Discovery and development of novel salicylate synthase (MbtI) furanic inhibitors as antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2018.06.033

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (K_i = 5.3 μM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC⁹⁹ = 156 μM), which is conceivably related to mycobactin biosynthesis inhibition.

Full text of DOI:10.1016/j.ejmech.2018.06.033

Accepted Manuscript
Discovery and development of novel salicylate synthase (MbtI) Furanic inhibitors as
antitubercular agents
Laurent R. Chiarelli, Matteo Mori, Daniela Barlocco, Giangiacomo Beretta, Arianna
Gelain, Elena Pini, Marianna Porcino, Giorgia Mori, Giovanni Stelitano, Luca
Costantino, Margherita Lapillo, Davide Bonanni, Giulio Poli, Tiziano Tuccinardi,
Stefania Villa, Fiorella Meneghetti
PII:
S0223-5234(18)30524-5
10.1016/j.ejmech.2018.06.033
EJMECH 10503
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 April 2018
Revised Date: 5 June 2018
Accepted Date: 13 June 2018
Please cite this article as: L.R. Chiarelli, M. Mori, D. Barlocco, G. Beretta, A. Gelain, E. Pini,
M. Porcino, G. Mori, G. Stelitano, L. Costantino, M. Lapillo, D. Bonanni, G. Poli, T. Tuccinardi,
S. Villa, F. Meneghetti, Discovery and development of novel salicylate synthase (MbtI) Furanic
inhibitors as antitubercular agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.06.033.
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ACCEPTED MANUSCRIPT
H2L
Database
Ki 5.3 µM
Anti-TB
VS
I
MbtI

ACCEPTED MANUSCRIPT
Discovery and Development of Novel Salicylate Synthase (MbtI) Furanic
Inhibitors as Antitubercular Agents
Laurent R. Chiarelli,a,‡ Matteo Mori,b,‡ Daniela Barlocco,b Giangiacomo Beretta,c Arianna Gelain,b Elena Pini,b
Marianna Porcino,b Giorgia Mori,a Giovanni Stelitano,a Luca Costantino,d Margherita Lapillo,e Davide
Bonanni,e Giulio Poli,f Tiziano Tuccinardi,e,* Stefania Villab,* and Fiorella Meneghettib.
a Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani", Università degli Studi di Pavia, via Ferrata
9, 27100 Pavia; Italy.
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via L. Mangiagalli 25, 20133
Milano; Italy.
c Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, Via Celoria 2, 20133
Milano; Italy.
d Dipartimento Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, via Campi 103, 41121
Modena; Italy.
e Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa; Italy.
f Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, via A. Moro 2, 53100 Siena; Italy.
KEYWORDS: Tuberculosis, Iron, siderophores, antimycobacterial drugs, virtual screening, drug design
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ABSTRACT. We report on the virtual screening, synthesis, and biological evaluation of new furan
derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based
virtual screening procedure was applied to screen the Enamine database, identifying two
compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active
compound I as starting point for the development of novel MbtI inhibitors, we obtained new
derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a
emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 µM). Moreover, compound 1a
showed a promising antimycobacterial activity (MIC99 = 156 µM), which is conceivably related to
mycobactin biosynthesis inhibition.
INTRODUCTION
Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), is one of
the deadliest infectious diseases worldwide, with about 1.7 million deaths reported in 2016
[1]. New TB drugs are needed because of the complexity and toxicity of the current TB
therapeutic regimens and to provide alternatives to contrast the emergence of multidrug-
resistant (MDR) and extensively drug-resistant (XDR) Mtb strains [2,3]. The investigation
of novel pharmacological targets, such as InhA, MmpL3, DprE1 and QcrB, plays a
fundamental role in the development of new potential antitubercular agents [4]. Since the
most promising targets are membrane proteins, the identified hit compounds may also
interfere with the integrity of the host membranes; therefore, the selection of different
targets to be tackled is still urgent [5-8]. In addition, there are a number of novel anti-TB
compounds in clinical trial, and two new drugs have been recently approved: delamanid
and bedaquiline. Despite these advances, many more TB drug candidates are necessary
to sustain an effective and productive drug pipeline [9]. In particular, more information on
the effectiveness, safety and tolerability of bedaquiline is urgently required, because its
severe side effects (mainly QT prolongation) could be potentially life-threatening [9].
