SAR analysis of the three lead compounds was performed to
identify related molecules with increased potency. Initially, we
Table 1
SrtA inhibition of the rhodanine lead compound (series 1) and its derivatives from ChemBridge (scaffold A) as well as synthesized derivatives (scaffold B)
R1
O
O
R2
R4
R1
O
N
N
R3
S
S
S
S
A
B
Compound
Scaffold
R1
R2
R3
R4
IC50 (lM)
SA SrtA
BA SrtA
1 (lead)
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
A
A
A
A
A
A
A
A
B
B
B
B
B
B
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–Me
–Me
–Pr
2,4-Me2
3-Cl
3-Me
4-NO2
2,4-Me2
3-Cl
3-Br, 2-OH, 5-NO2
3-Br, 2-OH, 5-NO2
3-Br, 2-OH, 5-NO2
3-Br, 2-OH, 5-NO2
3-Br, 4-OH, 5-NO2
4-Me, 3-NO2
3.7 0.1
17
15
12
6
4
3
20 1.6
35 11
>1000
119 30
2,4-Me2
3-NO2
3-Br, 2-OH, 5-NO2
14
4
13 1.8
53 34
–H
–H
–H
3-Cl
2-NO2
–H
405 69
186 22
492 129
109 10
104 10
199 23
1-9
1-10
1-11
1-12
1-13
–CH2Ph
–Et
–Et
74 58
–Allyl
27
9

N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
7177
Table 2
SrtA inhibition of the pyridazinone lead compound (series 2) and its derivatives from ChemBridge (compounds 2-1 to 2-9) as well as additionally synthesized derivatives
R1
(compounds 2-10 to 2-48)
N
N
R2
R4
O
R3
Compound
R1
R2
R3
R4
IC50 (l
M)a
SA SrtA
BA SrtA
2 (lead)
–SH
-OMe
–Ph
3-Cl
4.5 0.3
ChemBridge
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
–SH
–OEt
–OH
–OH
–Cl
–SH
–OCH2Ph
–OMe
–SEt
–Ph
–Ph
–Et
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–H
–H
—
–H
–H
–H
–H
–H
–H
0.20 0.06
>50b
>50
>50
9.3 0.6
>50
>50
>50
1.4 0.7
1.4 0.5
–SMe
–SMe
–SMe
–OMe
–OH
–OH
–OH
–SH
1.8 0.4
>50
>50
>50
0.3 0.1
2-9
–SEt
Ethoxy-thiol
2-10
2-11
2-12
2-13
–SH
–SH
–SH
–SH
–SH
–SH
–SH
–SH
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–SH
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–Cyclohexyl
–Ph
–Ph
–H
4-NO2
3-Br
3-F
3-Me
3,5-Cl2
13 1
3.2 1.7
6.7 0.6
30
3
>50
5.5 1.3
3.3 0.7
301 72
17.9 1.6
1.5 0.4
4.4 1.8
5.7 1.0
3.1 0.7
166 32
1.8 0.3
1.7 0.4
2-14
2-15
14
4
2-16
1.4 0.3
1.2 0.4
1.2 0.5
0.9 0.2
0.4 0.1
5.2 0.9
2-17c
2-18
–H
–H
3-F
3-Me
3,5-Cl2
2-19
2-20
2-21
–SH
–SH
–SH
–Ph
–Ph
–Ph
Methoxy-chloro
2-22
2-23
2-24
2-25
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–OMe
–OMe
–OMe
–OMe
–OMe
–OMe
–Ph
–Ph
–Ph
–Ph
–Ph
–Cyclohexyl
–H
3-Br
3-F
3-Me
3,5-Cl2
>50
>50
>50
>50
>50
>50
2-26
2-27
Ethoxy-chloro
2-28
2-29
2-30
2-31
2-32
2-33
2-34
2-35
2-36
2-37
2-38
2-39
2-40
2-41
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–OEt
–Cl
–Cl
–Cl
–Cl
–Cl
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–Cyclohexyl
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–H
4-NO2
3-Br
3-F
3-Me
3,5-Cl2
>50
>50
>50
>50
>50
>50
>50
1.0 0.3
219 74
>50
>50
>50
–H
4-NO2
3-Br
3-F
3-Me
3,5-Cl2
0.3 0.2
247 45
–Cl
–Cl
>50
>50
–Cyclohexyl
Dichloro
2-42
2-43
2-44
2-45
2-46
2-47
2-48
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Ph
–Ph
–Ph
–Ph
–Ph
–Ph
–Cyclohexyl
–H
4-NO2
3-Br
3-F
3-Me
3,5-Cl2
>50
>50
>50
>50
>50
61
>50
5
14
4
The compounds have been segregated into four subclasses.
Or Kai pp for values that are lower than 7.5
l
M as determined by the Morrison’s equation.
M of inhibitor concentration.
Compound 2-17 is a disulfide dimer of 2-10.
a
b
c
Inhibitory effect less than 50% at 100
l
with a much larger 5-phenyl furan moiety. Similar to the results
obtained for the scaffold A molecules, these variations result in
molecules with significantly elevated IC50 values. The most
dramatic difference can be seen by comparing compounds 1 and
1-10. Even though they are closely related on one side of the rho-
danine ring (Ph vs CH2Ph on the R1 position), the other side is sub-
stantially different as compound 1-10 does not have the
aforementioned 2-OH group. Taken together, none of the analogs

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N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
Table 3
was heated at reflux for 10 h using a Dean–Stark to afford the ana-
log 2-43 in 76% yield for the two steps. The regioselectivity of the
addition of oxygen nucleophiles to 2-42 to 2-48 was dependent on
the conditions: use of 1,4-dioxane as the solvent, with sodium eth-
oxide or methoxide, afforded cleanly the 4-alkoxy products 2-22 to
2-34 (83–95% yield) while the use of sodium hydroxide in ethanol
afforded cleanly the 5-ethoxy analogs 2-35 to 2-41 (75–94% yield).
