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org/jmc Perspective

Chemical Biology of Sortase A Inhibition: A Gateway to Anti-

infective Therapeutic Agents
Rachit Sapra, Amit K. Rajora, Pushpendra Kumar, Govind P. Maurya, Nalin Pant,* and V. Haridas*
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ABSTRACT: Staphylococcus aureus is the leading cause of

hospital-acquired infections. The enzyme sortase A, present on
the cell surface of S. aureus, plays a key role in bacterial virulence
without affecting the bacterial viability. Inhibition of sortase A
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activity offers a powerful but clinically less explored therapeutic

strategy, as it offers the possibility of not inducing any selective
pressure on the bacteria to evolve drug-resistant strains. In this
Perspective, we offer a chemical space narrative for the design of
sortase A inhibitors, as delineated into three broad domains:
peptidomimetics, natural products, and synthetic small molecules.
This provides immense opportunities for medicinal chemists to
alleviate the ever-growing crisis of antibiotic resistance.

1. INTRODUCTION nosocomial infections and pose a serious threat worldwide.

Staphylococcus aureus is a highly prevalent Gram-positive
bacterial species and is also known as the “all-time champion”
of microbial human pathogens.1 It is an opportunistic pathogen
that causes a wide number of diseases such as minor skin
infections, soft tissue infections, pneumonia, endocarditis,
osteomyelitis, and blood infections.2−4 Infections caused by S.
aureus are typically acute and aggressive by nature and also top
the list of both hospital and community-associated infections.
The β-lactam antibiotics are the most successful clinically
effective antibiotics used to treat bacterial infections in humans.5
Their wide therapeutic success is largely due to their broad-
spectrum activity, oral availability, excellent pharmacokinetics,
safety, and bactericidal action. They have been the drugs of
choice for the treatment of S. aureus-induced infections.
However, these antibiotics are often seen as ineffective due to
the development of antibiotic-resistant strains.6−12 They
prevent bacterial infections by targeting bacterial factors which
either inhibit bacterial growth or result in bacterial killing.13,14
These bacteriostatic and bactericidal properties of antibiotics
along with their extensive use and abuse have led to the rapid
emergence of drug-resistant strains, causing global concern. The
key progress toward the development of antibiotics and the
emergence of antibiotic-resistant strains is outlined in Figure
1.15−20
S. aureus has been included as one of the six high-priority
threatening pathogens collectively referred to as the “ESKAPE”
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiel-
la pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa,
and Enterobacter).15,21−23 These ESKAPE pathogens can evade
the action of antibiotics, resulting in morbidity and mortality.
They are among the most common opportunistic pathogens in
Across the globe, around 700 000 patients die every year due to
infections caused by drug-resistant strains.24 The death toll due
to antimicrobial resistance is estimated to rise to 10 million by
2050. According to recent statistical data from the U.S. Centers
for Disease Control and Prevention (CDC), in the United States
alone every year more than 2.8 million people suffer from
infections caused by drug-resistant bacteria, and 35 000 of them
die.25 As per the CDC data, out of 323 700 estimated cases of
infections caused by methicillin-resistant S. aureus (MRSA) in
hospitalized patients, an estimated annual death rate of 10 600
has been reported in 2017. Thus, there is an urgent need
worldwide to develop new and more effective anti-infective
agents against S. aureus.26
The development of new antibiotics is at present the most
promising option available for treating bacterial infections.
However, due to the emergence of antibiotic resistance, anti-
virulence agents have gained widespread interest as a promising
method for combating bacterial infections (Figure 1).27−30 This
shift to anti-virulence agents is due to an increasing prevalence of
drug-resistant strains and a shortage of new antibiotics for
clinical use.
Virulence factors are the secretory, membrane-associated, or
cytosolic molecules that bacteria use to infect hosts. They play a
Received: March 3, 2021
Published: September 13, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.jmedchem.1c00386

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Figure 1. Timeline showing milestones in the development of drugs against S. aureus and emergence of drug-resistant strains.
key role in the establishment of bacterial infections leading to
bacterial colonization, invasion of the host, adhesion, biofilm
formation, and evasion or suppression of the host’s immune
system. The anti-infective agents targeting the virulence factors
affect the bacterial cell’s ability for host recognition, thereby
preventing the infection. In contrast to the drugs which inhibit
cell growth or directly kill the bacteria, anti-virulence agents
disarm the pathogen rather than destroying it.27,31 Therefore, it
was believed that bacteria would generate weakened resistance
to such molecules compared to traditional antibiotics. However,
there are reports on the development of resistance to anti-
virulence drugs.32−34
The bacterial surface proteins involved in the recognition of
hosts’ extracellular matrix proteins are anchored to the S. aureus
cell wall via a transpeptidation reaction catalyzed by sortase A
(SrtA).35−44 SrtA plays a critical role in the bacterial patho-
genesis; however, it is not vital for the growth or survival of S.
aureus.38−40 SrtA-targeting anti-infective agents interfere with or
destroy the cell wall anchoring pathway and restrain the
virulence of pathogenic bacterial strains without affecting
bacterial cell viability. This would lead to a reduced possibility
of development of drug resistance; hence, SrtA is considered as a
potential anti-virulence therapeutic target to combat S. aureus-
caused bacterial infections. Moreover, the enzyme’s extracellular
location and the absence of SrtA homologs in humans render the
drugs with anti-SrtA activity specific to bacterial cells with low
off-target effects.
An excellent perspective article on this topic by Cascioferro
and co-workers summarized important developments in SrtA
inhibitors up to 2015.42 A recent review by Paek and co-workers
describes small synthetic molecules with SrtA inhibition ability,
primarily focusing on their structural and synthetic aspects.44
This Perspective provides a comprehensive treatment of S.
aureus SrtA inhibitors, with emphasis on recent developments.
In the first part, the potential of SrtA as an anti-virulence target,
its mechanism of action, and its structure are briefly discussed,
followed by a comprehensive discussion on natural and synthetic
inhibitors of SrtA. In addition to these, a section dealing with
commonly used assays to determine SrtA inhibition is also
included.
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2. S. AUREUS SORTASE A: AN IDEAL ANTI-VIRULENCE
TARGET
Host cell adhesion is a crucial step of virulence in both Gram-
positive and Gram-negative bacteria.45−48 S. aureus and other
Gram-positive bacteria produce a wide array of surface proteins
that mediate the attachment of bacterial cells to host
extracellular matrices such as fibrinogen, fibronectin, and
collagen, leading to adhesion.46,49,50 These surface proteins are
referred to as microbial surface components recognizing
adhesive matrix molecules (MSCRAMMs). These
MSCRAMMs play a critical role in bacterial colonization, host
invasion, and evasion of host’s immune system. These
extracellular proteins are synthesized in the cytoplasm and
displayed on the bacterial surface. Owing to the crucial role
played by these surface proteins in the infectious process, anti-
infective therapies that impair bacterial adhesion have emerged
as ideal strategies for the treatment of S. aureus-induced
infections.46
The covalent anchoring of surface proteins on the cell wall of
S. aureus is catalyzed by a membrane-bound transpeptidase
enzyme, namely sortase.36,37,51 The activity of the sortase
enzyme was discovered by Schneewind and co-workers in the
early 1990s (Figure 1).52−55 The purification and character-
ization of SrtA from S. aureus by Schneewind in 1999 marked a
new direction in chemical and biological research.56 Since then,
many other sortase isoforms including sortase B, C, D, E, and F
have been discovered from several other Gram-positive
bacteria.57−61
Among the various isoforms, SrtA is the most studied sortase,
as it plays a key role in the pathogenesis of Gram-positive
bacteria. It is referred to as “housekeeping” sortase as it is present
in the majority of Gram-positive bacteria and is involved in the
chemical ligation of a variety of cell-surface proteins. SrtA
recognizes and covalently anchors surface proteins containing
an N-terminal LPXTG motif (X = any amino acid) to the
bacterial cell wall. It is known to anchor about 20 S. aureus
surface proteins, including fibronectin-binding proteins (FnbA
and FnbB), clumping factors (ClfA and ClfB), serine-aspartate
repeat proteins (SdrC, SdrD, and SdrE), Staphylococcal protein
A (Spa), and iron-regulated surface determinant A (IsdA), onto
the bacterial surface.61−64 Their cell wall anchoring is essential
for the bacteria to exert pathogenic effect. S. aureus mutants
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Figure 2. SrtA-mediated cell wall anchoring pathway of surface proteins. This involves a surface protein recognition and thioesterification (a), further
nucleophilic attack by pentaglycine of lipid II to generate a lipid II-linked surface protein (b). The surface protein is incorporated into cell wall by
transglycosylation (c(i)) and transpeptidation (c(ii)). Abbreviations: Sec, secretory pathway; LPXTG, leucine, proline, any amino acid, threonine, and
glycine; GN, N-acetylglucosamine; MN, N-acetylmuramic acid; PP, undecaprenyl pyrophosphate; (Gly)5, pentaglycine; iGln, isoglutamine.

lacking SrtA cause virulence attenuation by the defective display
of surface proteins.65,66
SrtA plays an important role in the formation of biofilm;
hence, SrtA inhibitors reduce or destroy the biofilm formation
ability of S. aureus. Biofilms are organized microbial assemblies
encased in an extracellular matrix that are used by bacteria to
adhere to biotic or abiotic surfaces.67,68 They confer a favorable
growth environment to the bacteria and shield it from
antibiotics, disinfectants, preservatives, or host immune systems.
These also contribute to the evolution of bacteria with drug-
resistant strains by promoting the transfer of genes associated
with resistance.69−71
Initial attachment, cell aggregation, biofilm maturation, and
dispersal are the steps involved in S. aureus biofilm formation.72
S. aureus surface proteins, including fibronectin-binding proteins
(Fnbps), mediate bacterial attachment to fibrinogen, fibronec-
tin, and other hosts’ extracellular matrix proteins. These proteins
are anchored to the S. aureus cell wall through a SrtA-catalyzed
transpeptidation reaction. Thus, anti-virulence agents that target
SrtA are likely to suppress the biofilm formation ability of S.
aureus. Studies have revealed that overexpression of SrtA in
bacteria leads to an increase in the level of biofilm formation,
whereas the SrtA knockout S. aureus strains are devoid of biofilm
formation ability.73,74 Reduction in the biofilm formation ability
of various MRSA strains was observed as a result of mutations in
Fnbps. Therefore, SrtA inhibitors should reduce the binding of
the bacteria to the host cell-matrix proteins, reducing infection.
13099
Similarly, loss in bacterial adhesion was observed upon
inactivation of SrtA for other Gram-positive bacteria, including
Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus
mutans, and Streptococcus suis.75−79 Additionally, SrtA is not
required for bacterial viability, which would exert less pressure
on bacteria for developing drug resistance. The extracellular
location and absence of any eukaryotic homologs make SrtA
targeting easier and selective. Based on these advantages, SrtA is
considered an ideal target for developing anti-virulence drugs.41
The mechanism of SrtA-mediated surface protein anchoring
in S. aureus is well established (Figure 2).54,61,80 The bacterial
surface proteins are synthesized in the cytoplasm and consist of
an N-terminal globular domain and a C-terminal cell wall sorting
signal. The cell wall sorting signal comprises a conserved
LPXTG motif, a hydrophobic domain, and a tail of positively
charged residues.52 The LPXTG sorting sequence is secreted
from the cytoplasm, along with the N-terminal globular domain,
through the secretory (Sec) pathway. The hydrophobic domain
remains embedded in the bacterial cell membrane, with the tail
retained in the cytoplasm. The LPXTG motif of the protein is
recognized by SrtA, followed by nucleophilic attack of the
enzyme’s Cys184 on the carbonyl of Thr-Gly peptide bond.56
This results in the cleavage of the Thr-Gly peptide bond,
generating a thioester acyl-enzyme intermediate (Figure 2). The
thioester bond between SrtA and surface proteins is
subsequently attacked by the free amine group of the
pentaglycine unit within the lipid II precursor (undecaprenyl-
pyrophosphoryl-N-acetylmuramoyl-(L-Ala-D-iGln-Nε(NH2-
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Scheme 1. Schematic Representation of the Reverse Protonation Model for SrtA Catalysis
Gly5)-L-Lys-D-Ala-D-Ala)-β(1 → 4)-N-acetylglucosamine),
regenerating the active-site Cys184.61,81,82 Finally, the surface
proteins are incorporated into the peptidoglycan via trans-
glycosylation and transpeptidation reactions involved in
bacterial cell wall synthesis.83 This leads to covalent attachment
of the proteins to the cell wall peptidoglycan, to become true
surface proteins.
SrtA contains His120, Cys184, and Arg197 residues that are
found to be essential for the enzyme’s bioactivity.56,84−89
McCafferty and co-workers studied the catalytic mechanism of
SrtA based on mutational analysis, protein stability, isotope
effects, pH rate profile, and kinetic parameters.87 The
mechanism, namely, the reverse protonation model (Scheme
1), proposes that the active form of SrtA exists in a reverse
protonated state and represents a small fraction (0.06%) of the
total enzyme.87 These studies are in agreement with a Cys184-
His120 catalytic dyad and with Arg197 stabilizing the
tetrahedral intermediate.
