Discovery of Diarylacrylonitriles as a Novel Series of Small Molecule Sortase A Inhibitors

Article abstract of DOI:10.1021/jm0498708

On the basis of a hit from random screening, a novel class of small-molecule sortase A inhibitors was generated. The primary structure-activity relationship and the minimal structural requirements for potency were established through structural modifications and molecular modeling studies.

Full text of DOI:10.1021/jm0498708

2418
J . Med. Chem. 2004, 47, 2418-2421
Discover y of Dia r yla cr ylon itr iles a s a
Novel Ser ies of Sm a ll Molecu le Sor ta se A
In h ibitor s
Ki-Bong Oh,†,‡ Soo-Hwan Kim,† J aekwang Lee,†
Won-J ea Cho,§ Taeho Lee,† and Sanghee Kim*,†
F igu r e 1. Structure of the screening hit 1.
Natural Products Research Institute, College of Pharmacy,
Seoul National University, 28 Yungun, J ongro,
Seoul 110-460, Korea, Graduate School of Agricultural
Biotechnology, College of Agriculture & Life Sciences,
Seoul National University, Seoul 151-742, Korea,
and College of Pharmacy, Chonnam National University,
Kwangju 500-757, Korea
recognizes the surface protein substrate bearing the
NPQTN motif sorting signal, and anchors to the cell
wall envelope.9
Recent extensive biological studies9,11-14 have shown
that both SrtA and SrtB are critical virulent factors that
participate in the establishment and persistence of
infections. Staphylococcus aureus mutants lacking sor-
tase fail to display surface proteins and are defective
in the establishment of infections without affecting
microbial viability.5,11 Therefore, a sortase inhibitor has
the potential to be used in combination with an anti-
bacterial agent to provide a more effective treatment
for infectious diseases.1,15
Received February 11, 2004
Abstr a ct: On the basis of a hit from random screening, a
novel class of small-molecule sortase A inhibitors was gener-
ated. The primary structure-activity relationship and the
minimal structural requirements for potency were established
through structural modifications and molecular modeling
studies.
Currently, there have only been a few reports in the
literature describing inhibitors of sortase, due, in part,
to the fact that importance of sortase as a new target
has only recently been acknowledged. Initially, Schnee-
wind’s group tested many types of compound.16 Of these
compounds, methanethiosulfonates or organo-mercuri-
als displayed inhibitory effects on the sorting reaction,
presumably via interaction with the thiol group of the
active-site cysteine. In addition, they also observed that
vancomycin and moenomycin, inhibitors of cell wall
polymerization into peptidoglycan strands, slowed the
sorting reaction. However, these antibiotics did not
interfere directly with the sorting reaction, but rather
changed the physiological concentration of the pepti-
doglycan precursors. Recently, two groups indepen-
dently reported the synthesis and evaluation of substrate-
derived, irreversible peptidic inhibitors of SrtA. Walker
and co-workers17 replaced the scissile amide bond
between the threonine and glycine residues of the
LPXTG motif with a diazoketone or chloromethyl ketone
group. Clubb et al.18 synthesized a peptidyl-vinyl sulfone
substrate mimic that irreversibly inhibited SrtA.
Herein, we report our discovery of small-molecule
inhibitors of SrtA through the random screening and
structural modifications based on the screening hit. The
inhibitors with low micromolar inhibitory potencies, in
vitro, against the recombinant SrtA have been obtained.
At the start, to identify inhibitors of SrtA, we ran-
domly screened our diverse small molecular library of
1000 compounds, using a recombinant SrtA,19 expressed
and purified in our laboratory. This investigation af-
forded several hits with high micromolar inhibitory
potencies. Of these, compound 1 (Figure 1), with an IC50
of 231 µM, was the most attractive as a starting point
for a novel class of inhibitors because of its simplistic
structure and synthesis.
Gram-positive pathogenic bacteria display surface
proteins that play pivotal roles in the adhesion to
specific organ tissues, invasion of host cells, or the
evasion of host-immune responses.1 Some typical ex-
amples of these are Protein A, fibronectin-binding
proteins A and B, and collagen-binding protein. A
remarkably common feature of these functionally and
structurally diverse surface proteins is the possession
of a distinctive C-terminal ‘sorting sequence’, composed
of a hydrophobic domain, a positively charged tail, and
a conserved LPXTG (where X represents any amino
acid) motif.2-4
The surface proteins are covalently attached to the
cell wall peptidoglycan, by a mechanism ubiquitous
among the entire class of the Gram-positive bacteria.
Through a series of elegant experiments, Schneewind
et al.5-8 showed that sortase participates in the pathway
involved in the secretion and anchoring of surface
proteins. Sortase is a cytoplasmic membrane-bound
cysteine protease-transpeptidase that employs an ac-
tive-site cysteine residue for catalysis. Two different
sortase species have been identified, sortase A (SrtA)
and sortase B (SrtB), which recognize specific surface