The assignment of the regiochemistry of the products was based on
the observation of a strong NOE enhancement of the methylene of
the ethyl signal in the 5-ethoxy compounds with the C5 vinyl
hydrogen, an NOE which was absent from the 4-alkoxy com-
pounds. The displacement of the remaining chloride atom in either
the 4- or 5-alkoxy compounds was uneventful although we found
that the reaction worked best in DMF as solvent. In this way the
analogs 2-10 to 2-16 and 2-18 to 2-21 were formed. The symmet-
rical disulfide dimer, 2-17, could be formed by direct air oxidation
of the thiol 2-10. The other disulfides were prepared by the reac-
tion of the thiol 2-10 with methyl methanethiosulfonate (MMTS)
or Aldrichthiol (2-pyridyldisulfide) to give 2-49 and 2-50 in yields
of 88% and 65%, respectively. Finally the symmetrical disulfide 2-
17 could also be prepared in 85% yield by reaction of the thiol 2-
10 with the pyridyl disulfide 2-50.
SrtA inhibition of the pyrazolethione lead compound (series 3) and its derivatives
from ChemBridge
R4
H
N
N
N
R1
R2
X
R3
Compound
X
R1
R2
IC50
(l
M)a
BA SrtA
SA SrtA
3 (lead)
3-1
3-2
3-3
3-4
3-5
3-6
3-7
3-8
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
4-NO2
4-Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Me
4-Cl
5.2 0.1
39 3.8
6.8 0.3
8.9 0.3
9.6 1.3
14 1.1
68 12
52 9.6
42 8.4
2,4-(NO2)2
2-Br,4-NO2
2-OH,4-NO2
2-OH,5-NO2
2,4-Me2
3,4-Me2
4-I
4-N@N–Ph
2-Cl
2-OH
3-9
9
2
1.4 0.2
3-10
3-11
3-12
3-13
3-14
3-15
3-16
54 16
22
0.30 0.04 1.7 0.2
56 0.2
62
48 29
45 10
6
2,4,6-Br3
4-NO2
4-NO2
Substituents on the pyridazinone ring (R1 and R2) were sus-
pected to contribute greatly to the inhibitory activity, as replacing
the –SH with –OH at the R1 position dramatically reduces potency
(2 vs 2-7). Minor alteration of R2 (from –OMe to –OEt) and removal
of 3-Cl on the phenyl ring (R4) also increase the potency more than
20-fold (compare 2 with 2-1). These observations suggest that the
functional groups located on the pyridazinone ring may be as crit-
ical as those located on the phenyl ring. Therefore, we synthesized
analogs with different substituents on the pyridazinone ring to
optimize their potency further. Based on the substituent, these
compounds are segregated into 4 subclasses: ethoxy-thiol (2-10
to 2-21); methoxy-chloro (2-22 to 2-27); ethoxy-chloro (2-28 to
2-41); and dichloro (2-42 to 2-48) pyridazinone compounds. Addi-
tionally, we also varied the R3 and R4 positions of each subclass in
order to probe the importance of the phenyl ring. With the excep-
tion of compound 2-35, members of the ethoxy-thiol subclass are
the most potent molecules. Within this series, switching the rela-
tive positioning of the R1 and R2 groups does not dramatically af-
fect activity (compare 2-10 with 2-18, or 2-13 with 2-19, or 2-14
with 2-20). In contrast, varying the phenyl ring causes substantial
changes in potency, with the lowest IC50 obtained when all substit-
uents are eliminated or when only small substituents are present.
Interestingly, replacing entire phenyl ring with a cyclohexyl group
did not profoundly alter activity (2-10 vs 2-16). This suggests that
this portion of the ethoxy-thiol molecules may form non-specific
hydrophobic interactions with the enzyme, which can be disrupted
with groups larger than a phenyl or cyclohexyl ring are present.
Because the ethoxy-thiol compounds all contain a thiol group
that could potentially interact with the active site cysteine thiol
of SrtA (residue Cys184) we created a series of molecules that
are disulfide variants (compounds 2-17 in Table 2, and 2-49, 2-
50 in Fig. 3). Compound 2-17 is the symmetrical disulfide dimer
of 2-10 and exhibits an approximately twofold increase in its po-
tency. Interestingly, asymmetrical disulfide derivatives of 2-10 that
contain methyl (2-49) or pyridyl (2-50) groups are even more po-
9
4-NO2
2-Me,4-NO2 4-Cl
X
R3
3-17
3-18
S
S
0.76 0.03 1.4 0.3
115 16
N
–Cyclohexyl
R1
R4
O
N
N
3-19
3-20
3-21
4-NO2
17
26
51
2
4
6
O
N
O
4-COMe
4-NO2
NH
N
O
or Kai pp for values that are lower than 7.5
equation.
l
M as determined by the Morrison’s
a
of compound 1 showed improved activity against SrtA and were
not pursued further.
2.3.2. Synthesis and SAR of the pyridazinone compounds
(series 2)
Initial SAR studies of lead compound 2 made use of derivatives
purchased from ChemBridge (compounds 2-1 to 2-9) (Table 2).
