Inhibition of Nonessential Bacterial Targets: Discovery of a Novel Serine O-Acetyltransferase Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00627

In ?-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 ?243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC₅₀ <100 μM were identified via an indirect assay using Ellman's reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition.

Full text of DOI:10.1021/acsmedchemlett.9b00627

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Letter
Inhibition of Non-Essential Bacterial Targets: Discovery
of a Novel Serine O-acetyltransferase Inhibitor
Joana Magalhaes, Nina Franko, Samanta Raboni, Giannamaria Annunziato, Päivi Tammela, Agostino
Bruno, Stefano Bettati, Andrea Mozzarelli, Marco Pieroni, Barbara Campanini, and Gabriele Costantino
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.9b00627 • Publication Date (Web): 13 Feb 2020
Downloaded from pubs.acs.org on February 16, 2020
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Inhibition of Non-Essential Bacterial Targets: Discovery of a Novel Serine O-acetyltransferase
Inhibitor
a
b
b,f
a
c
Joana Magalhães, Nina Franko, Samanta Raboni, Giannamaria Annunziato, Päivi Tammela,
Agostino Bruno, Stefano Bettati, Andrea Mozzarelli,b,e,f Marco Pieroni, a,g,* Barbara
a,†
d,e,f
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Campanini, Gabriele Costantino
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P4T group, and Laboratory of Biochemistry and Molecular Biology Department of Food and Drug,
c
University of Parma, 43124 Parma, Italy; Drug Research Program, Division of Pharmaceutical
Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), Helsinki,
d
FI-00014, Finland; Department of Medicine and Surgery, University of Parma, 43125 Parma, Italy;
e
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National Institute of Biostructures and Biosystems, Rome, Italy; Institute of Biophysics, CNR, Pisa,
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Italy; Centro Interdipartimentale “Biopharmanet-tec”, Università degli Studi di Parma, Parma,
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Italy; Centro Interdipartimentale Misure (CIM) ’G. Casnati’, University of Parma, Parma, Italy.
†
Present address: Experimental Therapeutics Program, IFOM – The FIRC Institute for Molecular
Oncology Foundation, Via Adamello 16-20139, Milano, Italy.
This article is dedicated to the truly brilliant memory of Prof Maurizio Botta, who has inspired
generations of researchers with his vision and sympathy.
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Abstract
In ϒ-proteobacteria and actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence,
and become dispensable during growth or acute infection. The biosynthetic machinery required to convert
inorganic sulfur into cysteine is absent in mammals, therefore it is a suitable drug target. We searched for
inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-
cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91,243 compounds
was performed to identify potential SAT inhibitors. Scaffold similarity and analysis of the overall physico-
chemical properties allowed the selection of 73 compounds that were purchased and evaluated on the
recombinant enzyme. Six compounds displaying an IC below 100 μM were identified via an indirect assay
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using Ellman’s reagent and then tested on a Gram-negative model organism, with one of them being able to
interfere with bacterial growth via SAT inhibition.
Keywords
Adjuvant therapies; Antimicrobial resistance; Cysteine biosynthesis; Non-essential targets; Serine
acetyltransferase.