The crystal and NMR structures of SrtA in free and bound
states have been successfully elaborated in the litera-
ture.84−86,90S. aureus SrtA is a 206-amino acid long protein,
comprising unstructured and structurally ordered regions. The
unordered domain, containing nonpolar amino acid residues, is
responsible for embedding the enzyme in the bacterial cell
membrane, whereas the ordered domain is responsible for the
SrtA-catalyzed transpeptidation. Clubb and co-workers first
reported the structure of SrtAΔN59 using NMR spectroscopy.84
SrtAΔN59 is a SrtA mutant without 59 residues from the enzyme’s
N-terminal. The recombinant SrtAΔN59 is fully capable of
catalyzing in vitro cleavage of peptides bearing the LPXTG motif
and the transpeptidation reaction. The NMR studies revealed
that SrtA primarily possesses a unique 8-stranded β-barrel fold
(β1−β8) including two short helices and several loops (Figure
3a). The structure indicated that the β1 strand lies antiparallel to
the β2 strand, separated by a short hairpin structure. A 310 helix
(H1, Figure 3a) tethers the β3 strand to the β2 strand in a
parallel orientation. The β3, β4, and β5 strands are positioned
antiparallel to each other, with β4 and β5 strands connected by a
long loop and α-helix (H2, Figure 3a). The β6 and β7 strands are
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Figure 3. (a) NMR structure of SrtAΔN59 from S. aureus. The β-strands
and helices are colored blue and green, respectively. Adapted with
permission from ref 84. Copyright 2001 National Academy of Sciences,
USA. (b) NMR structure of covalent SrtAΔN59-LPAT complex. Image
from the RCSB PDB ID: 2KID.85
separated by another hairpin turn, followed by a long loop for
antiparallel pairing. The sulfhydryl group-containing Cys184 is
located toward the end of the β7 strand, followed by another
loop leading to the β8 strand. The β7 and β8 strands form the
base of a hydrophobic pocket, whereas the amino acid residues
connecting β3-β4, β2-β3, β6-β7, and β7-β8 strands constitute its
wall.
The X-ray crystal structure of SrtAΔN59 was reported by
Narayana and co-workers.90 The solid state structure revealed
that the unit cell comprises three molecules with different
conformations of amino acid residues in the connecting loop of
β6 and β7 strands. In contrast to the NMR structure, the crystal
structure revealed that one α and two 310 helices connect the
eight β strands, giving rise to the β-barrel structure. In addition
to this, conformational differences in the amino acid side chains
were also observed.
Experimental determination of the underlying molecular
interactions between LPXTG-containing substrates and SrtA is
difficult due to the transient existence of the Thr-Gly bond.
Clubb and co-workers synthesized an N-terminal carbobenzy-
loxy-protected tetrapeptide, LPAT, with the carbonyl group of
Thr replaced by −CH2SH.85 The LPAT peptide covalently
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binds to the Cys184 of SrtA via a disulfide bond. This could
structurally mimic the thio-acyl intermediate, formed by the
covalent attachment of LPXTG-containing surface protein to
SrtA. The results gathered from the NMR screening agree with
mutagenesis studies. The studies indicated that the recognition
of surface protein involves accommodation of the LPXTG motif
in a large groove that leads into the enzyme’s active site. The
base of the binding groove is constituted by β4 and β7 strands,
while the amino acids comprising loops connecting β6/β7, β7/
β8, β3/β4, and β2/H1 form its walls (Figure 3b).
Particularly, the amino acids constituting the loop connecting
β6 and β7 strands play a key role in catalysis, which is
demonstrated by a significant loss in SrtA bioactivity upon
mutations of these residues.85,89,91 Upon substrate binding, the
loop undergoes extensive structural changes and adopts a closed
conformation containing a 310 helix. The loop closure upon
substrate binding facilitates hydrophobic contacts of SrtA
residues with the leucine residue in the LPAT peptide. The
proline residue is in contact with the nonpolar side chains of
Ala92, Ala104, Ala118, Leu169, and Ile182 and buries the
peptide in the active-site binding groove. The covalently bound
LPAT peptide adopts an L-shaped kinky structure with the Ala-
Pro peptide bond in the trans conformation. Mutations of amino
acids including Leu97, Ala104, Ala118, Val168, and Leu169
resulted in significant losses of SrtA transpeptidation activ-
ity.85,89,91
The NMR studies on SrtA and SrtA−substrate complex
suggest that SrtA binds to a single calcium ion through side
chains of Glu and Asp residues within β3-β4 and β6-β7
loops.84,85 It is further indicated that the electrostatic interaction
between Glu171 and calcium ion upon substrate-binding leads
to structural and mobility changes in the β6-β7 loop. The
binding to calcium ion near the active site stimulates the catalytic
activity of SrtA by 8-fold.84
Molecular dynamics (MD) simulations revealed multiple
conformations for SrtA and LPATG sorting signals.92,93 Because
of the highly dynamic nature of SrtA, it is also suspected that the
catalytic site is not necessarily the binding site for at least some of
the inhibitors. Therefore, given the dynamic nature of this non-
covalent association, it is not surprising that very few
compounds with strong SrtA inhibition have been reported.
The catalytic mechanism and structural information gathered
on SrtA and its complex have been successfully utilized for
protein engineering and synthesizing designer macromole-
cules.94−101 This has also served as a basis for designing SrtA-
targeting anti-infective agents that reduce bacterial virulence.
3. EXPERIMENTAL METHODS FOR EVALUATING
SORTASE A INHIBITORS
For more than a decade now, research efforts have focused on
strategies to discover drugs that target bacterial virulence factors
that are not essential for cellular viability/survival. Since then, a
large number of natural or synthetic SrtA inhibitors have been
identified using diverse strategies. Several literature reports on
SrtA inhibitors are based on the identification of hit candidates
through high-throughput screening (HTS) of large compound
libraries. HTS allows the identification of molecules with drug-
like properties among several thousands of compounds
screened.
In combination with HTS, virtual screening of chemical
libraries against the structure of SrtA is another popularly
followed method for the identification of suitable drug
candidates. Preliminary experimental studies using various
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biochemical assays are then conducted on hit candidates
obtained from screening. These early-stage measurements,
including the determination of half-maximal inhibitory concen-
tration (IC50) and minimum inhibitory concentration (MIC),
help in analyzing the compound’s bioactivity.
The nature of data on the biological activity of a compound is
dependent on the type of the biological assay performed. Many
conventional assays such as fluorescence resonance energy
transfer (FRET),37,56,62,85,102−107 high-performance liquid
chromatography (HPLC),92,108,109 cell adhesion,110,111 clump-
ing,66 sodium dodecyl sulfate−polyacrylamide gel electro-
phoresis (SDS-PAGE),112,113 and fluorescein incorporation
assay,114 have been employed and reported in the literature to
assess the SrtA inhibitory activity of inhibitors. It is useful to
briefly survey these different techniques since it benefits the
medicinal chemistry community.
3.1. Fluorescence Method. The anti-SrtA activity of the
test compound is most commonly determined using fluorogenic
peptide cleavage assay. Kinetic parameters like catalytic rate
constants, catalytic efficiency, apparent rate constant, and IC50
values are evaluated by monitoring the fluorescence enhance-
ment of self-quenched fluorogenic substrate.56,103 The substrate
consists of a fluorophore−quencher FRET pair, EDANS (5-[(2-
aminoethyl)amino]naphthalene-1-sulfonic acid) and Dabcyl (4-
([4′-(dimethylamino)phenyl]azo)benzoic acid), suitably placed
at the ends of a peptide sequence containing the SrtA-sensitive
LPXTG motif.37,56,62,105 Peptide substrate Abz-LPETG-Dap-
(DNP)-NH2 is also used in FRET assay to evaluate SrtA activity
with 2-aminobenzoic acid (Abz) as the fluorophore.85,103 The
quencher, 2,4-dinitrophenylaniline (DNP), is linked to the
peptide sequence using 2,3-diaminopropionic acid (Dap). SrtA
inhibition by fluorescence measurements can also be deter-
mined by using commercially available assay kits.104,105 In the
absence of SrtA, the fluorophore and quencher are held closely,
leading to fluorescence quenching. Upon incubation of a peptide
with SrtA, Thr-Gly in the peptide sequence undergoes
enzymatic cleavage (Figure 4a). The peptide segment attached
to SrtA via a thioester bond can undergo hydrolysis (Figure
4b).56 This results in the formation of two peptides, with one
bearing the fluorophore and the other with the quencher. The
separation of fluorophore and quencher restores the fluores-
Figure 4. (a) Schematic representation of FRET-based enzymatic assay
using Dabcyl-EDANS appended peptide substrate. (b) Mechanism of
SrtA-catalyzed reactions: (i) initial cleavage of LPXTG substrate by
SrtA, generating a thioester acyl−enzyme intermediate, and nucleo-
philic attack on intermediate by (ii) water and (iii) pentaglycine unit as
nucleophilic substrate. Abbreviations: Dabcyl, 4-([4′-
(dimethylamino)phenyl]azo)benzoic acid; EDANS, 5-[(2-
aminoethyl)amino]naphthalene-1-sulfonic acid.
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cence of the donor molecule. The increase in fluorescence
intensity will be proportional to the amount of peptide which has
undergone cleavage. Treatment of SrtA with a test compound
(inhibitor) would reduce the enzymatic cleavage of peptides,
consequently resulting in lower fluorescence intensity. The
experimental protocol can be validated by performing blank
measurements including positive and negative controls.
Inhibitors including phenyl vinyl sulfone, 1-(3,4-dichlorophen-
yl)-3-(dimethylamino)propan-1-one, and p-hydroxymercuri-
benzoic acid with known SrtA-inhibition ability are used as
positive controls, whereas the bacterial solutions in the absence
of inhibitor or with SrtA knockout strains of S. aureus are used as
negative controls.56
The FRET-based enzymatic assay is a quick and effective
method for coarse/gross analysis of the anti-SrtA activity of test
compounds. It is the commonly used technique, since it is well
suited for the HTS approach. However, it can only provide
information on the SrtA-catalyzed initial cleavage of LPXTG-
containing peptides. The kinetic parameters associated with the
transpeptidation step (Figure 4b) cannot be quantified, since the
fluorophore has already regained its fluorescence in the first step.
Thus, no additional nucleophilic substrate is generally used to
resolve the thioester acyl−enzyme intermediate generated in the
first step. However, the intermediate can undergo hydrolysis due
to the presence of water molecules.56 Since the assay relies on
fluorescence measurements, it is likely that fluorescence
quenching interferes with the results due to the inner filter effect.
Recently, more sophisticated fluorescence-based assays have
been developed to measure the SrtA inhibition potency of a test
compound. The assay designed by Zhang and co-workers
exploited the use of FRET between cyanine dye−peptide
conjugates, Cy3-CLPETGG and GGGG-Cy5.106 In contrast to
Dabcyl-EDANS and Abz-DNP-based peptide cleavage assays,
this assay relies on SrtA-catalyzed chemical ligation of peptides
bringing donor (Cy3) and acceptor (Cy5) in proximity to each
other. This results in enhanced fluorescence emission of Cy5
which can be quantified by fluorescence imaging. The
measurements in this assay are less likely to be affected by the
inner filter effect over other fluorescence-based assays. A
fluorescence-based assay to determine SrtA inhibition efficacy,
based on the increase in fluorescence intensity of an externally
added biarsenical dye, has also been developed.107 The assay is
relatively simple and 24.7-fold more sensitive than peptide
cleavage assays. It can be successfully applied for identifying new
candidates with potent SrtA deactivation ability.
3.2. HPLC Method. To address discrepancies associated
with the fluorescence intensity attenuation method, an HPLC-
based assay has been devised to analyze SrtA-inhibition
activity.108,109 This protocol utilizes the same peptide substrate,
Abz-LPETG-Dap(DNP)-NH2, as in the FRET assay, and a
nucleophilic substrate, like pentaglycine or triglycine for the
transpeptidation reaction.92,108,109 The Abz and DNP motifs
facilitate UV/fluorescence detection of the unreacted peptide
substrate, or acylation and transpeptidation products. After
incubation of SrtA with the two substrates in the presence or
absence of the inhibitor, the reaction mixture is purified by
centrifugation to remove the enzyme. The mixture is subjected
to HPLC analysis, and the area under the peaks corresponding
to starting materials, intermediates, and product formed is used
to quantify the percentage conversion.108,109 Additionally, the
HPLC data can be verified by MALDI-TOF mass spectrometric
analysis. Unlike FRET-based enzymatic detection, the HPLC
assay enables monitoring the concentrations of reaction
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intermediates and products. In particular, the kinetic parameters
for the transpeptidation step can be accurately quantified.
Additionally, the identification of individual intermediates and
products might also be useful to decipher the mechanism of SrtA
inhibition.109 Although the HPLC-based assay is more time-
consuming, usage of fast-flow HPLC columns might enable
high-throughput applications of this assay.
3.3. Cell Adhesion and Clumping Methods. In addition
to FRET and HPLC assays already described, the SrtA
inhibition potency of test compounds can also be evaluated
using cell adhesion assay.110,111 Bacterial attachment to host
tissues occurs via cell wall anchored surface proteins like
fibronectin- and fibrinogen-binding proteins.72 These proteins
are displayed on the bacterial surface via the transpeptidation
reaction catalyzed by SrtA.37,38 Thus, SrtA inhibition reduces
the surface display of the fibronectin- and fibrinogen-binding
proteins on bacterial cell walls, which in turn would hinder S.
aureus attachment to host tissues. Therefore, the cell adhesion
assay can also be used to evaluate SrtA inhibition activity. This
assay involves monitoring the binding of bacterial cells to
fibronectin- or fibrinogen-coated surfaces. As the first step,
overnight-cultured bacterial cells are inoculated and incubated
with different concentrations of inhibitors under appropriate
conditions for cellular growth. The cellular growth in the
incubation medium is monitored by determining its optical
density (OD), generally measured at 600 nm. After attaining a
suitable population of bacteria, the cells are pelleted by
centrifugation, washed, and resuspended in phosphate-buffered
saline (PBS). The suspension is added and incubated in a well
plate coated with bovine fibrinogen or human plasma
fibronectin. The well plate after washing and cell fixation using
glutaraldehyde is stained with crystal violet. After washing and
subsequent drying, the absorbance of the well plates at 570 nm is
recorded. In the absence of test compound (inhibitor), more
immobilization of crystal-violet-stained S. aureus on the well
plate is expected, resulting in high absorbance. However, after
incubating bacterial cells with SrtA inhibitor, decreased enzyme
activity would lead to low absorbance, as the tendency of S.
aureus to adhere to protein-coated well plates is reduced. The
test results are validated by performing blank and control
experiments under identical conditions.