Therefore, the research of new anti-TB drugs that can shorten and simplify current
pharmacological therapy is pivotal [10].
One of the strategies adopted to fight bacterial pathogens is to target the biosynthesis of
mycobactins, high-affinity iron-chelating molecules called siderophores [6]. Iron is an
essential redox cofactor, involved in several cellular processes, allowing the survival and
growth of the mycobacterium in the host. Nevertheless, Mtb is capable of sustaining an
infection even in an iron-deficient environment by overcoming it by means of several iron
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acquisition pathways, among which the synthesis of siderophores plays a key role. Water-
soluble carboxymycobactins and lipophilic mycobactins, compounds that vary according to
the nature of their lipophilic moiety, are able to solubilize iron and compete with the iron-
binding proteins (e.g. transferrin, lactoferrin, ferritin) of the host. While mycobactins remain
associated within the cell wall, carboxymycobactins are released outside the outer
membrane, where they scavenge iron, making it available for transport into the Mtb
cytoplasm [11]. As the bacterial strains unable to produce siderophores lose their ability to
grow within macrophages and sustain the infection [12], there is a considerable interest in
deciphering the mechanism of siderophore assembly, with the goal of targeting selected
steps for antitubercular drug development. Among the enzymes involved in siderophore
biosynthesis [6], the Mg2+-dependent salicylate synthase (MbtI) has been validated as a
target for fighting TB [13,14]. Since MbtI catalyzes the first step of the process by
converting chorismate to salicylate via an isochorismate intermediate (Scheme 1), its
inhibition leads to a reduced mycobacterial pathogenicity without toxicity issues, due to the
absence of this target in the host [15]. The first structural and biochemical characterization
of MbtI was reported by Harrison et al. [16] and structural data highlights that the Mg2+ is
coordinated by E297 and E434, as well as the C1 carboxylate of chorismate [13]. Residue
K205 assists the attack of water at the chorismate C2 carbon for the isomerization reaction
and E252 provides acid catalysis for the elimination of the hydroxyl group from C4 carbon.
The positioning of the pyruvate side chain of the substrate is allowed by the interactions
with Y385, R405 and G419 [13].
Scheme 1. Reaction catalyzed by MbtI, the salicylate synthase of Mycobacterium tuberculosis.
With the aim of identifying new MbtI inhibitors, a receptor-based virtual screening (VS)
study was carried out and a preliminary optimization of one of the two identified hits was
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performed. This study has led to novel chemical entities that could represent an alternative
and/or complementary therapy for the treatment of multidrug-resistant strains, opening
new avenues to improved clinical outcomes.
RESULTS AND DISCUSSION
Virtual Screening. A VS study, focused on the ligand-MbtI key interactions, was
developed. A pharmacophore model was created based on the analysis of the crystal
structure of MbtI co-crystallized with 3-(1-carboxyprop-1-enyloxy)-2-hydroxybenzoic acid
(Methyl-AMT, PDB code 3VEH) [17]. Figure 1A depicts the interactions of Methyl-AMT
inside the MbtI binding site. The carboxylic group of the 2-hydroxy benzoic acid forms an
H-bond with the side-chain of Y385; the analysis of different MbtI crystal structures
suggests that the position of this residue is highly conserved, and it is important for the
interaction of the carboxylic function of the reported MbtI inhibitors. The aromatic ring of
the 2-hydroxy benzoic acid shows lipophilic interactions with I207, A362, L404 and a
cation-π interaction with K438. The methyl fragment of the 1-carboxyprop-1-enyloxy ligand
chain is inserted into a lipophilic pocket mainly delimited by P251, L268, H334 and T361,
whereas the second carboxylic group shows an ionic interaction involving K438 and it is
compatible with the interaction with the Magnesium ion. Based on these considerations,
we built a structure-based pharmacophore taking into account a) the H-bond with Y385, b)
the lipophilic interactions of the aromatic ring, c) the methyl fragment and d) the
coordination of the carboxylic group with the Magnesium ion. The model, shown in Figure
1B, was generated using the software Ligandscout [18] and comprised (1) an H-bond
acceptor feature representing the oxygen atom of the carboxylic fragment interacting with
Y385, (2, 3) two hydrophobic features representing the aromatic and methyl substituents
and (4) a negative feature representing the carboxylic group of the 1-carboxypropenyl-1-
enyloxy fragment. In addition, the pharmacophore model was refined by adding excluded
volume spheres mimicking the steric hindrance represented by the MbtI binding site (see
Experimental Section for more details).