This work revealed one of the most potent inhibitors of SrtA, com-
pound 2-1 Kai pp = 0.20 M, where Kai pp is the apparent dissociation
l
constant for the enzyme–inhibitor complex, as determined by
the Morrison’s equation)46 and its close analog 2-9 (Kai pp = 1.4
tent and exhibit Kai pp values of ꢁ0.4 and 0.03
lM, respectively. In
lM). This discovery led us to investigate variants of these com-
this series the pyridyl thiol is the best potential leaving group as
it can be transformed into a stabilized pyridine-2-thione. As this
derivative is the most potent inhibitor, this data suggest that these
molecules may inhibit the enzyme through a thiol–disulfide ex-
change reaction involving Cys184. However, the mechanism of
inhibition by these molecules remains unclear as compound 2
reversibly inhibits SrtA and does not modify the enzyme based
on mass spectrometry data (described above). Although the
pounds by synthesizing several analogs (2-10 to 2-50). These com-
pounds were prepared by an adaptation of the literature route,56
namely heating a mixture of an arylhydrazine, mucochloric acid,
and dilute HCl afforded the 2-aryl-4,5-dichloropyridazin-3-ones
2-42 to 2-48 in good yields (85–95%). The less reactive 4-nitro-
phenyl-hydrazine required more forcing conditions, namely a tol-
uene solution of the initial formed hydrazone cyclization toluene

N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
7179
and lipophilic tribromophenyl substituent. Replacing the thione
group with a ketone is detrimental (compare 3 with 3-13), while
changing substituents on the R2 phenyl ring does not significantly
restore potency (3-13 vs 3-14, 3-15, 3-16). We also examined the
effect of varying the phenyl ring attached via the amide (R3 and
R4). These results are obvious; replacement of the phenyl group
(R3) with a more electron-withdrawing pyridyl group enhances
the potency (compare 3 with 3-17), while a normal cyclohexyl
group dramatically reduces the potency (3-18). Variation of the
R4 group moderately influences inhibitory activity (3-19 to 3-21)
with the reduction in potency by a factor of 3–10 compared to
the lead, suggesting inhibition may prefer the pyrazolethione nu-
cleus and the phenyl ring on the nitrogen.
S
S
S
N
N
N
S
N
N
O
O
O
O
2-49
Kiapp = 0.4 0.2 μM
2-50
Kiapp = 0.03 0.06 μM
Figure 3. Additional asymmetric disulfide derivatives synthesized for the pyridaz-
inone series containing thiomethyl (2-49) or 2-thiopyridyl (2-50) groups. IC50
values against S. aureus SrtA are indicated.
ethoxy-thiol subclass contains several potent SrtA inhibitors, 2-35
within the ethoxy-chloro subclass is nearly as potent with an IC50
2.4. The pyrazolethione and pyridazinone compounds also
inhibit BaSrtA and minimally affect S. aureus growth
value of ꢁ1
lM. This molecule possesses a unique combination of
substituents on the pyridazinone ring as it has –OEt and –Cl groups
on the R1 and R2 position, respectively. Interestingly, the SAR inhib-
itory trend observed in the ethoxy-chloro and ethoxy-thiol sub-
classes differ markedly as variations at the R1 and R2 sites in the
ethoxy-chloro subclass result in large reductions in potency that
are not observed when similar modifications are made in the eth-
oxy-thiol subclass. This suggests that compound 2-35 may have a
different inhibitory mechanism from the ethoxy-thiol subclass.
The binding mode of each molecule was explored further using
docking calculations and is discussed later in the text.
In cell culture, srtAꢀ strains of S. aureus show no defects in their
growth. This suggests that highly selective SrtA inhibitors will
function as anti-infective agents that only prevent the bacterium
from thriving within the human host, but otherwise do not impair
growth outside of the host. SrtA inhibitors may therefore have
advantages over conventional antibiotics that generate selective
pressures that lead to their obsolescence. Using a microtiter broth
dilution method57 for lead compounds 1–3, we determined the
minimal inhibitory concentration (MIC) of each molecule that
prevented S. aureus growth. This work revealed that lead com-
pounds 2 and 3 only minimally impair bacterial growth as they
have MIC values >1 mM. In contrast, the rhodanine lead compound
2.3.3. SAR of the pyrazolethione compounds (series 3)
A series of pyrazolethione analogs of the lead compound 3 were
obtained from ChemBridge through a similarity search. Inhibitory
activities against SrtA were evaluated and are shown in Table 3.
Initially, substituents on the R1 ring were varied while we kept
the thione group on the pyrazole nucleus constant (compounds 3
to 3-12). This led to the discovery of the most potent compound
1 has an MIC value of ꢁ10
lM, suggesting that it inactivates other
reactions essential for bacterial viability. This finding is compatible
with recent studies that have shown that rhodanine compounds
inhibit class C b-lactamases in Gram-negative bacteria.58 Several
arylalkylidene rhodanines have also been reported that have high
bactericidal activity against non-resistant S. aureus and MRSA
in the 3-series, 3-12 (Kai pp = 0.3
lM). This molecule contains a bulky
Figure 4. Inhibition of S. aureus cell growth by the lead compounds and several potent inhibitor compounds identified in the SAR studies. Growth inhibition was measured
using the microtiter broth dilution method. In this procedure 180 L of the cell culture was plated into a 96-well plate and 20 L of inhibitor solution was added to a final
concentration of 500 M. Growth was then monitored overnight at 37 °C using a temperature-controlled plate reader. The% growth inhibition is relative to cultures grown in
the absence of inhibitor. Error bars are the standard deviation from three measurements.
l
l
l

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N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
strains. These compounds exhibit MIC values lower than ampicillin
and cefotaxime and it has been proposed that they non-competi-
tively inhibit penicillin-binding proteins.59
The finding that compounds 2 and 3 do not affect bacterial
growth is fortuitous, as nearly all of the potent SrtA inhibitors we
identified in the SAR analysis are analogs of these molecules. In or-
der to more rapidly ascertain SrtA inhibitory effects on microbial
growth. The most promising candidates for further development
are 2-9 and 2-20 as they inhibit SrtA with low micromolar IC50 val-
ues and do not significantly inhibit S. aureus growth in cell culture.