Cell clumping assay is another method to determine the
potency of a compound to inhibit SrtA activity.66 Similar to the
cell adhesion assay, culturing the bacterial cells and subsequent
incubation with test compound, followed by centrifugation are
the steps involved in the cell clumping assay. The pelleted cells
are suspended and incubated in a fibrinogen solution. Unlike the
cell adhesion assay, immobilization of bacterial cells to
fibrinogen is analyzed by measuring the OD of the suspension
at 600 nm. In the absence of an inhibitor, SrtA-anchored surface
proteins bind to fibrinogen in solution, forming visible
aggregates which settle down with time, resulting in a lower
OD value. In the presence of SrtA inhibitors, cells do not
aggregate upon incubation with fibrinogen and remain
suspended in the solution.
3.4. SDS-PAGE Assay. The inhibitory activities of SrtA
inhibitors can also be determined using SDS-PAGE assay.112,113
The method involves the separation of proteins from complex
protein mixtures and their complexes, based on molecular
weight, under the influence of an applied electric field. The
analysis of SrtA inhibition by this method involves the
incubation of the candidate molecule with SrtA, S. aureus
surface protein (IsdA), and nucleophilic substrate (triglycine),
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followed by quenching of the reaction. The reaction mixture is
then subjected to SDS-PAGE. After electrophoresis, the protein
components in the reaction mixture are separated, and the
intensities of the bands are analyzed to quantify the amount of
starting materials and transpeptidation products. The experi-
ments are validated by performing blanks in the absence of test
compound, SrtA, or S. aureus protein and by control experiments
using known SrtA inhibitors.
3.5. Fluorescein Incorporation Assay. Another fluores-
cence-based method used for quantification of SrtA inhibition
activity of an unknown compound involves the incorporation of
fluorescently labeled LPXTG-containing peptides into the S.
aureus cell wall.114 The peptide is ligated to the bacterial cell wall
by a SrtA-mediated transpeptidation reaction. This assay
requires culturing S. aureus in a suitable growth medium with
different concentrations of a test compound in the presence of
peptide bearing the fluorophore (5(6)-carboxyfluorescein).
After a suitable incubation time, cells are pelleted and washed
with cold PBS. The cells are subsequently washed with 5%
sodium dodecyl sulfate to remove non-covalently bound
molecules. The cells are washed twice with cold PBS,
resuspended, and subjected to fluorescence measurements.
Analysis of the fluorescence intensity in the absence of the test
compound or using a S. aureus SrtA knockout strain serves as a
control to validate the experimental results. In the absence of the
test compound, the extent of inclusion of the fluorescently
labeled peptide into the S. aureus cell wall is indicated by strong
emission from the fluorophore. Incubation of cells in the
presence of a SrtA inhibitor is expected to show low fluorescence
intensity due to inhibition of SrtA.
A rotor fluorogenic D-amino acid (RfDAA)-based inclusion
assay recently developed by Hsu et al. has been successfully
applied to track the transpeptidation reaction involved in
peptidoglycan biosynthesis.115 RfDAAs are terminal D-lysine-
linked fluorescent probes which show fluorescence turn-on
response upon integration into the bacterial cell wall. The assay
has also been demonstrated to track SrtA-catalyzed trans-
peptidation reactions and could facilitate HTS of SrtA-targeting
inhibitors.
4. INHIBITORS OF SORTASE A
Infectious diseases are re-emerging at an alarming rate globally
due to bacterial resistance against conventional antibiotics. To
win the battle against infectious diseases, our strategy for
combating bacteria needs to change. In this context, attacking the
bacterial virulence target in the host cell is perhaps the most
effective option.31 We have a reasonably good structural and
biological understanding of the SrtA enzyme which is involved
in the virulence of S. aureus. Molecules that target bacterial
virulence would avoid the emergence of drug-resistant strains.
The emergence of multidrug-resistant organisms is having a
huge impact on the human health sector and is the driving force
for the development of anti-virulence agents as therapeutics.
In this Perspective article, we have analyzed all the sortase
inhibitors reported in the literature and classified them into
three general classes: peptidomimetics, natural product-based,
and synthetic small molecules. Further, we have selected those
inhibitors that showed better inhibitory activities, and they are
presented in tabular forms under each class. This will provide
both an extensive and a quick overview of each class, allowing
medicinal chemists to further fine-tune their structural intuition
to generate more potent inhibitors.
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In 2002, two separate research teams reported inhibition of S.
aureus SrtA based on synthetic and natural sources.116,117 A
multitude of publications have been published since then
regarding the SrtA inhibitors. Although SrtA inhibition studies
seem to indicate that all three aforementioned classes of
molecules have great potential, many of them have only
demonstrated in vitro anti-SrtA activity, with very few of them
exhibiting strong inhibitory properties. Consequently, to date,
there remains a paucity of clinically relevant candidates.
A critical look at the inhibitors reveals that only a handful of
them belong to peptidomimetics, though enormous design
possibility exists. The same is true for natural product-based
sortase inhibitors. Screening of a limited number of marine and
plant-based natural products identified potent SrtA inhibitors,
while activities of plenty of natural products are unknown.
Synthetic small molecules are a treasure trove of inhibitors. The
enormous chemical space available for design and the power of
combinatorial and diversity-oriented approaches are not fully
exploited. The involvement of talented and creative medicinal
chemists is urgently needed.
Although HTS is used for the efficient discovery of synthetic
small-molecule SrtA inhibitors, leading to the identification of
many hits, some of them may be falsely positive.118,119
Compounds that are reactive to proteins, aggregates, or that
interfere with the assay are likely to show false-positive results.
Compounds such as rhodanines, hydroxyphenylhydrazones,
catechols, benzofurazans, quinones, pyrroles, and thiophenes are
some of the Pan Assay INterference compoundS (PAINS).120
Such problematic classes of compounds have also been reported
as sortase inhibitors. The removal of such compounds using a
PAINS filter is necessary before further optimization. It is further
possible that some of the real inhibitors might also be
inappropriately classified as nuisance compounds.121 Therefore,
an electronic filter to remove PAINS, and then a detailed
experimental follow-up is necessary.
Although the exact mechanism of SrtA inhibition remains
uncertain for many compounds, hypothetical mechanisms based
on docking and experimental data have been proposed. Broadly,
the inhibitors are found to inactivate SrtA through either
covalent attachment or non-covalent binding to the enzyme’s
active site (Figure 5). The non-covalent inhibitors can bind the
enzyme’s active site through hydrogen bonding, van der Waals,
electrostatic, and π−π interactions, while covalent inhibitors
react with the sulfhydryl group of Cys184 present at the active
site of the enzyme. Covalent inhibitors can thus irreversibly bind
the active site of the enzyme, thereby blocking the LPXTG-
containing bacterial proteins from binding SrtA. This con-
sequently leads to the failure of bacteria to present surface
proteins, leading to virulence attenuation. The following
sections outline various literature reports focused on the
identification and development of SrtA inhibitors.
4.1. Peptidomimetics as Sortase A Inhibitors. A large
number of bioactive peptides have been discovered and show
great promise as drug candidates. While peptide-based
molecular structures have several advantages from the point of
view of “molecular recognition”, they also have some inherent
limitations. These limitations are primarily in terms of their
stability toward proteolysis, poor selectivity due to inherent
flexibility being a short peptide, and rapid clearance from the
body, hindering pharmaceutical applications. Therefore, pepti-
domimetic compounds have been developed to address these
shortcomings.
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Figure 5. General schematic representation of SrtA-catalyzed trans-
peptidation reaction and SrtA inhibition.
Peptidomimetics could overcome the limitations associated
with bioactive peptides but at the same time mimic the
conformation and other structural features of the native
peptides.122,123 Peptidomimetic inhibitors are developed based
on the structures and conformations of the native peptide
ligands; hence, they could bind the biological targets with high
specificity. The strategy adopted to develop peptidomimetic
inhibitors for SrtA involves designing structural mimics of the
LPXTG motif, keeping the necessary parts of the ligand for
molecular recognition. The Thr-Gly peptide bond of the
LPXTG motif is replaced by a stable and cleavage-resistant
bond to block transpeptidation. Peptidomimetics are therefore
considered an excellent choice as SrtA inhibitors.
4.1.1. LPAT-Coupled Diazoketone, Chloromethyl Ketone,
and Vinyl Sulfone. Walker and co-workers synthesized
peptidomimetic inhibitors by replacing the scissile Thr-Gly
bond of the natural substrate (LPXTG) of SrtA with
diazomethyl ketone (1a) and chloromethyl ketone (1b) groups
(Table 1).116 Both compounds showed an irreversible time-
dependent inhibition, as a result of alkylation of active site
Cys184. Compounds 1a and 1b exhibited inhibitory constants
(Ki) around 0.2 μM. Their identical values of Ki indicated that
both inhibitors have an equal affinity of interaction toward SrtA.
However, it was observed that 1b exhibited ∼2-fold higher first-
order rate constant of inactivation (ki) (1.1 ± 0.1 × 10−2 min−1)
than 1a, which was attributed to the higher electrophilicity of the
chloromethyl group over the diazomethyl group. Thus, 1b
showed greater effectiveness as a SrtA inhibitor, with a second-
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order rate constant of inhibition (k2nd, ki/Ki) of (5.3 ± 0.6) × 104
M−1 min−1.
Similarly, an N-protected LPAT-containing peptidomimetic
has been designed with vinyl sulfone at the C-terminus (1c,
Supporting Information (SI) Table S1).124 Compound 1c
exhibited irreversible inhibition of SrtA by covalent attachment
through the vinyl sulfone group of 1c to the SrtA active-site thiol.
Its enzyme inhibition efficiency was quantified by evaluating the
kinetic constants Ki (9 μM), ki (4 × 10−4 min−1), and k2nd (44.4
M−1 min−1). The lower ki and k2nd of 1c over those of 1a and 1b
indicate slow binding to SrtA, which is in agreement with the low
electrophilicity of the vinyl sulfone moiety.
4.1.2. Phosphinic Peptides as SrtA Inhibitors. To get insight
into the mechanism by which the substrate-derived inhibitors
function, McCafferty and co-workers designed compound 2 (SI
Table S1), with the scissile Thr-Gly bond replaced by a non-
hydrolyzable phosphorus−carbon bond.125 Preliminary inhib-
itory analysis of 2 indicated a reversible and competitive SrtA
inhibition, with IC50 = 10 mM. The poor SrtA inhibition efficacy
of 2 indicated the need for an accurate design for the
development of more potent inhibitors.
4.1.3. Leu-Pro-Ala Tripeptide Derivative. Jung et al. reported
an N-protected Leu-Pro-Ala tripeptide linked to 3-amino-4-
mercapto-2-butanol via an amide linkage exhibiting SrtA
inhibition.126 Incubation of SrtA in pH 8 buffer with 5 M of
the tripeptide followed by reverse-phase HPLC analysis
indicated covalent modification of SrtA via the formation of a
disulfide bond involving the thiol group of the tripeptide
inhibitor and active-site Cys184.
4.1.4. Peptide Macrocycles. Driven by the high selectivity,
binding affinity, and enhanced molecular interactions of bicyclic
peptides, Heinis and co-workers screened bicyclic peptides for
SrtA inhibitory activity.114 These bicyclic peptides showed
substantially better SrtA inhibition than the many potent small-
molecule inhibitors. Phage display and HTS of peptides against
SrtA revealed that bicyclic peptide(s) containing the Leu-Pro-
Pro motif, similar to the LPXTG motif, were more potent
inhibitors of SrtA. In vitro inhibition studies performed on 13
shortlisted bicyclic peptides showed 3a (ACPQLPPCRVSCG)
and 3b (ACTQRCPQLPPCG) (Table 1) as the potent
inhibitors, with inhibitory constants of 4.3 ± 0.3 and 2.4 ± 0.3
μM, respectively. Substitution of amino acids in the second ring
of 3a by alanine shows no reduction in the inhibitory activity,
indicating that the second ring is not interacting with SrtA.
However, the substitution of Arg and Gln by Ala in 3b reduced
the activity, indicating that the amino acids of the first ring are
involved in the interaction with SrtA. Bicyclic peptide 3b was
shown to exhibit a high binding selectivity for SrtA over other
proteins possessing surface-accessible cysteine. The increased
activity of inhibition shown by bicyclic peptides
ACTSRCPQLPPCG and ACHSRCPQLPPCG over 3b in-
dicates that inhibitors can be made more potent by optimizing
the amino acids in the first ring. The treatment of S. aureus with
the bicyclic peptide ACHSRCPQLPPCG reduced the incorpo-
ration of the fluorescein-labeled LPETG substrate into the
bacterial cell wall, thus supporting SrtA’s inhibition activity.