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Introduction
Antimicrobial resistance (AMR) is one of the most threatening insults to public health worldwide. Features
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related to this upset are worrying, both in terms of loss of human lives and costs for hospitalization. Experts
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estimate that 10 million people will die every year due to antimicrobial resistance by 2050, unless proper
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measures are adopted to contain the emergence. AMR refers to the ability of microorganisms (not necessarily
bacteria, but also fungi, viruses, or protozoa) to nullify the therapeutic effects of antimicrobials, and it can
affect anyone, at any age, in any country. Generally, misuse of prescribed medicines, incorrect prescriptions
and poor prevention and control measures account for the development of AMR.3–5 The search for novel
druggable molecular targets different from those already exploited by the current antibacterial arsenal is crucial
to prevent the onset of cross-resistance for the newest molecules under investigation. Along with this
conventional approach, adjuvant strategies aimed at engaging non-essential targets, such as those related to
bacterial virulence and/or persistence after colonization of the host, are gaining growing interest.6–9 It has been
speculated that targeting those enzymes, which are dispensable during the normal bacterial life cycle, but that
become essential during host invasion, might have a lower impact on AMR because they facilitate the clearing
action of the immune system without stimulating the rise of resistance. In addition, by decreasing bacterial
fitness, adjuvants act as antibiotic activity enhancers. The majority of pathogens spend part of their life cycle
in extremely challenging environments like macrophages or the gastric mucosa, where survival and persistence
require a fine metabolic adaptation. In many cases, this adaptation is made possible through a series of sulfur-
containing biomolecules such as Fe-S clusters, thiamine, glutathione and biotin, which ensure bacterial
survival especially due to their detoxifying properties and reducing power.10–12 In this regard, blocking the
synthesis of cysteine, which is the most relevant building block for the above-mentioned biomolecules, holds
great promise as an adjuvant strategy to undermine bacterial fitness and promote eradication of the infection.
Cysteine metabolism has been indicated as a promising drug target in ϒ-proteobacteria (like Salmonella
enterica serovar Typhimurium) and actinomycetales (Mycobacterium tuberculosis),10,13 as it has been shown
that suppression or reduction of the cysteine biosynthesis led to a decreased bacterial fitness and infectivity.14,15
In both vegetative and swarm cell populations, inhibition of cysteine biosynthesis is associated with an
unpaired oxidative stress response, enhancing the antibacterial activity of common antibiotics and in some
cases restoring the activity of obsolete antibacterials.14,15 Finally, whereas mammals lack the biosynthetic
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machinery that leads to the de novo synthesis of cysteine from inorganic sulphur, bacteria and plants possess
highly conserved enzymes enabling its biosynthesis through the reductive sulphate assimilation pathway
(
Figure 1).10,16
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Sulphate and thiosulfate, the most abundant forms of extracellular sulphur, enter the cell through specific
transporters. Sulphate is then activated to be reduced and the entire process requires the consumption of one
mole of GTP, two moles of ATP and three moles of NADPH for each mole of cysteine produced. In E. coli
and Salmonella, after activation of sulphate by ATP with formation of adenosine 5´-phosphosulphate (APS),
3'-phosphoadenosine-5'-phosphosulfate (PAPS) is synthesized. Afterwards, PAPS is reduced to bisulfide that
is incorporated into cysteine by O-acetylserine sulfhydrylase (OASS) in the last step of cysteine
biosynthesis.17,18 This enzyme, present in two isoforms, OASS-A and OASS-B, depends on pyridoxal 5’-
phosphate and catalyses a -replacement reaction on O-acetylserine (OAS), synthesized by serine
acetyltransferase (SAT).19 It was demonstrated that inhibition of OASS was able to revert the bacterial
resistance to fluroquinolones and tetracycline.14,15 This has prompted us to undertake a medicinal chemistry
campaign aimed at discovering potent and selective OASS inhibitors, which culminated with the release of the
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most potent inhibitor of StOASS known so far for both the isoforms. Despite the remarkable inhibitory
activity, it was impossible to evaluate the efficacy for bacterial cells likely due to the scarce permeability of
the compound through the bacterial cell wall. After thorough investigation of the Structure-Activity