The ability of several of the compounds to inhibit the sortase A
protein from B. anthracis (BaSrtA) was tested to gain insights in their
selectivity. This enzyme shares 27% amino acid sequence identity
with S. aureus SrtA and also attaches proteins to the cell wall that
contain an LPXTG sorting signal.60 In addition, BasrtA-knockout
strains show defects in their ability to escape macrophages, suggest-
ing that BaSrtA may be useful in treating anthrax.14 IC50 measure-
ments against BaSrtA were made for the most potent S. aureus SrtA
inhibitors. For the series 2 molecules, the S. aureus SrtA and BaSrtA
enzymes show similar trends in their susceptibility. For example,
molecules that poorly inhibit S. aureus SrtA also are ineffective
against BaSrtA (compounds 2-6 to 2-8), while potent S. aureus SrtA
inhibitors also effectively inhibit BaSrtA. Interestingly, compounds
2-9 and 2-20, which significantly impair S. aureus SrtA activity and
are not bactericidal (Fig. 4), are even more potent BaSrtA inhibitors
growth, we grew S. aureus cultures in the presence of 500 lM of
each inhibitor and compared the rate of growth with control cul-
tures grown in 2.5% DMSO (the solvent used to solubilize the inhib-
itors). This method enables an estimate of MIC to be obtained as
molecules that do not affect bacterial growth can be assumed to
have MIC values >1 mM. Consistent with the MIC data, compound
1 is toxic, while compounds 2 and 3 only modestly perturb growth
(Fig. 4). An analysis of the growth data suggests that series 3 mol-
ecules are very promising anti-infective agents as four of its mole-
cules inhibit SrtA with an IC50 or Kapp <5
lM, but otherwise do not
i
substantially affect bacterial growth (compounds 3-1, 3-9, 3-12,
and 3-17). Interestingly, the most potent SrtA inhibitor (compound
3-12) shows no detrimental effect to bacterial viability, highlight-
ing its potential for further development as an anti-infective agent.
Compounds in the 2-series show a variation of effects on S. aureus
with Kai pp values of ꢁ0.3 and 0.4
lM, respectively. The most potent
non-bacteriocidal 3-series compounds, 3-9 and 3-12, are also prom-
ising, as they inhibit BaSrtA with Kai pp values of 1.4 and 1.7
respectively. Combined these data suggest that the mechanism of
lM,
Figure 5. Image showing the SrtA–inhibitor complexes generated by Induced-Fit Docking. Dock poses with the highest rank (lowest IFD score value) are shown. Compounds
1 (A), 2 (B), 2-1 (C), 2-35 (D), 3 (E), and 3-12 (F) were docked into the structure of S. aureus SrtA derived from the solution structure of the covalent complex between SrtA and
the LPAT sorting signal analog.64 Ligand structures are shown in a ‘ball and stick’ format. The solvent accessible surface of SrtA is shown and colored to indicate the
electrostatic properties from acidic (red) to basic (blue). The secondary structure of the protein is shown behind the surface and the important neighboring amino acids are
68
labeled. The figures were created using the program PYMOL
.

N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
7181
enzyme inhibition by compounds 2-9, 2-20, 3-9, and 3-12 is con-
served across species, and that they are unlikely to significantly alter
microbial processes other than surface protein display.
However, replacing the substituent with 2,4-Me2 or 3,4-Me2 re-
duced potency, suggesting shape complementarity may be critical
for binding. The docking solutions also suggest why the pyrazole
nucleus may be specific to the sortase active site as its methyl
and thione groups contact two highly conserved residues, Ala92
and Arg197, respectively (Fig. 5F). This feature, along with their
hydrophobic network, may be the reason why most of the com-
pounds within this series exhibit high potency against SrtA en-
zymes, but little or no bactericidal activity.
2.5. Biostructural analysis
To gain insight into the mode of binding of the SrtA inhibitors,
we modeled how they interacted with the S. aureus SrtA enzyme
using an Induced-Fit Docking (IFD) protocol (Schrödinger
Inc.).61–63 Compounds were docked into the recently determined
solution structure of SrtA bound to a LPAT peptide.64 After removal
of the peptide coordinates the remaining protein structure was pre-
pared for docking using the Protein Preparation Wizard, and LigPrep
was used to prepare the ligand compounds.63 The inhibitors were
then docked into the SrtA receptor using a standard IFD workflow.
Models of the SrtA–inhibitor complexes with the lowest negative
IFD value were chosen to represent the final docking solution. When
docked into the active site of SrtA, compound 1 inserts its hydro-
phobic moiety into the lipophilic pocket generated by the side
chains of Ile199 in strand b8 and residues Val166 to Val168 in the
adjacent b6/b7 loop (Fig. 5A). This may explain why altering the
2,4-Me2 groups at the R2 position reduces potency three–fivefold.
On the rhodanine nucleus, the carbonyl oxygen is positioned to-
ward the highly conserved side chain of Arg197, and its sulfide
group is positioned toward His120. On the benzylidene ring, its 2-
OH group is in close proximity to Trp194 and Tyr187 side chains,
and its 5-NO2 group is oriented toward His120, suggesting a poten-
tial hydrogen bonding network. This could explain the observed
dramatic reductions in inhibitory activity when functional groups
on the benzylidene ring are relocated (Table 1, alterations to R3).
For pyridazinone compounds (series 2), most of them bind to
the active site in a similar orientation such that the phenyl ring
is buried in the aforementioned lipophilic pocket. This is evident
by comparing the docking solutions of compounds 2 (Fig. 5B), 2-
1 (Fig. 5C) and 2-35 (Fig. 5D). These models provide a plausible
explanation for why compound 2-1 has a Kai pp value ꢁ40-fold low-
er than compound 2, since the chloro group on the ring of com-
pound 2 would seem to create a steric hindrance within this
lipophilic pocket. Analogous to the docking solution observed for
compound 1 (Fig. 5A), the carbonyl oxygen atom on the pyridazi-
none ring in the docked complexes of 2, 2-1 and 2-35 are all posi-
tioned towards the conserved Arg197 side chain. In addition, the
3. Discussion
In this study we have identified several promising small mole-
cules that reversibly inhibit the S. aureus SrtA sortase with Kai pp val-
ues in the high nanomolar range, rhodanine, pyrazolethione, and
pyridazinone compounds. SAR analysis has led to some of the most
promising anti-infective agents thus far reported as compounds
2-9 and 3-12 inhibit the enzyme with Kai pp values of 1.4 and
0.3 lM, respectively. Importantly, both of these molecules do not
impair microbial growth in cell culture, suggesting that they selec-
tively inhibit sortase. Molecules based on the pyridazinone frame-
work are quite promising, and can reach Kai pp values of ꢁ0.20
lM,
but in some cases were bactericidal. Intriguingly, the most potent
inhibitors for S. aureus SrtA also inhibit BaSrtA, suggesting further
that they are specific sortase inhibitors. Additional studies with
more distantly related enzymes will be needed to define the degree
of specificity.