4.1.5. Leu-Pro-Arg-Asp-Ala Peptide. Virtual screening of
various oligopeptides revealed that pentapeptide Leu-Pro-Arg-
Asp-Ala (4, Table 1) exhibited SrtA inhibition with IC50 = 10.6
μM.127 Moreover, treatment of S. aureus with peptide 4 hindered
bacterial adhesion, biofilm formation, and bacterial invasion. In
vivo studies on Bagg Albino C (BALB/c) mice indicated that S.
aureus-induced mastitis was effectively treated with 4. This
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Table 1. Chemical Structures of Selected Peptidomimetic SrtA Inhibitors and Their Binding Parameters
a
Second-order rate constant of inhibition/inactivation potency (M−1 min−1). bApparent dissociation constant (μM). cIC50(μM).
highlighted the potential of 4 as an effective anti-virulence agent.
To determine the structure of the enzyme−inhibitor complex,
molecular modeling studies were performed. The studies
revealed that 4 binds to the SrtA active site and interacts with
Asn114, Ser116, Gln172, Trp194, and Arg197 residues (Figure
6).
Future Prospects and Outlook. Only a limited number of
peptides have been screened for SrtA inhibitory activity. Within
this limited number of compounds screened, peptide 4 showed
promising activity; hence, efforts are needed in the design of
peptide-based SrtA inhibitors. Peptidomimetics are potential
drug candidates; hence, more efforts in this direction are
urgently needed for the discovery of potent inhibitors. The
progress toward the discovery of SrtA inhibitors depends on the
combined efforts of organic and medicinal chemists. Well-
developed synthetic methodologies available today along with
rational de novo design of inhibitors will provide hope for the
discovery of potent SrtA inhibitors.
4.2. Natural Products as Sortase A Inhibitors. Natural
products have been used as medicines throughout the human
history. This knowledge of natural products as medicines came
as a result of hundreds of years of trial and error experiments.
Many natural products are current drug candidates.128
Interestingly, less than 10% of natural products are evaluated
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for biological activity; hence, many potential drugs are possible
in the future. It is noteworthy that many natural products are still
unidentified, and enormous opportunities for drug discovery are
still open.
Natural products are widely distributed in chemical space and
display enormous structural and chemical diversity; hence, they
are a treasure chest for potential SrtA inhibitors. However, only a
few natural products have been investigated, many of which
show good inhibitory effects. In the following section, the
natural products that have been investigated for SrtA inhibition
are discussed.
4.2.1. Marine Natural Products. Oceans cover about 70% of
the Earth’s surface and provide diverse conditions such as low
oxygen and lack of light compared to the terrestrial environment.
Consequently, the physiological adaptation of organisms to
these conditions increases the likelihood of finding structurally
unique natural products.129,130 In the past decade, a plethora of
marine natural products with new structures and diverse
bioactivities have been discovered.131,132
4.2.1.1. Indole Alkaloids. Alkaloids containing indole units
are commonly found in marine invertebrates. The bis(indole)
alkaloids 5a−5i (Table 2 and SI Table S2) were isolated from
the bright yellow marine sponge Spongosorites sp., consisting of
two indole moieties connected via heterocyclic units.133 Such
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Figure 6. (a) Structure of SrtA−4 complex determined using docking studies. (b) Modeled structure showing interactions between 4 and SrtA. From
ref 127, CC BY 4.0.
heterocyclic compounds are abundant in marine sponges. The
compounds 5a−5d belong to the topsentin class, while 5e−5i
belong to the hamacanthin class. Among these compounds, 5c
showed the highest anti-SrtA activity, having IC50 = 39.6 ± 1.3
μM.
4.2.1.2. Psammaplin. A previously characterized natural
compound, psammaplin A (6, Table 2), isolated from the
sponge Aplysinella rhax showed SrtA inhibition with IC50 = 47.4
μM and MIC > 200 μg/mL.134 Treatment of S. aureus with 6
reduced the fibronectin-binding ability of bacterial cells. The
compound also exhibited moderate anti-SrtB activity.
4.2.1.3. Aaptamines. Four aaptamines isolated from the
marine sponge Aaptos aaptos showed strong inhibitory activity
against S. aureus SrtA.135 Among them, isoaaptamine 7 (Table
2) was found to be the most potent SrtA inhibitor, with IC50 =
16.2 ± 0.9 μM. Structure−activity relationship (SAR) studies
highlighted the crucial role of the methyl group at the N-1
position of 7 for SrtA inhibition. The MD-simulated structure of
the SrtA-7 complex revealed hydrogen-bonding interactions of
the hydroxyl and nitrogen of 7 with Glu105 and Lys196 of
SrtA.136 Strong hydrophobic interactions of the methoxy and N-
methylamine of 7 with the enzyme’s Pro91, Ala104, and Gly192
were also evident from the MD-simulated structure.
4.2.1.4. Discorhabdins. Twelve compounds, including
(−)-3-dihydrodiscorhabdin 8a and (−)-discorhabdin 8b
(Table 2), were isolated from the dark green sponge Sceptrella
sp.137 The pyrroloiminoquinone alkaloids 8a and 8b showed
SrtA inhibition with IC50 = 75.7 and 6.5 μM, respectively.
4.2.1.5. Halisulfate. Bioactive metabolites from marine
sponge Coscinoderma sp. showed inhibitory activity against
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SrtA.138 Two sesterterpenes, 9a and 9b (Table 2), showed IC50
= 36 and 98 μM, respectively.
4.2.1.6. β-Carboline Alkaloids. Six β-carboline alkaloids
10a−10f (SI Table S2) isolated from marine Korean ascidian
Synoicum sp. showed inhibitory activity toward SrtA.139 Several
derivatives of these compounds also showed weak to moderate
SrtA inhibition.
4.2.1.7. Cadiolides and Isocadiolides. Five tris-aromatic
furanones and four related bis-aromatic diesters were isolated
from the ascidian Synoicum sp. collected from off the coast of
Chuja-do, South Korea.140 The structures of these compounds
were determined by spectroscopic analysis. Many of these
isolated compounds exhibited SrtA inhibition. Among them,
cadiolide 11 (Table 2) was found to be the most potent SrtA
inhibitor, with an IC50 value of 78.8 μM. However, it also
displayed high antibacterial activity against S. aureus, with MIC =
3.9 μM, which is not a desirable property of an anti-virulence
agent.
Recently, polybrominated aromatic compounds extracted
from Korean Synoicum sp. exhibited moderate SrtA inhibition
activity.141 Among them, isocadiolide A 12a and isocadiolide H
12b (Table 2) moderately inhibited SrtA activity, with IC50 = 67
and 70 μM, respectively.
4.2.1.8. Naphthoquinone. Herqueidiketal 13 (Table 2) was
isolated from marine-derived fungus Penicillium sp. The
compound showed inhibitory activity against SrtA, with IC50 =
23.6 μM and with moderate cytotoxicity.142
4.2.1.9. Lumichrome. Marine-derived Streptomyces sp. is a
storehouse of novel antibiotics. Lumichrome 14 (SI Table S2)
was extracted from the Streptomyces sp. MBTH32 strain, which
was isolated from marine sediment of Shinjin Island, Republic of
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Table 2. Chemical Structures of Selected Marine Natural Products as SrtA Inhibitors with Their IC50 Values

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Table 3. Chemical Structures of Selected Plant-Based Natural Products as SrtA Inhibitors with Their IC50 Values

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Table 3. continued
a
IC50 in μg/mL. bSecond-order rate constant of inactivation (M−1 min−1).
Korea.143 The compound exhibited SrtA inhibitory activity, IC50
= 198.20 ± 0.94 μM, without affecting the viability of S. aureus.
4.2.1.10. Polyketide. Recently, a series of alkaloids and one
polyketide were isolated from Aspergillus sp. F452. Among them,
the polyketide Aspermytin A 15 (SI Table S2) showed higher
SrtA inhibition with IC50 = 146 μM.144 The SrtA inhibition by
Aspermytin A resulted in the reduction of bacterial adhesion to
the fibronectin-coated surfaces.
4.2.2. Plant-Based Natural Products. Plant-based natural
products continue to play an important role in the discovery of
compounds with medicinal properties, therefore a cornerstone
of drug discovery and development. Plants are found in every
habitable environment and are the storehouse of a wide variety
of natural products with diverse structures and high medicinal
properties. A large proportion of the world’s population relies on
plant-based natural products for medicines.145,146 A very small
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proportion of plant species have been evaluated for the discovery
of bioactive compounds. Thus, the future exploration of plants
as a source of novel SrtA inhibitors may lead to more potent
inhibitors or serve as pointers for developing highly active SrtA
inhibitors.
4.2.2.1. Isoquinoline Alkaloid. The powdered rhizome of
Coptis chinensis was extracted, and it showed inhibitory activity
toward SrtA.147 Chloride and sulfate salts of berberine (16a and
16b, Table 3), palmatine chloride 17, and β-hydrastine 18 were
found to be the components that showed SrtA inhibition.
4.2.2.2. Curcumin. Methanol extract from Curcuma longa L.
rhizome showed inhibitory activity toward SrtA. Chromato-
graphic separation of the components revealed the presence of
curcumin 19a, demethoxycurcumin 19b, and bisdemethoxy-
curcumin 19c (Table 3 and SI Table S3).148 Curcumin showed
higher potency for SrtA inhibition with IC50 = 37.5 ± 1.9 μM,
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Figure 7. MD-simulated structure of the 20b-Glc−SrtA complex. From ref 161, CC BY 4.0.
followed by 19b (IC50 = 70.3 ± 1.8 μM) and 19c (IC50 = 103.5
± 3.9 μM). All these compounds showed MIC > 543.3 μM,
which indicates no effect on the growth of S. aureus. Molecular
docking studies showed that these compounds bind to the
catalytic region of SrtA, making contacts with Pro163, Gln172,
Ile182, and Ile199.149
S. mutans is a Gram-positive bacterium associated with dental
cavities in humans.150 It is regarded as the major contributor of
tooth decay and is known for its ability to form biofilm on dental
surfaces, referred to as dental plaque.151,152 Biofilm formation
starts with the initial attachment of S. mutans to the tooth
surface. The adhesion can be mediated by surface proteins,
including protein antigen c (PAc).153,154 The PAc protein is
covalently anchored to the bacterial cell wall by SrtA-mediated
transpeptidation. Thus, targeting SrtA is a powerful approach for
the prevention of dental cavities.155 This is supported by the
decreased biofilm formation ability of SrtA knockout strains of S.
mutans consequently reducing caries formation.156 Curcumin
was found to inhibit SrtA with IC50 = 10.2 ± 0.2 μM, leading to
reduced biofilm formation by S. mutans.77,157
4.2.2.3. Flavonoids. Flavonoids constitute an important class
of natural products widely found in fruits, vegetables, and
beverages. These natural compounds having polyphenolic
structures can be classified into flavones, flavonols, flavanones,
isoflavones, and anthocyanidins. Flavonoids have been success-
fully demonstrated to possess many biological and pharmaco-
logical activities, including anti-cancer, anti-Alzheimer’s, anti-
inflammatory, and anti-oxidative effects.158 Several flavonoids
exhibiting SrtA inhibitory activity have also been reported.
Among the series of flavonoid-based inhibitors, a few promising
compounds are discussed below.
Oh and co-workers analyzed the SrtA inhibitory activity of a
series of naturally occurring flavonols and identified that morin
20a, quercetin 20b, and myricetin 20c are active against
recombinant SrtA with IC50 = 37.39 ± 3.14, 52.70 ± 1.56, and
44.03 ± 3.52 μM, respectively (Table 3).159 These compounds
exerted no growth inhibition effect on the S. aureus Newman
strain. Among these, compound 20a was found to be a more
potent SrtA inhibitor than 20b and 20c. Additionally, it also
inhibited SrtA from S. mutans with IC50 = 27.2 ± 2.6 μM and
reduced the biofilm formation of S. mutans without affecting the
bacterial viability.160
Compound 20b-Glc (Table 3), a 3-O-glycoside derivative of
20b, was also found to inhibit the activity of SrtA.161 In vitro
studies to determine SrtA inhibition efficacy revealed SrtA
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inhibition with IC50 = 71.8 ± 11.9 μM, with no influence on
bacterial cell viability. S. aureus Newman strain treated with 20b-
Glc revealed that it blocked adhesion of bacterial cells to
fibronectin-coated plates, supporting the inhibitory activity. MD
simulations indicated that 20b-Glc binds directly to the active
site of SrtA through multiple hydrogen bonding and hydro-
phobic interactions (Figure 7). The 4H-chromen-4-one moiety
of 20b-Glc showed strong interactions with Ala104 and Ser106.
In addition to these interactions, van der Waals interactions of
20b-Glc with Val166, Gly167, Val168, and Val193 stabilized the
SrtA−inhibitor complex.
Based on the beneficial pharmacological properties of
myricetin and its SrtA inhibition efficacy, Macedo and co-
workers investigated the anti-biofilm formation potency of 20c
and its 3-O-glycoside derivative 20c-Glc (SI Table S3).162 The
studies revealed that the inhibitor 20c-Glc has no influence on
bacterial cell growth but strongly inhibited its adhesion and
biofilm formation. MD simulations suggested that the inhibitor
20c binds to SrtA as its natural substrate. The SrtA inhibition
ability of myricetin was also evaluated using a FRET-based
enzymatic assay by Nitulescu and co-workers.104 The bioactivity
studies indicated strong SrtA-inhibition by 20c. Docking studies
revealed that myricetin non-covalently binds to the enzyme’s
active site and thereby hinders the interaction between SrtA and
LPXTG-containing natural substrate (Figure 8).104 The studies
indicated the involvement of SrtA residues, including Ala104,
Glu105, Asn114, Ser116, Gly167, Val168, Thr180, Ile182,
Arg197, and Ile199, in stabilizing the SrtA−20c complex.