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Figure 1. (A) Interactions of Methyl-AMT (green) inside the MbtI binding site and (B) receptor-based
pharmacophore model used for the VS study.
The pharmacophore model was used to screen the Enamine database, comprising about
1 500 000 commercially available compounds, retrieving only compounds matching all the
four pharmacophore features and respecting the volume constraints given by the MbtI
binding site. By applying this strict filter, only 2050 compounds were further considered
and subjected to docking studies. Recently, we have reported an evaluation of the
consensus docking approach [19]. Through this approach, one ligand is docked into the
target protein by means of multiple docking methods. Then, among the best-ranked poses
(originated by the different docking procedures), the binding pose predicted by the largest
number of docking methods is considered as the best docking pose. From a qualitative
point of view, previous results highlighted that consensus docking was able to predict
ligand binding poses better than the single docking evaluations [20]. Furthermore,
concerning the VS studies, the results suggested that this approach performed as well as
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the best available methods found in the literature [20], and it was also able to
experimentally identify new active molecules [21-23]. The 2050 compounds selected
through the pharmacophore filter were subjected to a consensus docking protocol
calibrated for the docking of MbtI inhibitors. The MtbI-tuned consensus docking protocol
included 5 different docking methods, i.e. Plants and Gold with the four different fitness
scoring functions, which were found to be the most reliable procedures among a total of 11
different docking methods, according to self-docking studies performed on six MbtI-ligand
co-crystal structures (see Section S1 of Supplementary material for more details). The five
selected docking procedures were then applied to the 2050 commercial compounds. For
each ligand, the five docking poses predicted by the five docking procedures were
clustered together to search for common binding modes. The analysis revealed that only
74 compounds presented a binding pose shared by all the five docking methods and thus
reached the maximum consensus level. Therefore, based on the consensus docking filter,
only 3.6% of the docked compounds were considered as potential active inhibitors. These
results are in agreement with our previous consensus docking calculations reported in
literature, which highlighted the high filtering strictness of this procedure. In 2014, we
tested the reliability of a consensus docking protocol comprising 10 different procedures by
applying this technique on three different targets of the Directory of Useful Decoys (DUD)
and at the end of the screening calculations only an average of 0.40% of the starting
databases was considered as potentially active [19]. More recently, the whole DUD
dataset has been analyzed by means of the same consensus docking approach and an
average of 0.44% of the starting databases was considered as potential actives [20].
Considering that the higher the number of docking methods included in the consensus
docking protocol, the higher its filtering strictness, the results obtained with the MtbI-tuned
consensus docking protocol are in line with those shown by our previous evaluations. The
analysis of the binding mode predicted for the 74 potential MtbI inhibitors identified by the
consensus docking protocol revealed that only five compounds still matched the receptor-
based pharmacophore model in their docking pose. On these bases, the five compounds
were purchased at the highest purity level available from the commercial source (≥ 95%)
and subjected to biological assays to evaluate their MbtI inhibitory activity.
MbtI inhibitory activity. Compound effects on MbtI enzyme activity were tested at
subsaturating concentrations of substrate (chorismic acid, CHA, 50 µM), at a final
concentration of 100 µM. The most potent MbtI inhibitors reported in the literature belong
to the class of the benzimidazole-2-thiones [24] and to the AMT-series, which is
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structurally related to isochorismate [14,15], showing IC50 values of about 10 µM. In
particular, Methyl-AMT, which emerged as the most powerful candidate of the AMT-series,
was used as a positive control, showing an IC50 value of 11.6 µM, in agreement with the
previously reported values [14,15]. As shown in Table 1, one compound was found to be
provided with a remarkable activity (compound I, IC50 = 21.1 µM), another one displayed a
modest activity (compound III, IC50 = 77.6 µM), while the three other compounds resulted
completely inactive.