The library screening also revealed several rhodanine related
compounds that are potent SrtA inhibitors. However, analogs of
the lead molecule did not show improved potency. The lead rhoda-
nine compound was also shown to be bactericidal, suggesting it
has polytrophic effects. This is consistent with recent studies
showing rhodanine compounds inhibit class C b-lactamases in
Gram-negative bacteria58 and penicillin-binding proteins in non-
resistant S. aureus and MRSA strains.59
Overall, the biostructural analysis of the inhibitors is in reason-
able agreement with the SAR results, and provides insights into the
mode of action of each inhibitor from the docking poses. This
agreement may in part be due to the use of the recently reported
NMR structure of SrtA bound to a (2R,3S) 3-amino-4-mercapto-2-
butanol analog of the sorting signal.64 The structure of the active
site in this protein differs markedly from previously reported
structures of the apo-form of the enzyme (PDB:1t2p)65 and may
be more biological relevant. This assertion is substantiated by trial
docking experiments using the apo-form of the enzyme that failed
to yield results consistent with the SAR data. The structure of the
enzyme in its substrate bound form may therefore be useful for
virtual screening experiments. In summary, we have discovered
potent S. aureus and B. anthracis SrtA sortase inhibitors that could
be useful anti-infective agents. Future studies will define their
inhibitory mechanism in detail and use structure-based ap-
proaches to discover compounds with even greater potency.
thiol group on both compounds
2 and 2-1 points towards
His120, which may explain the significant reduction in activity
when this group is replaced with a chloro group (compare eth-
oxy-thiol with ethoxy-chloro subclasses in Table 2). Interestingly,
the docking solution of compound 2-35 suggests that it positions
its ethoxy moiety toward another lipophilic region created by the
side chains of Pro94 and Ala92 located in helix H1. This structural
difference may explain the distinct SAR profiles observed within
the ethoxy-chloro and ethoxy-thiol subclasses. The ethoxy-thiol
subclass is more tolerant to alteration at this site, compatible with
the docked solution that projects this group towards an open
groove on the protein surface. In contrast, in the ethoxy-chloro ser-
ies its juxtaposition against the helix H1 may make it less tolerant
to alteration, which is compatible with our finding that only com-
pound 2-35 within the ethoxy-chloro series has a low IC50 value
(vide supra).
4. Experimental
4.1. Chemistry
Materials were obtained from commercial suppliers and
were used without purification. All the moisture sensitive reac-
tions were conducted under argon atmosphere using oven-dried
glassware and standard syringe/septa techniques. Most of reac-
tions were monitored with a silica gel TLC plate under UV light fol-
lowed by visualization with a p-anisaldehyde or ninhydrin staining
solution. Some reactions were monitored by a crude 1H NMR spec-
trum. 1H NMR spectra were measured at 400 MHz in CDCl3 unless
The docking calculations suggest that the elongated structure of
the series 3 compounds may be advantageous as it may enable
contacts to two hydrophobic pockets on the enzyme. One phenyl
ring (R2) is in contact with the b6/b7 loop Val166-Val168 residues,
while the other (R3) is closer to Trp194 and Pro94 side chains
(Fig. 5E). Changing substituents on this R3 position from 4-NO2 to
2,4,6-Br3 (compound 3-12) improved the potency ꢁ15-fold, indi-
cating a preference for a more lipophilic moiety at this position.

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N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
stated otherwise and data were reported as follows in ppm (d)
from the internal standard (TMS, 0.0 ppm): chemical shift (multi-
plicity, integration, coupling constant in Hz). 2D-NMR experiments
(NOESY, COSY, and TOCSY) at 500 MHz were performed to confirm
the regioselectivity of the substitution reactions. Melting Points of
solid compounds were observed on a Thomas Hoover capillary
melting point apparatus. Infrared (IR) spectra were recorded on a
Nicolet AVATAR 370 spectrometer using liquid films (neat) on NaCl
plates. The purity of the new compounds was assessed by several
methods: high-field proton and carbon NMR (lack of significant
impurities), Rf values on TLC (lack of obvious impurities), melting
point, and mass spectrometry.
10 h. The solution was concentrated and the solids were washed
with water to give 6.5 g of a yellowish solid, 2-43, 76% (two steps).
Mp 221 °C. 1H NMR 8.35 (2H, d, J = 9.2 Hz), 7.98 (1H, s), 7.90 (2H, d,
J = 9.2 Hz); 13C NMR 155.77, 146.99, 145.37, 136.99, 136.72,
135.65, 125.64, 124.16.
4.1.2. General procedure for the synthesis of 2-substituted 4-
alkoxy-5-chloropyridazin-3-ones, for example, 5-chloro-4-
ethoxy-2-phenylpyridazin-3-one (2-28)
To a solution of 2-42 (200 mg, 0.809 mmol) in 6 mL of 1,4-diox-
ane was added 1 mL of freshly generated NaOEt (0.8 M) in EtOH
(for methoxy substitution, NaOMe solution in MeOH was used)
at 0 °C. The suspension was stirred for 2 h as the solution was
OH
2-42,
2-44,
2-45,
2-46,
2-47,
R = Ph
Cl
Cl
HCl
H
N
Cl
Cl
N
N
R = 3-bromophenyl
R = 3-Fluorophenyl
R = 3-methylphenyl (m-tolyl)
R = 3,5-dichlrorophenyl
H2N
R
O
+
H2O,
reflux, 4 hr
85~95%
R
O
O
2-48, R = cyclohexyl
85 %
H
Cl
Cl
PTSA
Cl
Cl
N
N
N
N
↑↓
Toluene,
(Dean-Stark)
10 hr, 90%
OH
NO2
O
2-43
O
NO2
2-43P
4.1.1. General procedure for the synthesis of 2-substituted-4,5-
dichloropyridazin-3-ones, for example, 2-phenyl-4,5-dichloro-
pyridazin-3-one (2-42)