The search for more potent SrtA inhibitors led to the
identification of another series of flavonoids isolated from the
roots of Sophora f lavescens. Among the tested compounds,
kurarinol 21 (SI Table S3) exhibited SrtA inhibitory activity,
with IC50 = 107.7 ± 6.6 μM.163 It was demonstrated to possess
weak antibacterial activity against S. aureus, with MIC = 219 μM.
Among 50 natural compounds screened using FRET assay,
acacetin 22 (SI Table S3) was demonstrated to significantly
inhibit S. aureus SrtA activity with IC50 = 128.4 ± 16.5 μM and
MIC > 3.60 mM.164 Further studies revealed that acacetin
reduced the surface display of bacterial proteins such as Fnbps
by inhibiting SrtA-catalyzed transpeptidation.
The MD-simulated structure of the SrtA−22 complex (Figure
9) suggested the placement of acacetin’s phenyl ring in the
hydrophobic pocket of SrtA, surrounded with Ala34, Pro36,
Leu39, Ala60, Gly61, and Trp136. Other interactions
responsible for SrtA binding include CH−π interactions
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Figure 8. The binding of 20c to the active site of SrtA highlighting the
molecular interactions stabilizing the 20c−SrtA complex. The structure
is based on molecular docking studies. From ref 104, CC BY 4.0.
Figure 9. Structure of the SrtA−22 complex determined by molecular
modeling. From ref 164, CC BY 4.0.
Figure 10. Structure of the SrtA−23 complex based on MD simulations. From ref 165, CC BY 4.0.
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between the phenyl ring of 22 and Trp136 and hydrogen
bonding of the inhibitor’s carbonyl oxygen with Arg139 and
Lys140. Furthermore, treatment with 22 protected BALB/c
mice from S. aureus-induced renal abscess and reduced the
mortality rate.
A naturally occurring flavonoid named Dryocrassin ABBA 23
(Table 3), previously extracted from Dryopteridis crassirhizoma-
tis Rhizoma, was found to exhibit SrtA inhibitory activity with
IC50 = 24.17 μM.165 MD simulations showed that 23 binds to
the SrtA active site due to its strong interactions with Ala104,
Glu105, Val166, Gly167, Val168, Ile182, Val193, and Trp194
(Figure 10).
Flavonoids 24 and 25 (Table 3), isolated from Spatholobus
suberectus Dunn., possessed SrtA inhibitory activity.166 Fla-
vonoid 24 showed IC50 = 96.1 μM and MIC > 4.7 mM, while
IC50 and MIC for compound 25 were found to be 74.9 μM and
>477.5 μM, respectively. In another report, naturally occurring
flavonoid, isovitexin 26 (apigenin-6-C-D-glucopyranoside, Table
3), was shown to inhibit SrtA with IC50 = 67.02 μM.167
Thus, many flavonoids can be used to treat S. aureus infections
by reducing SrtA-mediated adhesion of bacterial cells to host
tissues. However, these molecules bind to many unrelated
enzymes, and hence the results need to be evaluated with care.
The promiscuous binding behavior and the aggregation-prone
nature of hydrophobic flavonoids could interfere with the
assay.168
4.2.2.4. Sterolglycosides. Liliaceae is a pharmacologically
important plant family containing many steroidal alkaloids. The
genus Fritillaria belongs to the family of Lilliaceae which have
been used as expectorant and anti-hypertensive drugs and are
also prescribed to treat cough, bronchitis, and tumors. The
medically relevant compound, namely β-sitosterol-3-O-gluco-
pyranoside 27 (Table 3) isolated from Fritillaria verticillata
showed SrtA inhibitory activity with IC50 = 31.8 μM.134,169
4.2.2.5. Phenylpropanoids. Traditional Chinese medicines
consist of a large number of biologically active natural products.
Lonicerae f los has been used for the treatment of fever, sores, and
arthritis for many years in China. Chlorogenic acid 28 (Table 3)
is a major component of this medicinal herb. Compound 28
inhibited S. aureus SrtA with IC50 = 95.6 ± 15.7 μM. A high MIC
value of 2.9 mM indicates its negligible effect on bacterial
viability.170 Upon treatment of S. aureus with 28, the cells’ ability
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Figure 11. Structure of SrtA−38 complex determined by MD simulations. From ref 177, CC BY 4.0.
to adhere to fibrinogen-coated plates was quantified using cell
adhesion assay. The studies revealed that 28 inhibits the
adhesion of S. aureus to fibrinogen, hence reducing bacterial
virulence. Docking studies and MD simulations were performed
to determine the structure of the 28−SrtA complex. The studies
indicated that the phenyl unit of 28 binds to the substrate
recognition site of SrtA through hydrophobic interactions with
Leu168 and Val169. In addition to this, hydrophobic
interactions between Ile182 and Cys184 of SrtA and the central
region of 28 stabilize the SrtA−28 complex. The studies also
indicated hydrogen bonding interactions of the guanidine
moiety of Arg197 with the hydroxyl groups on the cyclohexane
unit of 28.
In another study, five natural products structurally similar to
28 were examined for their SrtA inhibitory activity.171 The
bioactivity studies revealed that none of these compounds,
including caffeic acid 29a (SI Table S3; IC50 = 860.4 μM) and
ferulic acid 29b (SI Table S3; IC50 = 1323.5 μM), showed
improved SrtA inhibition over 28. High MIC values (>5.75
mM) for compounds 29a and 29b indicated their minimal effect
on S. aureus viability. However, the cyclized derivative of caffeic
acid 30 (Table 3) showed higher S. aureus SrtA inhibition, with
IC50 = 36.16 μM.104
Organic extract from dried roots of Pulsatilla koreana, based
on the Korean folk medicine, exhibited anti-SrtA activity against
S. mutans.172 Detailed chromatographic analysis of the extract
showed several lignans and phenylpropanoids with SrtA
inhibitory activity. Among the isolated 12 natural products,
compounds 29a and 31 (SI Table S3) exhibited the highest
inhibition against S. mutans SrtA, with IC50 = 20.2 and 20.1 μM,
respectively.
Recently another phenylpropanoid, 32 (SI Table S3), isolated
from the pine leaves of Cedrus deodara, inhibited S. aureus
biofilm formation without affecting bacterial growth.173 The in
vitro experiments indicated that 32 inhibited SrtA trans-
peptidation activity with IC50 = 161.02 μM. SrtA inactivation
by 32 reduced the display of surface-anchored proteins that are
required for biofilm development. The MD-simulated structure
of the SrtA−32 complex revealed that 32 non-covalently binds
to the active site of SrtA, involving hydrogen bonding and
hydrophobic interactions. The SrtA residues involved in
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interactions with 32 include Gln113, Val166, Val168, Leu169,
Lys173, Gln178, Thr180, Arg197, and Val201.
4.2.2.6. Quinone Derivatives. Using HTS, three naturally
occurring compounds 33a, 33b, and 34 (Table 3) possessing a
quinone skeleton were identified as efficient SrtA inhibitors.174
The naphthoquinones, namely shikonin 33a and alkannin 33b,
previously isolated from the roots of Lithospermum erythro-
rhizon, strongly inhibited S. aureus SrtA with IC50 in the
nanomolar range (Table 3). However, both compounds were
found to be toxic to the bacterial cell, which is not desirable.
Although 34 exhibited weaker SrtA inhibition activity (IC50 =
6.2 ± 1.1 μM) than 33a and 33b, it was found to have little
influence on the viability of S. aureus even at high
concentrations. Compound 34 irreversibly inhibits SrtA via a
Michael addition reaction involving the attack of the enzyme’s
reactive thiol group on the carbon−carbon double bond of
34.174 The values of ki, Ki, and k2nd were quantified to be 3.88 ×
10−2 min−1, 3.57 μM, and 1.1 × 104 M−1 min−1 (Table 3),
respectively. Reduction in biofilm formation upon treatment
with 34 was also observed. In another study, naturally occurring
naphthoquinones juglone 35a (Table 3) and plumbagin 35b
with strong SrtA inhibition potency were discovered.105
Naphthoquinones 35a and 35b covalently attach to SrtA, like
34.
4.2.2.7. Bisbenzyl Derivatives. A naturally occurring neutral
bisbenzyl compound, erianin 36 (Table 3), could inhibit SrtA
with IC50 = 65.7 ± 7.2 μM and MIC 1.6 mM.175 In silico analysis
revealed that 36 binds to the active site of SrtA, involving
interactions such as electrostatic interactions with Arg197,
hydrogen bonding interactions with Val193, and hydrophobic
interactions with Ala92, Ala104, Asn112, Ser116, Ile182,
Cys184, Trp194, and Ile199.
4.2.2.8. Chalcone. Chalcone 37 (Table 3) was demonstrated
to exhibit inhibitory activity against SrtA, with an IC50 = 53.15
μM.176 The compound 37 also reduced the surface display of the
Spa protein in S. aureus and altered the bacteria’s fibronectin-
binding ability in a concentration-dependent manner. In
addition to this, 37 reduced the biofilm formation and the
host-invading ability of S. aureus. In vivo studies showed a lower
mortality rate in S. aureus-infected mice upon treatment with 37.
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Table 4. Chemical Structures of Selected Synthetic Small Molecule SrtA Inhibitors and Their Binding Parameters

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Table 4. continued

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Table 4. continued

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Table 4. continued

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Table 4. continued
a
Apparent dissociation constant (μM). bSecond-order rate constant of inhibition (M−1 min−1). *Compound is a disulfide dimer.
4.2.2.9. Salvianolic Acid A. Salvianolic acid 38 (Table 3),
isolated from dried roots and rhizomes of Salvia miltiorrhiza
Bunge, was tested to determine its SrtA inhibition efficacy using
the FRET-based enzymatic assay.177 The bioactivity studies
revealed that 38 possesses potent anti-SrtA activity with IC50 =
11.6 μM. The compound was found to suppress the surface
expression of the Spa protein and reduced S. aureus adhesion to
fibrinogen and biofilm formation. The MD-simulated structure
of the SrtA−38 complex (Figure 11) and mutational analysis on
SrtA revealed that Ala92, Ala104, Ile182, and Arg197 play
indispensable roles in the binding of 38 to SrtA.
In vivo studies on S. aureus-infected C57BL/6J mice indicated
an 80% enhancement in survival rate upon treatment with 38.
Moreover, a 100% survival rate in infected mice was observed
upon co-administration of the antibiotic latamoxef along with
38.
4.2.2.10. Coumarins. A series of coumarins, 39a−39e (Table
3), isolated from Poncirus trifoliata showed SrtA inhibitory
activity in the range of 19.1−34.5 μM and had no influence on S.
aureus growth.178 Further studies revealed that treatment of S.
aureus with the coumarin-based inhibitors 39a−39c diminished
their adhesion ability toward fibronectin- and fibrinogen-coated
surfaces. This supported the anti-SrtA activity of coumarins, due
to a reduction in the surface display of fibronectin- and
fibrinogen-binding proteins.
Future Prospects and Outlook. Although more screening of
natural products has been performed compared to that of
peptide-based molecules, in comparison to the available natural
product resources, this number is very low. The screening of
marine and plant-based natural products provided promising
candidates such as 8b, 34, 35, and 38; however, a large chunk of
natural products still available provides high hope for discovery.
Testing many more natural products for inhibitory activity is
necessary and will provide more potent candidates. From our
survey of the available data, it merits to mention that
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hydrophobicity seems to be a dominant component of the
molecular recognition. It is capable of attenuating SrtA binding
across the chemical space domains sampled by these diverse
structural motifs.
4.3. Synthetic Small-Molecule Sortase A Inhibitors.
HTS and virtual screening of small-molecule chemical libraries
have been used to discover novel SrtA inhibitors. This has led to
the evolution of more potent synthetic inhibitors with high
selectivity, fewer off-target effects, and higher anti-virulence
activities. As indicated by the structure of SrtA, residues Ala92,
Ala104, Val168, Leu169, Ile182, Val193, and Trp194 with
lipophilic side chains constitute a hydrophobic binding pocket in
the proximity of the enzyme’s active site. Nonpolar groups of the
synthetic inhibitors including aromatic rings and alkyl chains can
interact with the SrtA residues in the hydrophobic pocket.
Inhibitors can interact via π−π interactions with aromatic side
chains and the polar functionalities of SrtA and can also form
hydrogen-bonding and electrostatic interactions. Inhibitors can
reversibly or irreversibly inactivate SrtA by non-covalent and/or
covalent interactions.
4.3.1. Vinyl Sulfones. Based on the inactivation of cysteine
proteinases by vinyl sulfones,179 McCafferty and co-workers
examined the SrtA inhibition activity of a series of vinyl
sulfones.180 Among them, vinyl sulfones 40a and 40b (SI Table
S4) were identified as attractive candidates as SrtA inhibitors
with IC50 = 736 and 190 μM and k2nd = 20.1 and 90 M−1 min−1,
respectively. Mass spectrometric analysis revealed that 40a
reacted with Cys184 present at the enzyme’s active site. The
covalent attachment of the inhibitors 40a and 40b through vinyl
sulfone groups to SrtA leads to the formation of a thioether
adduct. Compound 40b was more active than 40a due to the
presence of electron-withdrawing −CF3 group. The influence on
the adhesion of the S. aureus Newman strain treated with 40a to
fibronectin-coated plates was also quantified. The studies
revealed that 40a reduced bacterial adherence to fibronectin-
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coated surfaces, indicating the potential of compound 40a as an
effective SrtA inhibitor.