Table 1. In vitro activity of compounds I-V.
Co
de
Residual activity at
Chemical structure
IC50 (µM)
100 µM (%)
I
21.3 ± 4.3
21.1 ± 4.1
II
101.3 ± 3.6
-
III
38.4 ± 11.4
77.6 ± 5.7
IV
V
104.0 ± 13.2
108.8 ± 12.0
-
-
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Compounds I and III were also subjected to molecular dynamics (MD) simulations with
explicit water, in order to further analyze their predicted binding mode. Magnesium ion was
also included at the entrance of the MbtI active site in order to analyze its interaction with
the ligands. Figure 2A shows the predicted binding mode of compound I into the MbtI
binding site. The Magnesium ion presents an octahedral coordination geometry: it is bound
by a water molecule, one oxygen of the carboxylate groups of E294, E297 and E434 and
the two oxygens of the carboxylate of compound I. The furan ring shows an H-bond with
K438, whereas the benzene ring, as for Methyl-AMT, exhibits lipophilic interactions with
I207, L404 and a cation-π interaction with K438. Finally, the nitro group strongly interacts
with Y385 and, furthermore, it forms a second H-bond with the nitrogen backbone of R405.
Figure 2B shows the average minimized structure of the MtbI-III complex. The two
oxygens of the carboxyl group of the compound interact with the Magnesium ion, the
benzoic acid ring shows lipophilic interactions with I207, whereas the 2-methoxy-5-
nitrophenyl fragment loses the H-bond with the hydroxyl group of Y385 during the initial
steps of the MD. However, the loss of the H-bond with Y385 could be partially
compensated with the presence of H-bonds between the sulfonamide group and the
backbone oxygen of T361 and the side chain of K438.
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Figure 2. Minimized average structures of I (A) and III (B) docked into MbtI binding site.
In order to further assess the solidity of our VS workflow, a retrospective evaluation was
carried out. In particular, as shown in Supplementary material (Sections S2, S3) simpler
ligand-based approaches such as fingerprint similarity searches or 3D ligand similarity
screenings showed a lower performance in retrieving the most active identified compound
(I) from the initial commercial database. Moreover, as shown in Supplementary material
(Section S4), the application of the consensus docking approach was found to outperform
the single docking methods in identifying compound I from the 2050 ligands selected
through the pharmacophore filter.
Structure-activity relationship (SAR) studies. In this study we considered the most
active compound I as starting point for the development of novel MbtI inhibitors to obtain
new derivatives showing potent in vitro inhibition. In order to support the importance of
each pharmacophoric feature disclosed by the modeling studies, we designed and
synthesized analogs to explore the chemical space and to study the structural and
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functional requirements for the activity against MbtI. We took into account two main points,
namely the substitutions at the phenyl ring and the acidic moiety; the results are
summarized in Table 2. By alternatively removing the chlorine atom or the para nitro group
from I, we obtained the active derivative 1a and the inactive compound 1b, respectively.
When both these substituents were removed (1c), a complete loss of activity was
detected. The replacement of the phenyl ring with pyridine (1d) afforded a partially active
compound. The corresponding isomeric derivatives having the nitro substituent in meta
(1e) and in ortho (1f) positions lost potency, both inhibiting MbtI activity by
̴ 50%, at a
concentration of 100 µM. The inactive compound 1g, bearing in para position of the phenyl
ring a group unable to simultaneously accept two hydrogen bonds in the suitable
geometry, supported the hypothesized binding mode for this class of compounds; likewise,
the substitution of the nitro group with the CF3 group (1h) led only to a retention of a
modest activity (66% residual activity). As a further attempt, the para-nitro group of 1a was
replaced with the methylsulfonyl (1i) and sulfonamide (1j) substituents. With respect to the
nitro moiety, they are larger fragments; however, they could potentially interact through
two H-bonds with the protein by means of the two oxygens, as the nitro moiety. In the first
case, the replacement resulted in complete loss of activity, whereas compound 1j retained
a low activity (84% residual activity). The substitution of the para-nitro group with other
bioisosteres led to weak (1k,l) or inactive (1m-o) compounds. In order to assess if the
carboxylic group linked to the furan conferred potency in the most active candidate 1a, the
corresponding ester derivative (2a) was tested and it was found to be only slightly active
(71% residual activity), thus confirming the importance of the interaction between this
function and MbtI. Finally, when the furan was substituted with a phenyl moiety (3) a partial
activity was observed, while the removal of the furan led to an inactive compound (4).