Relationships for these OASS inhibitors,21–24 we have deemed of interest to investigate the effects of SAT
inhibition, which is the upstream enzyme of the cysteine synthase complex.25–28 Of notice, inhibition of SAT
is expected to exert partially distinct effects with respect to those brought about by OASS inhibition. Indeed,
inhibition of OASS is expected to induce accumulation of OAS, which is converted to N-acetylserine (NAS),
a natural inducer of the biosynthetic operon,29 and cysteine depletion. Conversely, inhibition of SAT is
expected to deplete the cell not only of cysteine, but also of the natural inducer. SAT catalyses the rate-limiting
step of L-cysteine biosynthesis, where the acetyl group of acetyl-CoA is transferred to the hydroxyl group of
L-serine to generate OAS. From the structural point of view, SAT is a dimer of trimers, with an -helical N-
terminal domain and a carboxyl-terminal left-handed -helix domain ending with a flexible, disordered C-
terminal sequence that is fundamental for its function and regulation. The C-terminal peptide binds OASS-A
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at the active site and it is responsible for its competitive inhibition. Even though exploitation of SAT inhibition
holds promise as antibacterial adjuvant strategy, only a limited amount of literature about potential inhibitors
is reported. In one case, after a virtual screening based on E. coli SAT crystal structure, a compound with an
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ICSAT50 of 72 µM, (figure 2) was identified. However, in the following studies, the compound was found to
inhibit E. histolytica proliferation at a concentration of 0.61 µM, ruling out the possibility that inhibition of
SAT was the main mechanism of action. In another case, a small library of natural products was screened for
their inhibitory activity toward methicillin-resistant Staphylococcus aureus (MRSA) SAT, and six compounds
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inhibiting SAT activity (IC ranging from 29.83 μM to 203.13 μM) were found. Interestingly, in this case
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two of the hits (Figure 2, compounds 1 and 2) showed an inhibitory effect on MRSA growth (MIC at 12.5
μg/ml and 25 μg/ml). Inspired by these encouraging preliminary data, we herein report the virtual screening of
a library of compounds in order to find potential inhibitors of SAT. As the most relevant result, we were able
to identify a hit compound showing weak antibacterial properties, consistent with inhibition of SAT.
[insert Figure 2 here]
Results and discussion
Virtual Screening
A crucial step of a VS is the selection of the chemical library. In general, to cross bacterial membrane,
antibiotics must possess a series of peculiar physico-chemical characteristics, such as molecular weight (MW),
LogD and polar surface area (PSA), which are poorly represented in commercial chemical libraries.31–33
Therefore, compounds structures were retrieved from the ChemDiv Anti-infective (8,523), anti-bacterial
(5,460), and antiviral (77,260) focused library collections, for a total of 91,243 compounds. Ligand 3D
structures were prepared along the initial stage of the Schrödinger virtual screening workflow (vsw), where
compounds were (i) filtered according to the Lipinsky’s rule of five, (ii) removed when the stereochemistry
was undefined, (iii) protonated at pH 7.4 using “epik”, and (iv) clustered into conformers generated using the
enhanced sampling methods. The structure of EcSAT in complex with cysteine (1T3D) and the structure of
HiSAT in complex with CoA (1SSM) were used as protein structures for the VS (see Experimental section).
The VS identified a pool of 1,409 compounds, which was reduced to 1,391 hits after visual inspection. The
analysis of commercial antibacterials with activity towards Gram-negative pathogens suggests an average
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ClogD of -2.8 and a polar surface area of 165 Å as preferred characteristics. Therefore, 37 molecules with
PSA lower than 200 and ClogD lower than 2 were initially cherry-picked to undergo activity assays.
[Insert Figure 2 here]
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The analysis of the remaining 1.354 compounds was carried out using Chemgps software,34,35 in order to
evaluate scaffold similarity and purchase scaffolds as diverse as possible. Principal component (PC) analysis
was carried out and in Figure 3 the representation of the 1.354 molecules is depicted using three PCs: PC1
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(shape, size and polarizability), PC2 (aromaticity and conjugation) and PC3 (lipophilicity, polarity and H-bond
capacity). This process allowed for the selection of 36 compounds with diverse chemical structures. At the end
of the VS process, 73 compounds were purchased and evaluated for their ability to inhibit SAT in biochemical
assay.