4.3.2. Diarylacrylonitriles. Based on the random screening of
a small-molecules library of 1000 compounds and further in vitro
activity, Kim and co-workers reported diarylacrylonitrile 41a,
with IC50 = 231.09 ± 6.53 μM (SI Table S4), as a hit
compound.181 SAR studies on 41a revealed that replacement of
the ester group by carboxylic acid (41b), amide (41c), or
hydrogen (41d) led to a reduction in SrtA inhibitory activity.
However, substitution of the ester by nitrile (41e) showed
enhancement in SrtA inhibition with IC50 = 187.40 ± 3.96 μM.
The loss in inhibition tendency of 41f, a saturated analog of 41e,
outlined the role played by a double bond for effective
inhibition. The (Z)-isomer 41g (Table 4) exhibited ∼7-fold
more anti-SrtA potency than the (E)-isomer 41e, thus
suggesting that the presence of a nitrile group and suitable
orientation of phenyl rings are important for diarylacrylonitriles
to interact with SrtA. To further improve SrtA inhibition
potencies, compounds 41h−41l (Table 4) were synthesized.
Among them, compound 41j showed the highest activity against
SrtA (IC50 = 9.24 ± 0.47 μM).
Molecular modeling studies revealed non-covalent interac-
tions between SrtA and compound 41j (Figure 12). The phenyl
Figure 12. Structure of the SrtA−41j complex highlighting the
underlying molecular interactions. Adapted from ref 181. Copyright
2004 American Chemical Society.
rings of 41j interacted with side chains of Ala92, Ala104, Val168,
Leu169, Ile182, Val193, and Trp194 present in the enzyme’s
hydrophobic cavity. In addition to this, hydrogen bonding
interaction between the nitrile group of 41j and guanidine unit
of Arg197 facilitated enzyme binding.
In another study, inhibitor 41j was demonstrated to prevent
bacterial infections by inhibiting SrtA and thus reducing
fibronectin-binding activity.134 Cytotoxicity analysis on the S.
aureus Newman strain revealed no influence on cell viability
upon treatment with 41j. Further, the in vivo investigations
Scheme 2. Mechanism of SrtA Inhibition by AAEKs 43a and 43b
a
Deprotonation. bβ-Elimination. cMichael-type conjugate addition.
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conducted on BALB/c mice showed that intravenous
administration of 41j to S. aureus-infected mice reduced kidney
and joint infections and increased mice survival rate.182
4.3.3. Prolinated Vinyl Sulfones. Based on SrtA inhibition by
vinyl sulfones180 and the importance of the nitrile group in
enzyme binding,181 Kudryavtsev et al. synthesized a series of
compounds with vinyl sulfone or nitrile groups functionalized on
a pyrrolidine scaffold.183 Inhibitors appended with a nitrile
group showed partial SrtA inhibition even at high concentration.
Among the inhibitors evaluated, compounds 42a−42c (SI Table
S4) exhibited SrtA inhibition, with IC50 ranging from 0.85 to 1.3
mM and k2nd around 104 M−1 min−1. Kinetic and mechanistic
studies revealed irreversible time-dependent enzyme inhibition
by covalent attachment of the inhibitors through the vinyl
sulfone group to the active-site Cys184. The high potency of
prolinated vinyl sulfones over simple alkyl-vinyl sulfones180
outlined the importance of additional non-covalent hydro-
phobic interactions in stabilizing the enzyme−inhibitor
complex.
4.3.4. Aryl(β-amino)ethyl Ketones. Schneewind and co-
workers screened a library of 135 625 small molecules and
shortlisted 6154 compounds displaying >20% SrtA inhib-
ition.184 Subsequent shortlisting based on their reactivity,
toxicity, and drug-like properties gave 407 compounds. The
promising hits were further analyzed, which led to the
identification of aryl(β-amino)ethyl ketones (AAEKs) 43a and
43b (Table 4) exhibiting Bacillus anthracis and S. aureus SrtA
inhibitory activities. The IC50 values of AAEKs 43a and 43b for
S. aureus SrtA were found to be 47 and 15 μM, respectively.
AAEK 43a was shown to irreversibly inhibit SrtA by covalent
attachment to the enzyme’s active site. This involves
deprotonation of AAEK, followed by β-elimination and attack
by active-site Cys184 on the generated electrophilic olefin
intermediate (Scheme 2).
4.3.5. Rhodanines, Pyrazolethiones, and Pyridazinones.
HTS of a small-molecule library containing ∼30 000 com-
pounds and sequential elimination of compounds based on the
percentage of SrtA activity inhibition gave 44 compounds as
potential inhibitors.185 Among these, only 10 compounds were
subjected to further studies based on their inhibitory activity and
similarity in physicochemical properties to known drugs. The
IC50 evaluation of these compounds yielded three lead
compounds belonging to families of rhodanines, pyrazole-
thiones, and pyridazinones. These lead compounds were
demonstrated to reversibly bind the enzyme’s active site, as
indicated by kinetic studies and mass spectrometric analysis.
SAR studies on lead inhibitors were performed to discover more
potent SrtA inhibitors.
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The IC50 values of 13 analogs of hit compound 44 (Table 4)
revealed that none of the structural modifications resulted in
higher SrtA inhibition.185 Induced-Fit Docking studies of 44
into the S. aureus SrtA active site indicated hydrophobic
interactions of the two methyl groups of 44 with Val166 to
Val168 and Ile199. The enzyme−inhibitor complex was
stabilized by hydrogen-bonding between the nitro and the
hydroxyl groups of 44 with SrtA His120, Tyr187, and Trp194. In
the docked molecular structure of the complex, the carbonyl
oxygen of 44 was oriented toward Arg197 and the sulfide toward
His120. Rhodanine 44 influenced the bacterial cell viability and
thus is not a promising candidate for the development of anti-
infective agents against S. aureus infections.
Based on the SAR analysis of 45a, compound 45b (Table 4)
was synthesized and found to exhibit higher SrtA inhibition
efficacy. Molecular docking studies revealed hydrophobic
interactions of the inhibitor’s phenyl rings with the side chains
of Pro94, Val166, Val168, and Trp194 residues in the enzyme’s
hydrophobic pockets (Figure 13). Significant enhancement in
Figure 13. Modeled structures of (a) 45a and (b) 45b in the active site
of S. aureus SrtA, generated by Induced-fit docking. Adapted with
permission from ref 185. Copyright 2009 Elsevier.
SrtA inhibition potency was observed upon replacement of the
nitrophenyl group in 45a with a more lipophilic tribromophenyl
substituent. The methyl and thione groups of the pyrazole
nucleus were found to be in contact with Ala92 and Arg197 in
the 45b−enzyme complex (Figure 13). Replacement of thione
in 45a by ketone yielded 45c, with significantly reduced potency
of inhibition, whereas replacing phenyl group by an electron-
withdrawing pyridyl group (45d) increased the SrtA inhibition
efficacy. The compound 46 (Table 4) was found to exhibit lower
anti-SrtA activity over 45a. Compounds 45a, 45b, and 45e
exhibited high SrtA inhibitory activity, and no detrimental effect
on bacterial growth was observed, thus indicating the potential
of pyrazolethione compounds as SrtA-targeting anti-virulence
agents.
Among the pyridazinone inhibitors, lead compound 47a
(Table 4) and its derivatives, 47b−47g, were identified as
potentially active SrtA inhibitors. The SAR studies performed on
hit compound 47a highlighted the significant influence of
substitution pattern on both phenyl and pyridazinone rings.
Replacement of the methoxy group by an ethoxy along with the
removal of chlorine positioned on the phenyl ring of 47a gave
inhibitor 47e. The ethoxy-thiol pyridazinone inhibitor 47e was
found to be one of the efficient SrtA inhibitors, with IC50 = 13 ±
1 μM.
Replacement of thiol by hydroxyl and removal of the chlorine
unit in 47a gave 47h, which significantly reduced the inhibitory
activity. The symmetrical (47g) and asymmetrical (47i and 47j)
disulfide derivatives of 47e were also found to be potent SrtA
inhibitors.
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Docking studies revealed that the phenyl ring of 47a is
suitably placed in the enzyme’s hydrophobic pocket. The
carbonyl oxygen in 47a is positioned toward Arg197, whereas
the thiol group points toward His120 at the SrtA active site. The
observed decrease in inhibition ability of 47k over 47e indicates
the importance of the suitable placement of the thiol group.
Among the compounds tested, 47c and 47f do not exert
bactericidal activity and hence are the most promising
pyridazinone inhibitors.
Representative compounds belonging to the rhodanine,
pyrazolethione, and pyridazinone series were subjected to
combined pharmacophore and 3D quantitative SAR studies to
identify pharmacophoric features.186 In later studies, to outline
the mechanism of SrtA inhibition by 47e and to gain insight into
the SrtA−47e complex, the water-soluble sodium thiolate
analog 47l (Table 4) was synthesized.187 Mass spectrometric
analysis indicated the formation of a disulfide bond between
SrtA active-site Cys184 and the thiolate of 47l. The NMR
solution structure of the SrtA−47l complex revealed hydro-
phobic interactions between phenyl ring of 47l and Leu97,
Val166, Val168, Leu169, Ile182, and Val193 of SrtA (Figure 14).
Figure 14. Structure of the SrtA−47l complex (a, b) showing
interactions of 47l with residues in the hydrophobic pocket of SrtA.
Adapted with permission from ref 187. Copyright 2017 John Wiley &
Sons.
Stronger inhibition was observed upon substituting R4 with a
fluoro (47m) or methoxy (47n) group or positioning an
amidine substituent at the R3 position (47o, Table 4). The rate
constant, k2nd, for 47o was determined to be 3.6 × 104 M−1
min−1. The improved inhibitory activity is due to additional
interactions such as multipolar contacts (47m), hydrophobic
interactions (47m and 47n), and electrostatic interactions
(47o) with SrtA. The length of the hydroxyalkoxy moiety (47p
vs 47q) at the solvent-exposed R2 position influenced binding
toward SrtA. The compounds with hydroxyalkoxy moiety at R2
and fluorine at R4 position (47r and 47s) showed strong SrtA
inhibition with apparent dissociation constants in the nano-
molar range (Table 4). The high value of k2nd (1.8 × 105 M−1
min−1) for 47s indicated high SrtA inhibition potency. The
terminal hydroxyl of the 3-hydroxypropoxy group of 47s acted as
a hydrogen bond donor to the carbonyl oxygen of Gly192 and as
a hydrogen bond acceptor from the hydroxyl of Tyr187 (Figure
15). However, due to the shorter length of the alkyl chain at the
R2 position of 47r, the inhibitor did not form favorable
interactions with Tyr187 and Gly192; instead, it formed a single
hydrogen bond with Ala92, resulting in lower SrtA-binding
ability.
4.3.6. Morpholinobenzoate Derivatives. A commercial
small-molecule library comprising 150 000 compounds was
subjected to in silico virtual screening by Velu and co-
workers.188,189 Hit compounds were identified upon applying
limiting factors, such as molecular weight, hydrophobicity, and
distance from the SrtA active site. The 108 hit compounds
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Figure 15. 3D structures of SrtA−47r (a, c) and SrtA−47s (b, d)
complexes generated by docking the inhibitors to the NMR structure of
SrtA. Adapted with permission from ref 187. Copyright 2017 John
Wiley & Sons.
identified were further screened for inhibition against SrtAΔN59.
This led to the discovery of 48a (Table 4) as a lead candidate for
the development of SrtA inhibitors.
The FlexX docking model revealed hydrogen bonding
interactions of the morpholine ring oxygen, amide carbonyl
oxygen, and NH of 48a with Trp194, Ser116, and Glu105,
respectively.188 A salt bridge between the carboxylic acid of 48a
and guanidine side chain of Arg197 was also observed. The
orientation of thiophene and phenyl unit of 48a suggested that
appropriate substitutions on these rings could facilitate stronger
SrtA binding. The ester derivative 48b was found to exhibit
improved SrtA inhibition, with IC50 = 71 ± 2.5 μM.
Replacement of the thiophene ring in 48b by furan (48c)
showed enhancement in enzyme inactivation, whereas hydro-
genation of the double bond of 48b was found to destroy
inhibitory activity. Replacement of the carboxylic acid or methyl
ester by −CH2OH (48d), −CHO (48e), or −CONH2 resulted
in lower binding to the enzyme. The corresponding acetylenic
derivatives of 48a and 48b were found to possess less SrtA
inhibitory activity. In addition to the above structural
modifications, double bond configuration, amide hydrogen,
and morpholine ring oxygen, and carbonyl group influenced
SrtA inhibition efficacy. Among the series of morpholino-
benzoate derivatives, compound 48c turned out to be a
promising candidate for further development.
4.3.7. Dihydro-β-carbolines. Guided by the inhibitory
activities of indole-containing compounds 49a and 50a (SI
Table S4) against isocitrate lyase from Candida albicans,190
structurally analogous compounds were synthesized and
evaluated for their SrtA inhibitory activity.191 SAR studies on
49a revealed that retention of one of the two indole units and
both of the α-oxocarbonyl groups are critical to enzyme
inhibition. Among the series of compounds synthesized, 49b
(Table 4) and 49c showed the highest SrtA inhibition potency
with IC50 = 61 and 69 μM, respectively.