Table 2. In vitro activity of compounds 1a-o, 2a, 3,4.
Residual activity at 100 µM
code
R1
R2
(%)
1a
1b
4-NO2-Ph
2-Cl-Ph
H
H
18.2 ± 5.1
92.4 ± 13.8
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1c
1d
Ph
H
102.0 ± 8.5
45.0 ± 5.1
H
1e
1f
3-NO2-Ph
2-NO2-Ph
H
H
H
H
H
H
50.2 ± 13.0
55.1 ± 22.7
96.3 ± 19.2
66.3 ± 9.7
98.5 ± 14.4
84.6± 16.2
1g
1h
1i
4-OH-Ph
4-CF3-Ph
4-SO2CH3-Ph
4-SO2NH2-Ph
1j
1k
1l
H
H
46.8 ± 8.3
58.0 ± 8.9
O
O
1m
1n
4-CN-Ph
H
H
125.6 ± 17.9
92.4 ± 11.8
4-COOH-Ph
1o
2a
H
104.4 ± 7.8
71.1 ± 18.4
4-NO2-Ph
CH3
3
4
36.8 ± 7.6
89.3 ± 16.1
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The SAR studies allowed to evidence that compound 1a shows an improved potency
with respect to our initial hit I, as it displayed an IC50 value of 7.6 ± 1.6 µM (Figure 3A).
Moreover, the MbtI kinetic analysis in the presence of 1a proved that this compound is a
potent competitive inhibitor towards the chorismate binding site, displaying a Ki value of
5.3 ± 0.6 µM (Figure 3B and 3C).
Figure 3. Effects of the compounds against MbtI enzyme activity. (A) Graph comparing I (▼) and Methyl-
AMT (¢) with 1a (ò). IC50 values were determined at subsaturating concentrations of CHA (50 µM). (B)
Steady state kinetics analysis towards CHA of MbtI in the presence of different concentrations of 1a (0, 5,
10, 20, 50 and 100 µM) highlights the competitive behavior of the inhibitor. (C) Global reciprocal plot of data
in panel B. All data are mean SD of three replicates.
Mtb minimal inhibitory concentration determination (MIC99). In order to assess its
potency, the inhibitor 1a was investigated for its antibacterial activity against M.
tuberculosis H37Rv, together with 2a, on the basis of the higher activity of esters with
respect to the carboxylic acids on this parasite, probably owing to a better ability to cross
the mycobacterial cell wall [15]. Compound 1a showed a MIC99 of 156 µM, whereas for 2a
we did not detect an improvement of the cellular potency, probably due to its low solubility
in the assay medium. It is noteworthy that, to the best of our knowledge, 1a can be
considered as one of the MbtI inhibitors showing the best antimycobacterial activity.
Mycobactin biosynthesis inhibition. To assess if the antitubercular activity is indeed
related with iron-uptake inhibition, we used the non-pathogenic M. bovis BCG, whose
siderophores closely resemble the structure of Mtb mycobactins [26,27]. The compound,
when assayed against cells grown in the low iron containing Chelated Sauton’s medium,
showed a MIC value 3-fold lower than against mycobacteria grown in 7H9 medium (80 µM
vs 240 µM), suggesting that the mechanism of action of 1a involves iron uptake. Thus, in
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order to support the correlation of the antitubercular activity of 1a with MbtI inhibition, the
effects of the compound on siderophore production were assessed in M. bovis BCG cells,
grown in iron-depleted medium and in the presence of different sub-lethal concentrations
of compound 1a. To this purpose, the levels of siderophore production were measured
using Universal CAS liquid assay [28] and through the mycobactins isolation and
quantification. As shown in Figure S94, the CAS assay performed on the medium of the
cells grown in different concentrations of 1a, showed a removal of iron inversely related to
the concentration of the compound. Similarly, the concentration of mycobactins,
determined in the same cells, decreased at higher concentrations of 1a, confirming that
the inhibitory effect towards Mtb growth could be due to mycobactin biosynthesis inhibition.