Determination of StSAT inhibition
While StSAT three-dimensional structure has not been solved yet, our group has a long-standing expertise in
the characterization of cysteine biosynthetic enzymes from Salmonella, as above reported.11,17,23 Since our
ultimate goal is to develop molecules effective in blocking cysteine biosynthesis in Salmonella, we choose to
test the compounds identified in the VS on StSAT. The very high overall sequence identity between the two
proteins (i.e. EcSAT and StSAT, with 100% identity of the active site residues) supports the use of StSAT in
the activity assays. SAT catalyses the transfer of the acetyl group of acetyl-CoA to serine, with release of
CoASH. SAT activity can be determined by measuring the decrease in absorbance at 232 nm due to hydrolysis
of the thioester bond of acetyl-CoA.36,37 However, this methodology is not suitable for screening of compound
libraries since most organic molecules display high absorbance at 232 nm.
[Insert Figure 4 here]
[Insert Figure 5 here]
[Insert Scheme 1 here]
Since the transfer of the acetyl group of acetyl-CoA to serine occurs with release of the free thiol CoASH, we
used an indirect assay for the determination of SAT activity. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, also
known as Ellman’s reagent)) has a highly oxidizing disulfide bond that is stoichiometrically reduced by free
thiols originating a mixed disulfide and a molecule of 5-thio-2-nitrobenzoic acid (TNB) (Scheme 1). The
-1
-1 38
product TNB at pH higher than 7 absorbs strongly ( = 14,000 M cm ) and was thus used to follow the
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catalytic activity of the enzyme. The method was optimized and validated under our experimental conditions
through the determination of the kinetic constants for L-Ser and acetyl-CoA and the IC for the known
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inhibitor glycine (Figure 4). The results obtained agree with data in literature determined using the direct
assay.39 In particular, the IC50 for glycine is in very good agreement with the K 13 ± 4 mM previously
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reported. The method was adapted and validated to a 96-well plate format to allow higher throughput. At the
primary screening phase, the inhibitory activity of the 73 compounds from VS was determined at 100 µM
(
Figure 5). The obtained values for assay quality parameters, Z', SW and S/B, were robust enough to conclude
that this assay can be used for screening procedures.40,41 Inhibitory effects were first assessed by kinetic
measurements with 27s intervals, since the total number of compounds allowed such measurements.
Nevertheless, the 180s time-point was selected as the optimal endpoint measurement for calculation of
%
inhibition since it provided higher robustness of the parameters Z', SW and S/B. 13 compounds, displaying
a percentage of inhibition ≥ 40% in the primary screen, were re-tested at 100 µM and, after the confirmatory
testing, dose-response experiments were performed for 6 compounds displaying a percentage of inhibition
higher than 50% at 100 µM concentration. These 6 compounds (Table 1) displayed micromolar IC values
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towards StSAT with compound 7 being the most potent inhibitor with an IC of 14 µM (Table 1). Being aware
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that many Gram negative antibacterial drug discovery programs fail because of the scarce aptitude for
numerous small molecules to cross the cellular membrane, before establishing the most promising hit
compound to more forward we decided to investigate the in-cell antimicrobial activity of compounds 3-8.
[Insert Table 1 here]
Inhibitory effects on E. coli growth
The inhibitory activity of compounds 1-6 against the Gram-negative E.coli ATCC25922 was determined by
using broth microdilution assays. As mentioned, testing SAT inhibitors in rich medium would be of poor
significance for the purpose of this study. In fact, the cysteine present in the growth medium would be
transported inside the cells thus reversing the inhibition of its biosynthesis. Therefore, minimum inhibitory
concentrations (MICs) were evaluated both in conventional Muller-Hinton broth (MHB) and in minimal media
(LB 20%) with limited amount of nutrients, where cysteine is absent and thus the effect of inhibiting its
biosynthesis would lead to growth inhibiting effects. Compound 3 was able to interfere with bacterial growth
in LB20% (Table 1), although at a fairly high concentration of 64 µg/mL. In addition, it can be speculated that
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SAT inhibition is indeed accountable for the activity of compound 3, as in rich medium, MHB, the MIC was
not reached even at the highest concentration tested (128 µg/mL). Results were confirmed upon re-synthesis
of compound 3, previously used from a commercial source.