protein sorting signals.9
SrtA is constitutively expressed, and cleaves the
amide bond between the threonine and glycine of the
above-mentioned LPXTG motif, via the nucleophilic
attack of the thiol group of Cys184 on the carbonyl group
of the threonine.6,7,10 The resulting acyl-enzyme inter-
mediate is subsequently captured by the peptidoglycan
amino group found at the end of the cell wall cross-
bridge. Conversely, SrtB, which was identified only very
recently, is only transcribed under the condition of low
iron and has different substrate specificity to SrtA. SrtB
* To whom correspondence should be addressed. Tel: 82-2-740-
8913. Fax: 82-2-762-8322. E-mail: pennkim@snu.ac.kr.
† Natural Products Research Institute, College of Pharmacy, Seoul
National University.
To understand the structure-activity relationship, we
initially prepared compounds 2-5 (Figure 2),20 in which
the methyl ester group of compound 1 was replaced with
a carboxylic acid, primary amide, and nitrile groups, and
‡ Graduate School of Agricultural Biotechnology, Seoul National
University.
§ Chonnam National University.
10.1021/jm0498708 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/30/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2419
F igu r e 3. Lineweaver-Burk plot of SrtA inhibition by com-
pound 14c. [S], substrate (Dabcyl-QALPETGEE-Edans) con-
centration [×10-3 µM]; V, reaction velocity (µM/min). Each
data point represents the mean ( SD of three experiments.
F igu r e 2. Structure of the prepared compounds 2-11.
Ta ble 1. Inhibition of SrtAa
entry
compound
IC50 (µM)b
Sch em e 1. Synthesis of (Z)-Diarylacrylonitrile 14
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
10
11
14a
14b
14c
14d
14e
231.086 ( 6.530
>1000
476.034 ( 11.083
187.403 ( 3.958
>1000
>1000
>1000
>1000
908.928 ( 26.682
27.892 ( 0.490
>1000
36.222 ( 0.942
17.376 ( 0.490
9.244 ( 0.474
22.957 ( 0.643
25.463 ( 0.948
and the exact location of the two aromatic rings are
crucial for effective inhibition of the enzyme activity.
a
Enzyme inhibitory activities were measured as described in
b
the Supporting Information. Data represents the mean ( SEM
(n ) 5).
Having established the primary structure-activity
relationship, we then investigated the effects of different
aromatic substituents on the new lead compound 10.
We prepared a series of compounds with the general
structure 14 (Scheme 1), where one phenyl ring was
fixed with a methoxy group at the 4-position, and the
other phenyl ring was modified with a methoxy group
at various positions. Other hydrophilic functional groups
were not considered at this point, as the recent NMR
structure of SrtA8 revealed this enzyme to have a
hydrophobic pocket suitable for sorting signal binding.
The syntheses of this series of compounds was ac-
complished by the well-known condensation21 of 4-meth-
oxyphenylacetonitrile 12 with benzaldehyde 13 in the
presence of t-BuOK.
All the synthesized compounds 14a -e showed inhibi-
tion of SrtA, with IC50 values of less than 40 µM, as
shown in Table 1 (entry 12-16). In particular, com-
pound 14c was the most active, with an IC50 of 9.2 µM,
which could be the first small-molecule inhibitor of SrtA,
with single-digit micromolar inhibitory potency, identi-
fied. The other compounds exhibited almost equipotent
activities, suggesting that the potency of these com-
pounds might not be highly dependent on the substitu-
tion pattern of the modified phenyl ring.
a hydrogen atom, respectively, with the retention of the
relative stereochemistry of the two phenyl rings. Of
these, compounds 2 and 5 did not show any inhibitory
activities to SrtA up to 1000 µM (Table 1). Conversely,
the nitrile compound 4 showed only a slightly increased
inhibitory activity (IC50 ) 187 µM), while amide 3
exhibited a decrease in the potency of the SrtA inhibi-
tion (IC50 ) 476 µM) than ester 1.
To test the influence of the double bond on the
activity, saturated analogues 6-8 were prepared from
their corresponding compounds (1, 4, and 5) by hydro-
genation. However, none of the three compounds ex-
hibited measurable activity against SrtA (entry 6-8).
To understand the influence of the double bond config-
uration further, we tested compounds 9-11 in which
two benzene rings were located in the trans orientation.
As shown in Table 1, the inhibitory potential of (Z)-ester
9 was diminished substantially as compared to its (E)-
isomer 1. The trans-stilbene 11 showed no activity as
its cis isomer 5. However, to our delight, we found that
(Z)-diarylacrylonitrile 10 (IC50 ) 28 µM) was almost 7-8
times more potent against SrtA than the screening hit
1 or the corresponding (E)-isomer 4. In comparison with
trans-stilbene 11, which was inactive, compound 10
differed structurally by only the nitrile group at the
ethylene bridge of two benzene rings. These results
strongly suggested that the presence of a nitrile group
To determine the type of inhibition, kinetic studies
were performed with compound 14c at the concentration
where approximately 50% of enzyme activity was in-
hibited. Inhibitory kinetics in Figure 3 show that
compound 14c only affect the slope of the Lineweaver