slowly warmed to 25 °C. The suspension was concentrated and
the mixture was subjected to flash column chromatography on sil-
ica gel to give 189 mg of 2-28, 92%. Mp 78 °C. 1H NMR 7.84 (1H, s),
7.54 (2H, m), 7.48 (2H, m), 7.41 (1H, m); 13C NMR 163.88, 156.01,
140.09, 140.96, 138.17, 128.89, 128.56, 125.46, 123.62, 69.34,
15.94. For the other analogs, the yields varied from 70% to 96%.
To a solution of phenyl-hydrazine (2.9 mL, 30 mmol) in diluted
HCl (4 M, 60 mL) was added mucochloric acid (5 g, 30 mmol) at
25 °C. The solution was refluxed for 3 h. The suspension was fil-
tered and washed with water. The solids were dried under high
vacuum to give 7 g of the yellowish white solid, 2-42, 94%. Mp
158 °C. 1H NMR 7.91 (1H, s), 7.57 (2H, m), 7.48 (2H, m), 7.42 (1H,
m); 13C NMR 156.15, 140.86, 136.39, 136.14, 135.33, 128.95,
128.89, 125.17.
Cl
Cl
R'O
Cl
N
N
N
N
NaOH(1M)
R'OH, 0 °C
to 25 °C
R
R
O
O
4.1.1.1. 2-(4-Nitrophenyl)-4,5-dichloropyridazin-3-one, 2–43.
To a solution of 4-nitrophenyl-hydrazine (4.6 mL, 30 mmol) in di-
luted HCl (4 M, 60 mL) was added mucochloric acid (5 g, 30 mmol)
at 25 °C. The solution was refluxed for 3 h. The suspension was fil-
tered and washed with water to give the crude 2-43P. The yellow
solids were subjected to the following cyclization reaction without
further purification. The suspension of the crude 2-43P and p-tol-
uenesulfonic acid (500 mg) in 200 mL of toluene was refluxed for
75~94%
2-35, R = Ph, R' = Et
2-36, R = 4-nitrophenyl, R' = Et
2-37, R = 3-bromophenyl, R' = Et
2-38, R = 3-Fluorophenyl, R' = Et
2-39
2-40
2-41
, R = 3-methylphenyl (m-tolyl), R' = Et
, R = 3,5-dichlrorophenyl, R' = Et
, R = cyclohexyl, R' = Et
Cl
Cl
NaOR'
N
N
N
N
1,4-Dioxane,
0 °C to 25 °C
Cl
R
R'O
R
O
O
83~95%
2-28
2-29
2-30
, R = Ph, R' = Et
, R = 4-nitrophenyl, R' = Et
, R = 3-bromophenyl, R' = Et
2-22
2-23
2-24
2-25
, R = Ph, R' = Me
, R = 3-bromophenyl, R' = Me
, R = 3-Fluorophenyl, R' = Me
2-31, R = 3-Fluorophenyl, R' = Et
2-32, R = 3-methylphenyl (m-tolyl), R' = Et
2-33, R = 3,5-dichlrorophenyl, R' = Et
2-34, R = cyclohexyl, R' = Et
, R = 3-methylphenyl (m-tolyl), R' = Me
2-26, R = 3,5-dichlrorophenyl, R' = Me
2-27, R = cyclohexyl, R' = Me

N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
7183
4.1.3. General procedure for the synthesis of 2-substituted 5-
alkoxy-4-chloropyridazin-3-ones, for example, 4-chloro-5-
ethoxy-2-phenylpyridazin-3-one (2-35)
at 25 °C. The solution was stirred for 30 min and concentrated.
The residual mixture was subjected to flash column chromatogra-
phy on silica gel to give 6.1 mg of 2-49, 88%. 1H NMR 8.26 (1H, s),
7.57 (2H, m), 7.48 (2H, m), 7.40 (1H, m), 4.63 (2H, q, J = 7.0 Hz),
2.52 (3H, s), 1.40 (3H, t, J = 7.0 Hz); 13C NMR 155.42, 150.01,
141.15, 134.82, 128.69, 128.21, 127.79, 125.36, 68.78, 23.42, 15.85.
To a solution of 2-42 (200 mg, 0.809 mmol) in 6 mL of EtOH was
added 0.8 mL of NaOH (1 M) at 0 °C. The suspension was stirred for
2 h as it was allowed to warm to 25 °C. The suspension was con-
centrated and the mixture was subjected to flash column chroma-
tography on silica gel to give 195 mg of 2-35, 95%. Mp 110 °C. 1H
NMR 7.91 (1H, s), 7.57 (2H, m), 7.47 (2H, m), 7.40 (1H, m), 4.38
(2H, q, J = 7.2 Hz), 1.54 (3H, t, J = 7.2 Hz); 13C NMR 154.13,
141.22, 132.68, 128.66, 128.32, 127.74, 125.24, 117.34, 66.64,
14.81. For the other analogs, the yields varied from 75% to 95%.
HS
N
N
S
aldrithiol
N
S
N
N
EtO
MeOH
25 °C
EtO
2-50
O
O
2-10
Cl
HS
NaSH
N
N
N
N
DMF
25 °C
45-90%
R'O
R
R'O
R
4.1.7. 4-Ethoxy-5-(2-pyridyldithio)-2-phenylpyridazin-3-one
(2-50)
O
O
To a solution of 2-10 (6 mg, 0.024 mmol) in 2 mL of MeOH was
added aldrithiol (7.9 mg, 0.036 mmol) at 25 °C. The solution was
stirred for 2 h and concentrated. The residual mixture was sub-
jected to flash column chromatography on silica gel to give
5.6 mg of 2-50, 65%. 1H NMR 8.51 (1H, d, J = 4.0 Hz), 8.08 (1H, s),
7.68 (1H, ddd, J = 8.0, 8.0, 1.5 Hz), 7.61 (1H, d, J = 8.0 Hz), 7.54
(2H, m), 7.47 (2H, m), 7.38 (1H, m), 7.16 (1H, ddd, J = 7.0, 5.0,
1.0 Hz), 4.70 (2H, q, J = 7.0 Hz), 1.45 (3H, t, J = 7.0 Hz); 13C NMR
157.60, 155.42, 150.51, 149.97, 141.06, 137.36, 135.34, 128.65,
128.22, 126.80, 125.29, 121.55, 120.30, 69.04, 15.91.