Structural optimization on compound 50a (SI Table S4)
highlighted that introduction of phenyl 50b (Table 4) and
methoxy group 50c led to dramatic increase in SrtA inhibition,
as indicated by their IC50 values. However, their corresponding
tetrahydro-β-carboline derivatives showed a loss of inhibitory
activity.
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4.3.8. Benzo[d]isothiazol-3(2H)-one-adamantane Amine
Derivatives. HTS of a small-molecule library containing 50 240
compounds against a catalytically active recombinant SrtAΔN59-
6His (SrtAΔN59 with six His units at the C-terminal) gave 41
active compounds with SrtA inhibition >65%.192 Subsequently,
to evaluate binding reversibility and to exclude false positives,
NMR studies on hit compounds were performed. The studies
confirmed that 28 compounds inhibited SrtA activity by
covalently modifying the enzyme’s active site. Among them,
compounds 51a and 52a (Table 4) were found to be promising
leads for further development of anti-infective agents.192,193
Compound 51a binds to the enzyme’s active site with high
affinity and inhibits SrtA with IC50 = 6.11 ± 0.34 μM. However,
it displayed significant bactericidal activity against S. aureus and
was found to be cytotoxic toward NIH 3T3 cells. To improve
SrtA inhibition efficacy and to reduce toxicity toward S. aureus
and NIH 3T3 cells, compound 51a was subjected to structural
optimization. This led to the identification of 51b and 51c
(Table 4) as active candidates. NMR spectroscopic studies
indicated the formation of the enzyme−inhibitor complex by
covalent attachment of 51a to the enzyme’s active-site thiol. The
formation of a disulfide bond by benzo[d]isothiazol-3(2H)-one
ring-opening of 51a by SrtA Cys184 was primarily responsible
for the observed inhibition. A complete loss in SrtA inhibition
was observed after the removal of the heterocyclic benzisothia-
zole nucleus or sulfur (51d and 51e, SI Table S4). The NMR-
based structure of the SrtA−51a complex revealed the
interaction of the phenyl ring with Leu97, Ala118, and
Trp194, while the adamantyl unit is suitably located in the
SrtA hydrophobic pocket, interacting with Val166, Val168,
Leu169, Thr180, Ile182, and Ile199. The linker connecting the
adamantyl and heterocyclic units plays a crucial role in SrtA
inhibition. It performs the dual function of strengthening SrtA
binding by forming a hydrogen bond with Arg197 and suitably
orienting the adamantyl group and the heterocyclic ring in a
conformation that maximizes SrtA binding.
Compounds with extended linkers (51a vs 51b and 51c)
showed slightly higher inhibition activity. In comparison to 51a,
inhibitors 51b and 51c reduced the unfavorable influence on
bacterial cell growth and showed ∼10-fold decrease in
cytotoxicity against NIH 3T3 cells.
In another study, the authors demonstrated the potential of
compound 52a (Table 4) as a SrtA inhibitor due to its fast
enzyme binding kinetics and low cytotoxicity.193 Results based
on NMR spectroscopic and mass spectrometric analyses
revealed that the inhibition mechanism involved nucleophilic
attack of Cys184 thiol on the chiral carbon of 52a. The reaction
proceeds via the formation of an olefin intermediate generated
after β-elimination of the 2-aminopyridine unit. The NMR
studies also indicated hydrophobic interactions of the thiophene
unit of 52a with Leu97 and Ala118 and π−π stacking
interactions with His120 and Trp194 (Figure 16). Replacement
of the thiophene by a substituted phenyl ring (52b−52e) led to
small changes in their inhibitory activity with little influence on
cytotoxicity.
4.3.9. 2-Phenyl-2,3-dihydro-1H-perimidine Derivatives.
Virtual screening of a library containing 55 789 compounds by
molecular docking led to the identification of 53a (Table 4) as a
SrtA inhibitor with IC50 = 47.2 ± 5.9 μM.194 The mechanism of
SrtA binding by 53a was investigated using MD simulations and
SAR analysis. The molecule 53a was shown to function by
blocking the SrtA active site through non-covalent interactions.
MD simulations revealed hydrogen bonds between the carboxyl
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Figure 16. Structure of SrtA−52a complex showing hydrogen bonding
and π−π stacking interactions. The structure was determined by
molecular docking studies. Adapted from ref 193. Copyright 2019
American Chemical Society.
group of 53a and His120 and Arg197 (Figure 17). They also
indicated the existence of a hydrogen bond between the
Figure 17. Structure of the SrtA−53a complex generated by molecular
docking. From ref 194, CC BY-NC-ND 4.0.
dihydroperimidine moiety and SrtA Pro163, providing addi-
tional stability to the SrtA−inhibitor complex. The naphthyl ring
of 53a interacts with amino acids in the enzyme’s hydrophobic
pocket that led to further enhancement in SrtA binding affinity.
The suitably oriented polar substituents on the phenyl ring
facilitated hydrogen-bonding with Gly119, Thr183, and Cys184.
The in vitro inhibition potency of various 53a derivatives 53b−
53e (Table 4 and SI Table S4) supported the binding
mechanism.
4.3.10. 3,6-Disubstituted Triazolothiadiazoles. Virtual
screening of a small-molecule library containing about 300 000
drug-like molecules revealed 105 compounds exhibiting SrtA
binding.112 Further, evaluation of the inhibition activity of the
shortlisted compounds against SrtAΔN24 (SrtA with 24 residues
truncated from N-terminus) led to the identification of the 3,6-
disubstituted triazolothiadiazole derivative (54a, Table 4) as a
SrtA inhibitor. Structure optimization of the enzyme−substrate
complex with hit compound 54a afforded compounds 54b and
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54c with higher SrtA inhibition potency. Due to the high in vitro
SrtA inhibitory activity and water solubility, inhibitor 54b was
subjected to surface plasmon resonance studies to unravel the
SrtA inhibition mechanism. The studies indicated that 54b
binds to the active site of SrtA, resulting in reversible inhibition.
Treatment of S. aureus with 54b was found to have no influence
on bacterial growth, thus highlighting the efficacy of 54b as an
anti-infective agent. To evaluate its in vivo SrtA inhibition
activity, BALB/c mice infected with S. aureus Newman strain
were injected with 54b. The studies demonstrated that 54b
effectively prevented bacteremia caused by S. aureus.
4.3.11. 2-Phenylhydrazinylidene Derivatives. Maggio and
co-workers screened 200 randomly selected compounds for
SrtA inhibition and identified 55a (SI Table S4) as a lead
compound.195 The SAR studies performed on 55a indicated
that the presence of electron-withdrawing groups on the phenyl
ring increased the anti-SrtA activity of 2-phenylhydrazinylidene
derivatives, whereas the activity decreased with electron-
donating substituents. Among the derivatives, compounds
with chlorine (55b, Table 4) and nitro (55c) substituents
emerged as strong SrtA inhibitors, with IC50 = 50 and 57 μM,
respectively. The anti-biofilm formation ability of synthesized
derivatives was examined against S. aureus ATCC 25923, ATCC
29213, and ATCC 6538 strains. Among these, compound 55d
was found to reduce the biofilm formation ability of S. aureus by
60% for all three bacterial strains. Taking the studies further,
Cascioferro and co-workers synthesized another series of 2-
phenylhydrazinylidene derivatives, including 55e−55i, and
evaluated them for anti-biofilm and anti-SrtA activity.196 The
compounds 55e and 55f (Table 4) were found to be active
against SrtA, with IC50 = 75 and 76.5 μM, respectively. However,
compounds 55g−55i (SI Table S4) showed weak SrtA
inhibition, with IC50 > 200 μM. The ability of these compounds
to suppress biofilm formation by S. aureus ATCC 25923, ATCC
29213, and ATCC 6538 strains was also examined. The studies
indicated that 55e significantly reduced the biofilm formation
ability of S. aureus ATCC 6538 (IC50 = 0.9 μM). Compounds
55f, 55g, and 55h inhibited biofilm formation by the S. aureus
ATCC 29213 strain, with IC50 = 29, 0.8, and 1.7 μM,
respectively. In addition to this, compounds 55h and 55i
decreased the biofilm formation ability of all three S. aureus
strains. The comparison of bioactivities revealed no correlation
between SrtA inhibition and anti-biofilm formation activity for
the phenylhydrazinylidene derivatives, which can be attributed
to the completely different assays employed for the evaluation.
4.3.12. 2-Phenyl-benzo[d]oxazole-7-carboxamide Deriva-
tives. A series of 2-phenyl-benzo[d]oxazole-7-carboxamide
derivatives were synthesized and evaluated for their activity as
SrtA inhibitors.197 Compounds 56a−56e (Table 4) showed
SrtA enzyme inhibition, with the lowest IC50 values among the
series. The replacement of −OH with −OCH3 (56c) had
minimal influence on their SrtA inhibition potency. However,
replacement of −OH by −NO2 in 56a and 56f (Table 4) gave
56g and 56h (SI Table S4) with lower SrtA inhibitory activity.
The studies also indicated that the isobutyl group is
indispensable for good inhibition (56b vs 56i−56k, SI Table
S4). The docking studies revealed that 56d interacts with
residues Ala118, Val166, Val168, Val169, and Ile182 through
hydrophobic interactions, with Cys184 and Arg197 through
hydrogen-bonding interactions, and with Tyr194 through π−π
stacking interactions.
4.3.13. 2-Phenyl-benzofuran-3-carboxamide Derivatives.
Based on the discovery of 2-phenyl-benzo[d]oxazole-7-carbox-
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amide as potent SrtA inhibitors,197 Fu and co-workers designed
a series of 2-phenyl-benzofuran-3-carboxamides derivatives
57a−57n as SrtA inhibitors.198 A total of 37 compounds with
L-shape similar to the LPXTG motif were synthesized. The
higher IC50 value of compound 57a (SI Table S4) over 57b
(Table 4) indicates that the isobutyl group plays a crucial role in
binding with SrtA. SAR studies indicate a correlation between
the number of hydroxyl groups and the potency of inhibition.
Compounds 57c, 57d, and 57j bearing two hydroxyl groups are
more active than 57a, 57b, 57e, 57f, and 57i. The nature of the
substituents at the terminal phenyl ring was found to
significantly influence the inhibitory activity. The higher IC50
of 57g and 57h (SI Table S4) over 57f and 57j (Table 4)
indicated that replacement of the −OH group(s) by −CF3 or
−NO2 reduced the SrtA inhibition activity. In addition to this,
substitution of −OH by −SH, −NH2 (57b vs 57k and 57l, Table
4), or −F (57b vs 57m, SI Table S4) also reduced the efficacy of
inhibition. Replacement of the ester group as the linker (57e) by
an ether (57n) leads to a decrease in inhibitory activity.
Compounds 57c, 57d, and 57j are the most potent inhibitors
among the series of compounds evaluated. Molecular docking
simulations revealed that the isobutyl group of 57d interacts
with Thr164, Val166, Val168, Leu169, and Ile182 SrtA residues.
A hydrogen bonding interaction of the ester carbonyl of 57d
with Cys184 and Arg197 is also indicated (Figure 18).
Figure 18. Structure of the SrtA−57d complex determined using
molecular modeling. Adapted with permission from ref 198. Copyright
2017 Elsevier.
4.3.14. Thiadiazole Derivatives. HTS of two commercially
available small-molecule libraries containing ∼28 500 com-
pounds gave 110 hits. Further concentration-dependent studies
and applying the PAINS filter led to the identification of
thiadiazole 58a (Table 4) as an inhibitor of SrtA.199 NMR
experiments revealed that 58a binds to SrtA, and so it was
selected as the starting point for further structural optimization.
A series of structural modifications on 58a, such as removal and
addition of different groups, were done to optimize the structure
for obtaining good inhibitors. Structure optimization of 58a led
to the identification of inhibitors 58b and 58c. Among them, 58c
with IC50 = 3.8 ± 0.3 μM was found to possess the strongest SrtA
inhibition activity without influencing S. aureus viability.
Molecular dockings using standard and induced fit approaches
showed that 58c occupies the active site (Figure 19). The
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Figure 19. MD-simulated structure of the covalent adduct formed by
58c and Cys184 of SrtA. Adapted with permission from ref 199.
Copyright 2019 Elsevier.
docking studies revealed Arg197 and His120 make non-covalent
interactions with the p-nitrophenyl and the pyridine rings of 58c.
The liquid chromatography−mass spectrometry (LC-MS)
analysis showed the formation of covalent disulfide adduct,
supporting the irreversible inhibition.
4.3.15. Thiadiazolidinone Derivatives. Thidiazolidinone
scaffold is a well-known pharmacophore in drug discovery. It
is present in tideglusib (59a, Table 4), a candidate under clinical
trial for myotonic dystrophy. Tideglusib is an irreversible
inhibitor of glycogen synthase kinase 3β and is also under clinical
investigation for the treatment of Alzheimer’s disease.200 HTS of
2400 drug candidates using FRET assay led to the identification
of tideglusib with strong in vitro SrtA inhibition.113 SAR studies
gave compounds 59b−59i, showing inhibition potency
comparable to that of 59a, with minimal influence on S. aureus
viability.
The SrtA inhibitory activity of the derivatives was determined
using the FRET and PAGE-based assays. The orthogonal
PAGE-based assay could rule out any false positives from the
FRET assay. Further studies revealed that 59a showed reversible
inhibition by non-covalently binding to the enzyme’s substrate-
binding pocket (Figure 20). The docked structure of the SrtA−
Figure 20. Modeled structure of the SrtA−inhibitor complex showing
potential binding modes between tideglusib (59a) and SrtAΔN59.
Adapted from ref 113. Copyright 2020 American Chemical Society.