Binding modes of compound 3
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The binding modes of the substrate cysteine and compound 3 are reported in Figure 6. Cysteine is bound inside
EcSAT active site through a network of H-bonds with Asp92, Asp157, His158, Arg192, and His193 (Figure
6
a, dashed black lines). Interestingly, 3 establishes: (i) similar interaction with Asp92, Asp157, Arg192, and
His193; (ii) a hydrophobic interaction with His158; (iii) further H-bond interaction with Gln133 and Gln258
(Figure 6b). The comparison of the binding modes of both cysteine and 3 reveals how compound 3 is able to
mimic the interactions observed for cysteine (Figure 6c, d).
[Insert Figure 6 here]
In summary, antimicrobial resistance (AMR) is currently one of the most serious threats to public health, and
developing strategies for its containment is a crucial pharmaceutical challenge. Adjuvant strategies, such as
targeting the biosynthesis of molecular building blocks that are crucial for bacterial adaptation upon host
invasion, represent an intriguing approach to face the onset of bacterial resistance. Among these strategies,
inhibition of the biosynthesis of cysteine, an amino acid involved in the synthesis of detoxifying biomolecules
such as glutathione, holds promise for further investigation. The VS of three focused libraries from ChemDiv
containing 91,243 compounds was carried out in order to deliver an inhibitor of bacterial SAT, an enzyme
responsible for the penultimate step of cysteine biosynthesis. The process led to the disclosure of 73 virtual hit
compounds that were purchased and tested against StSAT. A screening methodology using Ellman's reagent
to indirectly measure SAT activity was developed in 96-well plate format, yielding 6 compounds displaying
an IC below 100 μM that were further evaluated for growth inhibitory effects against E. coli. To rule out off-
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target effects, MIC assays were carried out in both complete and minimal media, and we were pleased to notice
that compound 3 retained an encouraging inhibitory activity towards Gram-negative E. coli in cell-based assay.
Although aware that 2-aminothiazoles may cause a false positive, and taking into consideration the call made
by some editors of the ACS journals with regard to the early identification of PAINS, we would like to point
out that derivatives 3-8 were not recognized as PAINS using the software “False Positive Remover”
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http://advisor.bkslab.org/).
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Supporting Information The Supporting Information is available free of charge athttps://pubs.acs.org/.
Description of methods used to set up the virtual screening, protein expression and purification,
determination of StSAT inhibition, and evaluation of MIC are reported in the supporting information.
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Corresponding Author information marco.pieroni@unipr.it
Author Addresses Food and Drug Department, University of Parma, Parco Area delle Scienze 27, 43124,
Parma
Author Contributions JM: synthesis, virtual screening, set up of the StSAT assay and preliminary
microbiological investigation; NF: data collection, data analysis, SR: data collection and analysis, manuscript
revision; GA: data analysis, manuscript revision; PT: Supervision of StSAT screening and MIC determination,
manuscript revision; AB: computational analysis, manuscript revision; SB: study design, resources,
manuscript revision; AM: resources, study design and supervision, manuscript revision; MP: supervision,
manuscript writing and revision; BC: study design, supervision, manuscript writing and revision; GC:
resources, supervision, manuscript revision.
Acknowledgments: This work was funded under the MSCA-ITN-2014-ETN project INTEGRATE [grant
number 642620]. PT thanks the Academy of Finland (Grant. no. 277001) for financial support. The “Centro
Interdipartimentale Misure “G. Casnati” is kindly acknowledged for the contribution in the analytical
determination of the molecules synthesized.
Abbreviations AMR, antimicrobial resistance; CDI, 1,1'-Carbonyldiimidazole; DTNB, 5,5'-dithiobis-(2-
nitrobenzoic acid); MHB, Muller-Hinton broth; MRSA, methicillin-resistant S. aureus; NAS, N-acetylserine;
OASS, O-acetylserine sulfhydrylase; PSA, polar surface area; SAT, serine acetyltransferase; VS, virtual
screening.