2420 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Letters
Ala 92, Ala 104, Leu 169, Val 168, and Ile 182. These
hydrophobic side chains have relatively strong lipophilic
interactions with the phenyl rings of 14c. However, it
seems that the side chain of Cys 184, which is crucial
for the transpeptidation activity, does not interact with
the ligand, even though the methoxy group of 14c is
positioned near the Cys 184 (2.463 Å). Of note, in this
model, the nitrile group of compound 14c has a hydro-
gen bonding interaction with two guanidyl NH of Arg
197, with a distance of 1.662 and 2.579 Å. Our model
predicts the effective inhibition of the enzyme activity
by the compounds containing a nitrile group.
In conclusion, based on a hit from random screening,
we have discovered, for the first time, a novel class of
small-molecule SrtA inhibitors. We have established the
primary structure-activity relationship, and defined the
structural requirements for potency, through structural
modifications and molecular modeling studies. These
results open the avenue toward novel antivirulence
chemotherapeutic agents against Gram-positive patho-
genic infection. Further structure-activity relationship
and mechanistic studies on this newly disclosed class
of inhibitors are ongoing and will be reported in due
course.
Ack n ow led gm en t. This study was supported by a
grant of the Korea Health 21 R&D Project, Ministry of
Health & Welfare, Republic of Korea (HMP-00-CH-15-
0014). K.-B. Oh is recipient of fellowship from the
Ministry of Education through the Brain Korea 21
Project.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails corresponding to the synthesis of the compounds de-
scribed in this paper, spectral data for all relevant compounds,
details of the biological assay protocol, and molecular modeling
methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
F igu r e 4. Docking model of compound 14c (yellow) within
the SrtA is shown. The structure of SrtA was obtained from
the Protein Data Bank (1IJ A). (up) The H-bond between the
CN of 14c and the hydrogen of the NH of Arg 197 backbone is
shown as a yellow dotted line. Two substituted aromatic rings
can establish hydrophobic interaction with the alkyl side
chains of Val 168, Leu 169, Ile 182, Ala 104, Ala 92, Trp 194,
and Val 193. (down) 14c is positioned to a MOLCAD surface
representation of binding site of SrtA. The colors represents
as follows: red for oxygen atoms, blue for nitrogen atoms, and
cyan for hydrogen atoms.
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behaved as a competitive inhibitor (Ki ) 6.81 ( 0.41
µM). Moreover, the binding of compound 14c to enzyme
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J M0498708