Chemistry. Compounds 1a,e and 4 were purchased and their purity was assessed using
HPLC (≥ 95%). Details are provided in Supplementary material. The methyl ester (2a) of
1a, as well as compounds 5 and 6, were obtained according to a common procedure
(Fischer-Speier esterification). Compounds 1b-d,f-o, 3 were synthesized by a Suzuki-
Miyaura coupling reaction [29], followed by a base-catalyzed hydrolysis of the ester
function. The synthetic pathways are reported in the Scheme 2, the general procedures
are described in the experimental section, while all the details concerning the specific
synthetic steps and the analytical data are provided in Supplementary material.
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Entry
R1
Entry
R1
1,2b
2-Cl-Ph
1,2j
4-SO2NH2-Ph
1,2c
Ph
1,2k
1,2d
1,2f
1,2l
2-NO2-Ph
1,2m
4-CN-Ph
1,2g
1,2h
1,2i
4-OH-Ph
4-CF3-Ph
1n
1,2o
3,7
4-COOH-Ph
4-SO2CH3-Ph
4-NO2-Ph
Scheme 2. Reagents and conditions: (a) conc. H2SO4, CH3OH, reflux, 24 h; (b) i-PrMgCl, Bis[2-(N,N-
dimethylamino)ethyl]ether, B(OCH3)3, THF, 15°C 20 min → 20°C, 30 min → 0°C, 10 min, N2; (c) appropriate
reagent, Pd(PPh3)2Cl2, 2M Na2CO3, 1,4-dioxane, 90°C, overnight, N2, (for 2k,l,o 60°C, 1 h, M.W.); (d) 1M
NaOH, CH3CH2OH/THF 1:1, reflux, 5 h (for 1d: LiOH·H2O THF/H2O, 20°C, 2 h; for 1l: 0.5M NaOH,
CH3CH2OH/THF 1:1, 20°C, 30 min); (e) KOH, H2O, reflux, 6 h.
CONCLUSIONS
MbtI is a validated and attractive target for the treatment of tuberculosis, and its inhibition
leads to a reduced mycobacterial pathogenicity without toxicity issues, due to the absence
of this target in the host. Starting from the computational analysis of the interaction
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between MbtI and methyl-AMT, a VS study was carried out, identifying the hit compounds I
and III as interesting inhibitors. Considering the most active compound I, we described the
synthesis and biological evaluation of new MbtI inhibitors, based on the furan scaffold and
having different substituents on the phenyl ring. Initial SAR studies applied on I allowed the
identification of a potent competitive MbtI inhibitor, compound 1a. Remarkably, 1a exerts a
slightly better in vitro inhibitory activity (IC50 = 7.6 µM) compared to Methyl-AMT (IC50
=
11.6 µM), which is the best MbtI inhibitor discovered to date. In addition, 1a displayed a
MIC99 value of 156 µM, currently being the first potent MbtI inhibitor endowed with a
promising antitubercular activity. In addition, siderophore assay suggested that the
inhibitory effect of 1a towards Mtb growth could be due to mycobactin biosynthesis
inhibition, thus highlighting the importance of this pathway as target for the development of
therapeutic interventions. It is noteworthy that compound 1a contains a nitrophenyl moiety
as a bioisoster of the salicylic moiety of Methyl-AMT. Even if the nitro group is a cause for
concern in medicinal chemistry, for the first time we demonstrated the effective
replacement of one carboxylic group of Methyl-AMT without loss of MbtI inhibitory potency,
allowing, at the same time, a better permeability through the Mtb cell wall. Our results are
the starting point to expand the SAR study among this class of derivatives, exploiting other
hitherto unconsidered pharmacophoric features, with the aim of disclosing new lead
compounds lacking the nitro group, making our series more attractive for further
developments.