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Reductive sulphate assimilation pathway in E. coli and Salmonella. OASS and SAT are highlighted. The
enzymes that catalyse sulfate reduction are: E1, ATP sulfurylase; E2, APS kinase; E3, PAPS sulfotransferase;
E4, NADPH sulfite reductase.
Figure 2
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H
OH
H
NCI 6535443
1
2
Chemical structure of some SAT inhibitors reported in literature.26,28
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Figure 3
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Graphical representation of the principal components of the 1,354 compounds. PC1 (shape, size and
polarizability), is represented in the x axis, PC2 (aromaticity and conjugation) is in y axis and PC3
(lipophilicity, polarity and H-bond capacity) is represented in z axis.
Figure 4
0.03
0.03
0.02
0.01
0.00
1.0
0.8
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0.0
A
B
C
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.00
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0
30
60
90
120
[
L-Ser] (mM)
[acetyl-CoA] (mM)
[Gly] (mM)
Validation of the indirect assay for the screening of potential SAT inhibitors. Panel A: initial reaction rate as
a function of L-Ser concentration. The line through data points represents the fitting to the Michaelis-Menten
-1
-1
-1
equation with kcat = 3813 ± 169 min , K = 1.07 ± 0.15 mM, k /K = 3563 min mM . Panel B: initial
m
cat
m
reaction rate as a function of acetyl-CoA concentration. The line through data points represents the fitting to
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the Michaelis-Menten equation with kcat = 3128 ± 186 min , K = 0.17 ± 0.04 mM, k /K = 18400 min
m
cat
m
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-
1
mM . Panel C: relative activity as function of glycine concentration. The line through data points represents
the fitting to a hyperbolic equation with IC = 15 ± 1 mM.
5
0
Figure 5
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Determination of the inhibitory potential of the 73 purchased compounds against StSAT at time point 180s.
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Figure 6
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a) X-ray crystal structure of EcSAT in complex with Cys (PDB code: 1T3D). The two monomeric chains of
SAT are depicted as blue and pink transparent cartoon, amino acid side chains are depicted as sticks and color
coded depending on the chain they belong to, the feedback inhibitor Cys is depicted in cyan sticks, while
relevant H-bonds are depicted as dashed black lines; b) Binding mode of 3 (orange sticks) into SAT active
site, the color code is the same reported in a); c) Superposition of the SAT-Cys and SAT-3 complexes,
highlighted with dashed line are H-bonds conserved in both complexes (cyan dash lines for Cys, orange ones
for 3); d) Superposition of 3 (orange sticks) and Cys (cyan sticks).
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Scheme 1
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Indirect assay of SAT activity. CoA released by SAT activity spontaneously react with DTNB with the release
of one molecule of TNB for each molecule of CoA produced.
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Table 1 – StSAT inhibition, IC50StSAT, and MIC values against E. coli of the hits derived from Virtual
Screening.
MIC
MIC
%
@
Inhibition
100µM
IC50
Cmpd
3
Structure
(µg/mL) in (µg/mL)
LB 20%
(µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in MHB
N
N
O
N
S
NO2
NO2
S
68
48.6±8.43
47.8±5.35
64
>128
HN
N
O
S
HN
N
S
4
5
72
57
>128
>128
>128
>128
N
NH2
O
O
O
O
84.1±5.39
HN
S
N
N
H
O
NH2
O S
O
2
False
positive
6
52
>128
>128
a
O
O
N
H
O
N
N
NH
NH
NH2
O S
2
O
7
8
75
79
13.6±2.28
52.8±2.38
>128
>128
>128
>128
N
H
O
NH2
O
O S
2
O
N
H
N
NH
aChanging the dose between 200 and 50 µM always caused the same inhibition. At higher concentration, the
compound was not soluble.
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