59a complex indicated π−π stacking of the naphthyl ring and
hydrogen bonding interactions of the sulfur atom of 59a with the
active-site His120 and Arg197. The in vivo experiments revealed
that 59a is a potential candidate for developing anti-virulence
agents.
4.3.16. Alkaloid Derivatives. Diana and co-workers synthe-
sized a series of topsentin analogs133 and evaluated their biofilm
inhibition efficacy.201 Many of the synthesized 1,2,4-oxadiazole
topsentin analogs inhibited biofilm formation in Gram-positive
S. aureus ATC25923 and Gram-negative P. aeruginosa
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ATC15442. Among them, compounds 60a−60c (Table 4)
displayed higher biofilm inhibition in S. aureus ATCC25923
with BIC50 (concentration that showed 50% inhibition of
biofilm formation) of 9.7, 0.7, and 2.2 μM, respectively. These
compounds were also demonstrated as strong SrtA inhibitors
with IC50 = 2.2 μM (60a), 10.4 μM (60b), and 2.2 μM (60c),
with minimal influence on bacterial growth.
4.3.17. Disulfanylbenzamides. Based on the previously
reported benzisothiazolinones inhibitor 51a,192 Schirmeister
and co-workers designed a series of disulfanylbenzamide
derivatives 61a−61h (Table 4) containing disulfide warhead
chemotype and evaluated their SrtA inhibition efficiencies.202 In
vitro and in silico studies revealed that the majority of
disulfanylbenzamide derivatives showed time-dependent and
irreversible inhibition of SrtA. The irreversible inhibition is
confirmed by LC-MS analysis, which revealed the disulfide
adduct. The presence of reducing agents like 1,4-dithiothreitol
(DTT) or tris(2-carboxyethyl)phosphine (TCEP) during
inhibition was also investigated to find the mode of inhibition.
Disulfide reducing agents DTT and TCEP could typically regain
the enzymatic activity in the presence of covalent inhibitors.
Molecular modeling studies using the NMR structure of SrtA
(Figure 21) indicated that the side-chain carbonyl oxygen of 61a
Figure 21. The structure of SrtA−61a complex indicating the
molecular interactions. The structure was generated by docking 61a
in the NMR structure of SrtA. From ref 202, CC BY-NC-ND 4.0.
is hydrogen bonded to Arg197. The hydrophobic interactions
and π−π stacking interactions assisted the formation of
enzyme−inhibitor complex. The biochemical studies indicated
that 61h showed significant SrtA inhibition, efficient reduction
in fibrinogen binding, weak bactericidal activity, and low
cytotoxicity toward mammalian cells. In addition to these,
compound 61h showed selectivity toward S. aureus SrtA over
other structurally related proteases.
Future Prospects and Outlook. Various research groups have
attempted to find small-molecule SrtA inhibitors. However, only
a few promising candidates have been identified that could serve
as a starting point for further development of SrtA inhibitors
with therapeutic potential. Pyridazinone-based small-molecule
covalent inhibitor 47s is a highly promising candidate with high
SrtA inactivation efficiency.187 The in vivo studies on diary-
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lacrylonitrile derivative 41j showed decreased mortality in S.
aureus-infected mice.182 The triazolothiadiazole derivative 54b
showed good SrtA inhibition and reduced mortality in S. aureus-
infected mice. The compound 54b is under preclinical
evaluation.112,203 Diana and co-workers reported 1,2,4-oxadia-
zole derivatives 60a−60c that showed very promising activity
against biofilm formation, and their non-toxicity against human
cells is worth mentioning.201
5. CONCLUSIONS AND FUTURE PERSPECTIVES
Antibiotics that are available today act due to their bacteriostatic
or bactericidal properties. The indiscriminate use of antibiotics
resulted in the emergence of drug resistance that has wreaked
havoc worldwide. It is an imminent threat to public health, and
therefore new strategies for combating antibiotic resistance are
urgently needed.
The widespread and dangerous S. aureus infections demand
effective anti-infective therapy. SrtA as a biological anti-virulence
target for therapeutic intervention is promising, since the
inhibition against SrtA may not put selective pressure on
bacteria resulting in the emergence of drug-resistant strains. In
addition, the extracellular location of SrtA and the absence of a
eukaryotic homolog make SrtA an attractive target. Therefore,
SrtA inhibition appears to be an excellent anti-infective
therapeutic strategy.41
Although a sufficient number of SrtA inhibitors are known
today, many inhibitors are not further pursued. The majority of
the SrtA inhibitors were evaluated in vitro, and the interactions
are rationalized based on in silico studies. Only a limited number
of SrtA inhibitors, for example, 41j and 54b, have been studied in
vivo,182,112 and none of the reported SrtA inhibitors are currently
under clinical evaluation. This suggests that there is an urgent
need to follow up on the potent inhibitors for translation into
drugs.
A large number of the SrtA inhibitors have limited potency
and poor selectivity. Many of the inhibitors may be falsely
positive or promiscuous. Introduction of the PAINS filter might
improve the situation by removing bad ones and selecting the
true hit compounds.121 Optimization of the true SrtA inhibitors
from the present list of inhibitors will be a starting point for drug
development.118
Peptidomimetics are an important class of compounds in
medicinal chemistry since they are closer to the native ligands,
but with enhanced proteolytic stability, improved bioavailability,
and pharmacokinetics.122,123 Only a very limited number of
peptidomimetics SrtA inhibitors have been developed. Given
their central importance in drug discovery, more investigations
in this direction are needed. A diversity-oriented synthesis that
enables combinatorial variation of amino acid residues at various
positions of the native ligand will be a powerful approach for
finding peptidomimetic SrtA inhibitors.
Natural products or their derivatives constitute almost half of
the best-selling drugs.204 Many natural products have been
tested and showed promising SrtA inhibitory properties; hence,
more extensive screening is a way forward. Natural products
represent an untapped reservoir of drugs and hence are good
starting points for designing ligands for SrtA. Natural products
are biologically validated starting points in structural space for
library design.205 Focused libraries based on natural products
are, therefore, another way to obtain highly active inhibitors.
Synthetic molecules provide a rich alternative to natural
products and leverage the power of organic chemistry to invent
required molecular shape for a given chemical space. Natural-
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product-inspired library design is a promising approach for the
discovery of SrtA inhibitors. The available structural space for
synthetic molecules is high and unending. Though considerable
efforts have been put into the discovery of small-molecule SrtA
inhibitors, many potent structural units are not yet discovered.
Fine-tuning of the promising scaffolds and new entities still
awaits discovery.
The anti-virulence drug discovery is in its infancy. Though
anti-virulence drugs are urgently needed for combating
antibiotic-resistant bacteria, there are many challenges ahead.
The broad chemical diversity of SrtA inhibitors provides
important cues for the future design of effective SrtA inhibitors,
but of course, there are many more hurdles ahead.
Anti-virulence drugs act by attenuation of virulence; hence, it
is believed that no resistant strains will emerge. However, recent
studies have shown that resistance does exist, challenging the
evolution-proof hypothesis.32−34 Therefore, more effective
therapies are needed. In a recent report, salvianolic acid A, a
natural product that inhibits SrtA (IC50 = 11.6 μM), along with
the antibiotic latamoxef, protected mice against lethal pneumo-
nia caused by MRSA.177 This study indicates that the use of an
anti-virulence drug along with a known antibiotic compound is a
promising approach for combating antibiotic resistance.30,206
The good functioning of the host immune system is a
requirement for the effective functioning of the SrtA inhibitors
as drugs. This will be a problem for immunocompromised
patients; hence, the use of immune-stimulating agents along
with SrtA inhibitors is a viable option.
Finally, while the discovery of anti-virulence agents is still in
its infancy, the detailing of the many promising SrtA inhibitors
elaborated in this Perspective should galvanize the research
community to explore the possibilities on offer.
Concerted and focused efforts from synthetic organic
chemists and biologists are urgently needed to discover
molecules with high SrtA inhibition and their eventual
transformation to useful drugs. As this Perspective has shown,
based on a very limited screening and rather elementary small-
molecule design efforts, potent SrtA inhibitors have already been
discovered. In the future, the identification of peptide-based
molecules, natural products, and designed small molecules as
potent SrtA inhibitors will create a paradigm shift in the
treatment of bacterial diseases.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00386.
Chemical structures of peptidomimetic (Table S1),
marine (Table S2), and plant-based natural products
(Table S3), and synthetic small-molecule (Table S4) SrtA
inhibitors with IC50 > 100 μM (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
V. Haridas − Department of Chemistry, Indian Institute of
Technology Delhi, New Delhi 110016, India; orcid.org/
0000-0002-2931-0585; Phone: +011-26591380;
Email: haridasv@chemistry.iitd.ac.in
Nalin Pant − Department of Chemistry, Indian Institute of
Technology Delhi, New Delhi 110016, India; Phone: +011-
26591525; Email: nalinp@chemistry.iitd.ac.in
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Authors
Rachit Sapra − Department of Chemistry, Indian Institute of
Technology Delhi, New Delhi 110016, India
Amit K. Rajora − Department of Chemistry, Indian Institute of
Technology Delhi, New Delhi 110016, India; Present
Address: Pharmaceutical Sciences Laboratory, Faculty of
Science and Engineering, Åbo Akademi University, 20520
Turku, Finland
Pushpendra Kumar − Department of Chemistry, Indian
Institute of Technology Delhi, New Delhi 110016, India
Govind P. Maurya − Department of Chemistry, Indian Institute
of Technology Delhi, New Delhi 110016, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00386
Notes
The authors declare no competing financial interest.
Biographies
Rachit Sapra completed his Bachelor’s degree in 2014 from St.
Stephen’s College, University of Delhi, India. He completed his
Master’s degree from the same college in 2016. Currently, he is
pursuing his Ph.D. from the Department of Chemistry, Indian Institute
of Technology Delhi (IITD), under the supervision of Prof. V. Haridas.
His research interests include the design and synthesis of peptide-based
molecular sensors and nanoparticles for biomedical applications.
Amit Kumar Rajora has completed his Master of Science in Organic
Chemistry from Jamia Millia Islamia University, New Delhi, India, and
Ph.D. in Pharmacy (pharmaceutics) from the University of Reading,
United Kingdom. He has also worked as Research Associate at the
Indian Institute of Technology, New Delhi, India, and as a National
Postdoctoral Fellow at the Regional Centre for Biotechnology,
Faridabad, Haryana. Currently, he is working as a Postdoctoral Fellow
at Åbo Akademi University, Turku, Finland.
Pushpendra Kumar received his Bachelor’s (2017) and Masters’s
degrees in Chemistry (2019) from Dayalbagh Educational Institute,
Agra, India. He is interested in the design and synthesis of self-
assembling peptide molecules for biomedical and material applications.
Govind P. Maurya received his M.Sc. degree in Synthetic Organic
Chemistry from M. L. K. PG College, Balrampur (affiliated with RMLA
University, Faizabad, Uttar Pradesh, India), in 2014. He is currently a
Ph.D. student in the research group of Prof. V. Haridas in the
Department of Chemistry, IITD. His research interest is in the design
and synthesis of self-assembling peptides for biological applications.
Nalin Pant is a Professor in the Department of Chemistry at IITD,
India. His research group is involved in various aspects of molecular
structures, bioorganic chemistry, and constrained molecular systems.
V. Haridas is a Professor in the Department of Chemistry at IITD,
India. His research group is involved in the design and synthesis of a
variety of peptidic and pseudopeptidic molecules for biomedicinal
applications. In addition, the group is also working on various aspects of
synthetic and bioorganic chemistry.
■
ACKNOWLEDGMENTS
We thank DST-SERB (Project: EMR/2017/003192/OC) for
funding. R.S. and P.K. thank CSIR for the doctoral fellowships.
G.P.M. thanks University Grants Commission (UGC), New
Delhi, for the doctoral fellowship.
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■
ABBREVIATIONS USED
AAEK, aryl(β-amino)ethyl ketone; Abz, 2-aminobenzoyl; CDC,
U.S. Centers for Disease Control and Prevention; Dabcyl, 4-
([4′-(dimethylamino)phenyl]azo)benzoic acid; Dap, 2,3-dia-
minopropionic acid; DNP, 2,4-dinitrophenyl; Fnbp’s, fibronec-
tin-binding proteins; DTT, 1,4-dithiothreitol; EDANS, 5-[(2-
aminoethyl)amino]naphthalene-1-sulfonic acid; FRET, fluores-
cence resonance energy transfer; GN, N-acetyl glycosamine;
HPLC, high-performance liquid chromatography; HTS, high-
throughput screening; IC50, half-maximal inhibitory concen-
tration; IsdA, iron-regulated surface determinant A; k2nd,
second-order rate constant of inhibition; ki, first-order rate
constant of inactivation; Ki, inhibitory constant; LC-MS, liquid
chromatography−mass spectrometry; MD, molecular dynam-
ics; MIC, minimum inhibitory concentration; MN, N-
acetylmuramic acid; MRSA, methicillin-resistant S. aureus;
MSCRAMMs, microbial surface components recognizing
adhesive matrix molecules; OD, optical density; PAc, protein
antigen c; PAINS, pan assay interference compounds; PBS,
phosphate-buffered saline; SAR, structure−activity relationship;
SDS-PAGE, sodium docdecyl sulfate−polyacrylamide gel
electrophoresis; Spa, Staphylococcal protein A; SrtA, sortase
A; S. aureus, Staphylococcus aureus; S. mutans, Streptococcus
mutans; Sec, secretory; TCEP, tris(2-carboxyethyl)phosphine
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