2-10, R = Ph, R' = Et
2-11
2-12
2-13
2-14
2-15
2-16
, R = 4-nitrophenyl, R' = Et
, R = 3-bromophenyl, R' = Et
, R = 3-Fluorophenyl, R' = Et
, R = 3-methylphenyl (m-tolyl), R' = Et
, R = 3,5-dichlrorophenyl, R' = Et
, R = cyclohexyl, R' = Et
4.1.4. General procedure for the synthesis of 2-substituted 4-alk-
oxy-5-mercapto-pyridazin-3-ones, e.g., 4-ethoxy-5-mercapto-2-
phenylpyridazin-3-one (2-10)
O
OEt
Ph
To a solution of 2-28 (63 mg, 0.25 mmol) in 2 mL of DMF was
added 70 mg of NaSH at 25 °C. After TLC showed complete con-
sumption of starting material, the solution was concentrated under
high vacuum and diluted with 10 mL of water. The aqueous layer
was washed with ethyl acetate and then pH of the aqueous layer
was adjusted to 5–6 by addition of 1 M HCl (aq). Ethyl acetate
(20 mL, two 10 mL portions) was added to the aqueous layer to ex-
tract the desired compounds. The organic layers were combined
and dried over magnesium sulfate and concentrated to give
45 mg of 2-10 as a white solid, 73%. Mp 101 °C. 1H NMR 7.72
(1H, s), 7.54 (2H, m), 7.46 (2H, m), 7.38 (1H, m), 4.63 (2H, q,
J = 7.2 Hz), 4.04 (1H, s), 1.42 (3H, t, J = 7.2 Hz); 13C NMR 155.76,
148.54, 141.16, 137.02, 128.80, 128.30, 125.51, 125.47, 68.73,
16.12. For the other analogs, the yields varied from 40% to 91%.
S
N
N
2-10
N
S
N
N
S
MeOH
25 °C
S
N
N
EtO
2-50
O
EtO
2-17
Ph
O
4.1.8. Bis(4-ethoxy-2-phenyl-5-pyridazyl)disulfide (2-17)
To a solution of 2-50 (10 mg, 0.028 mmol) in 2 mL of MeOH was
added 15 mg of 2-10 at 25 °C. The solution was stirred for 3 h then
concentrated and subjected to flash column chromatography on
silica gel to give 11.9 mg of 2-17, 85%. 1H NMR 8.13 (1H, s), 7.55
(2H, m), 7.48 (2H, m), 7.39 (1H, m), 4.73 (2H, q, J = 7.2 Hz), 1.43
(3H, t, J = 7.2 Hz); 13C NMR (DMSO) 155.36, 150.61, 141.44,
136.57, 128.97, 128.57, 126.09, 121.58, 68.81, 16.03.
R'O
R'O
NaSH
N
N
N
N
For additional information and the spectral data on specific
compounds, please see the Supplementary data.
DMF
25 °C
Cl
R
HS
R
O
O
55-90%
4.2. High-throughput screening
2-18
, R = Ph, R' = Et
2-19, R = 3-Fluorophenyl, R' = Et
2-20, R = 3-methylphenyl (m-tolyl), R' = Et
2-21, R = 3,5-dichlrorophenyl, R' = Et
A total of 30,000 chemical compounds (DiverSet Chemically
Diverse Library and Combichem Library, ChemBridge Corp.) were
screened for S. aureus SrtA
(residues 60–206) inhibition using
D
N59
an automated robotic system at the UCLA Molecular Screening
Shared Resource facility. A fluorescence resonance energy transfer
(FRET) assay was used in high-throughput screening in multi-well
4.1.5. General procedure for the synthesis of 2-substituted 5-
alkoxy-4-mercapto-pyridazin-3-ones
The procedures for 2-18 to 2-21 are same as that of 2-10 with
the corresponding starting materials. Yields: 45–85%.
plates (384 wells per plate).64 The assay monitors the SrtA N59-cat-
D
alyzed hydrolysis of an internally quenched fluorescent substrate
analog (o-aminobenzoyl (Abz)-LPETG-diaminopropionic acid-dini-
trophenyl-NH2 (Dap(Dnp)), SynPep Corp. Dublin, CA).53 Briefly,
S
HS
MeS
N
N
N
N
MMTS
20
confirmed by 1D 1H NMR, final assay concentration of 0.4
FRET buffer: 20 mM HEPES, 5 mM CaCl2, 0.05% v/v Tween-20, pH
7.5) was incubated with 0.5 L of test compound solution (dissolved
in Me2SO, final assay concentration of 10 M) for 1 h at 25 °C.
Thirty-two wells of each plate were dedicated to positive and nega-
tive controls (1 L of Me2SO or 2 mM p-hydroxymercuribenzoic
l
L of purified SrtA (>95% homogeneity and proper folding was
MeOH
25 °C
EtO
EtO
lM in
O
O
2-10
2-49
l
l
4.1.6. 4-Ethoxy-5-(methyldithio)-2-phenylpyridazin-3-one (2-49)
To a solution of 2-10 (6 mg, 0.024 mmol) in 2 mL of MeOH was
added methyl methanethiosulfonate (MMTS, 4.5 mg, 0.036 mmol)
l

7184
N. Suree et al. / Bioorg. Med. Chem. 17 (2009) 7174–7185
acid was added alternatively to the test compound solution). Subse-
quently, 30 L of fluorescent substrate solution (15 M final assay
STR Biospectrometry Workstation (Applied Biosystems). An equal
amount (1 L) of DMSO was used instead of the inhibitor solution
l
l
l
concentration in FRET buffer) was added to the mixture to initiate
the catalysis. Final Me2SO concentrations were less than 2% in all as-
say mixtures. The FRET assays were monitored by a Flex Station II
plate reader (Molecular Devices) with an excitation and emission
wavelengths of 335 and 420 nm, respectively. The assay mixture
was measured again after 5 h for end-point reading.
as a negative control. Cbz-LPAT* (where Cbz is a carbobenzyloxy
protecting group and T* is a threonine derivative that replaces
the carbonyl group with –CH2–SH) was used as a positive control,
as it readily forms a disulfide bridge with the Cys184 thiol group
of the enzyme.41,42
4.6. Determination of S. aureus MIC
4.3. Secondary assays
The minimal inhibitory concentration (MIC) was determined
using the microtiter broth dilution method.57 An overnight satu-
rated culture of S. aureus strain Newman (provided by Dr. Lloyd
Miller, Division of Dermatology, David Geffen School of Medicine,
UCLA) was diluted to an OD600 of 0.01. After additional incubation
For the top ten lead compounds, the concentration that is
required for a 50% reduction in enzymatic activity (IC50) was deter-
mined using well established methods.34,46,53 Briefly, 20
l
L of puri-
M in FRET buffer:
20 mM HEPES, 5 mM CaCl2, pH 7.5) was incubated with 1 L of test
compound solution (dissolved in Me2SO, final assay concentration
of 0.08–400 M) for 1 h at 25 °C. Subsequently, 30 L of substrate
solution in FRET buffer (37.5 M final assay concentration for
SaSrtA, and 100 M for BaSrtA) was added to the mixture and the
fied SrtA (final assay concentration of 1.5–15
l
l
at 37 °C and dilution to an OD600 of 0.005, 180
plated into a 96-well plate. Twenty microliters of inhibitor solution
at varied concentrations (final concentrations of 0.1–100 M) were
lL of the culture was
l
l
l
l
then added to the culture. Cell growth was monitored by measur-
ing the OD600 during an overnight growth at 37 °C using a temper-
ature-controlled plate reader. The cell growth percentage was
calculated relative to cultures gown in the absence of inhibitor as
l
fluorescence was then monitored as described above. IC50 values
were calculated by fitting three independent sets of data to Eq. 1:
well as in the presence of 10 lg/mL erythromycin. MIC measure-
ments were performed in triplicate.
mi
m0
1
¼
ð1Þ
h
1 þ ð½Iꢂ=IC50
Þ
4.7. Molecular docking
where mi and m0 are initial velocity of the reaction in the presence
and absence of inhibitor at concentration [I], respectively. The term
h is Hill coefficient.46
Some of the inhibitors tightly bind to the enzyme such that their
IC50 values are lower than the enzyme concentration used in the as-
Molecular docking of each inhibitor was performed using
Schrödinger Suite 200863 with an Induced-Fit Docking (IFD) work-
flow.61,62 Calculations were run on a PC equipped with 3.8 GHz In-
tel Hyperthreading CPU, 2.0 GB SDRAM memory, and a LINUX
operating system. The IFD protocol can be summarized as follows.
First, the Glide docking module scales the van der Waals radii for
both ligand and receptor binding site atoms by 50%. Second, the
Prime module restores, predicts, and energy minimizes 20 struc-
tures of the given ligand–receptor complex generated by the first
step. Finally, the ligand conformations are redocked into the in-
duced-fit receptor structures generated by the second step. Com-
plex structures possessing energies that are within 30 kcal/mol
were then ranked and the IFD scores determined. The poses pre-
sented in the paper are those conformations with the best score.
The receptor protein structure was prepared by the Protein Prepa-
ration Wizard in Maestro user interface (Schrödinger, LLC).63 The
bond orders were assigned, and the charges and hydrogen bonds
were optimized by using the default protocol. All inhibitor ligands
were prepared by the LigPrep63 module in a comparable manner.
say (1.5–15 lM). To accuratelydefinetheirpotencytheIC50 valuesof
these compounds were measured at different enzyme concentra-
tions.46 If a linear relationship between total enzyme concentration
[E]T and IC50 values was observed, the apparent dissociation con-
stant for the enzyme–inhibitor (Kai pp) was calculated by fitting the
data to Morrison’s quadratic equation (Eq. 2).66,67
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ð½EꢂT þ ½Iꢂ þ KappÞ ꢀ ð½EꢂT þ ½Iꢂ þ KappÞ ꢀ 4½EꢂT½Iꢂ
mi
m0
i
i
¼ 1 ꢀ
ð2Þ
2½EꢂT
4.4. Inhibitory binding reversibility study
The reversibility of inhibition was determined by measuring the
recovery of enzymatic activity after a sudden large dilution of the
enzyme–inhibitor complex.46 11.25
l
L of purified SrtA at a concen-
M was mixed with 1.25 L of each inhibitor such
that the final inhibitor concentration was 10-fold greater than its
IC50. After incubation at 25 °C for 1 h, 737.5 L of FRET buffer
was added. Thirty microliters of the diluted enzyme–inhibitor mix-
ture were then plated and 20 L of the fluorescent substrate
(37.5 M stock concentration) was added to initiate the cleavage
tration of 150
l
l
Acknowledgments
l
We thank Dr. Joseph A. Loo for assistance with the mass spec-
trometry experiments, and technical support for the molecular
docking studies from Schrödinger, LLC. This work was supported
by NIH Grant AI52217 to R.T.C. and M.E.J. N.S. acknowledges
support from DPST and JSTP scholarships from the Royal Thai
Government. N.S. was also supported by a UCLA Dissertation Year
Fellowship.
l
l
reaction. The reaction progress curve was monitored as described
above. Recovery of enzymatic activity after rapid dilution (100-
fold) was calculated by comparing these progress curves with mea-
surements of the reaction performed in the absence of inhibitor.
4.5. Mass spectrometry
Supplementary data
Thirty microliters of purified SrtA (1.5
tration, dissolved in 5 mM CaCl2, 20 mM HEPES, pH 7.5 buffer)
were incubated with 1 L of inhibitor such that the final inhibitor
lM final assay concen-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.067.
l
concentration was 1- and 10-fold higher than its IC50 value. After
incubating for 1, 48, or 96 h at 25 °C, the enzyme–inhibitor mix-
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