In conclusion, the analysis of the hypothetical binding mode of this class of compounds
suggested potential points of modification that led to significant developments, and that
could be further explored to increase the potency of these derivatives.
EXPERIMENTAL SECTION:
Pharmacophore model generation. One of the main drawbacks of the VS consensus
docking approach is the required computing time, because by using this method a whole
data set of molecules should be subjected to different docking procedures [19]. Therefore,
in order to reduce the number of compounds to be analyzed, a pharmacophore screening
was used as a prefilter. The pharmacophore hypothesis was built on the basis of the 3D
structure of MbtI co-crystallized with methyl-AMT (PDB code 3VEH) [17]. The software
LigandScout 4.08 [18] was used for the creation of the receptor-based pharmacophore
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models. An exhaustive pharmacophore model including all the possible features identified
by the program was generated and, subsequently, only the desired features were retained
in the final pharmacophore model, for a total of four features. The excluded volume
spheres were defined on the basis of the receptor structure as implemented in the default
LigandScout configuration.
Database Generation and Pharmacophore Screening. The Enamine database,
comprising about 1 500 000 commercially available compounds was used as the
screening database. The idbgen-gui utility of LigandScout was employed to create the
LigandScout 3D database. The iCon Best conformational sampling method was applied.
The 3D database was then screened using the previously created receptor-based
pharmacophore model and the receptor excluded volume, imposing that only the
compounds matching all the four pharmacophoric features of the model were retrieved.
Docking procedures and pose filtering. Prior to the docking analysis, a self-docking
evaluation of the main MbtI-inhibitor X-ray complexes (PDB codes 3RV6, 3RV7, 3RV8,
3RV9, 3ST6 and 3VEH) was carried out using Autodock, Autodock vina, Dock, Fred, Gold
with the four fitness functions implemented (i.e., GoldScore, ChemScore, Astex Statistical
Potential and ChemPLP), Glide with the standard and extra precision methods and Plants.
The use of Gold, with the four different fitness functions, and Plants resulted to be the
most reliable ones as they showed an average RMSD lower than 2.0 Å. For all the docking
procedures the region of interest for the docking studies was defined in such a manner
that it contained all residues that stayed within 10 Å from the ligand in the X-ray structure.
Gold. The ‘‘allow early termination’’ command was deactivated, while the possibility for
the ligand to flip ring corners was activated. For all other parameters, Gold defaults were
used and the ligands were subjected to 30 genetic algorithm runs. Four docking analyses
were carried out, corresponding to the four implemented fitness functions.
Plants. This docking software uses Ant Colony Optimization, a state-of the-art global
optimization algorithm to find minima of a scoring function representing favorable complex
structures [30,31]. ChemPLP scoring function was employed to score protein-ligand
interactions as well as intra-ligand clash terms. Standard settings for all parameters were
used for the scoring function and the optimization algorithm (search speed setting:
‘‘speed1’’).
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Consensus docking evaluation. By applying the five docking methods, five different
binding dispositions (best-scored docking pose) resulted from the docking of each ligand
into each protein binding site. The RMSD of these docking poses against the remaining
four was evaluated by using the rms_analysis software of the Gold suite. On this basis, for
each ligand docked into the protein binding site, a 5 × 5 matrix was generated reporting
the RMSD results. By using an in-house program [18], these results were clustered, so
that among the five results, all of the similar docking poses were clustered together. As a
clustering algorithm, we used the complete-linkage method, which is an agglomerative
type of hierarchical clustering. We selected an RMSD clustering threshold of 2.0 Å;
therefore, the so-obtained clusters contained the group of poses that were less than 2.0 Å
away from all others poses belonging to the same cluster. All of the ligands showing a
consensus level of five were taken into account.
Filtering of docking results. The filtering of the docking results was carried out by
superimposing the docked compounds to the pharmacophore directly from the supplied
poses, without changing their coordinates. The retrieval of compounds matching all the
four pharmacophoric features of the model was imposed in this search.
MD simulations. All simulations were carried out using AMBER 14 [32]. MD simulations
were performed using the ff14SB force field at 300 K. The complex was placed in a
rectangular parallelepiped water box. Magnesium ion was inserted analyzing its disposition
and interaction into the 2FN1 PDB code [33]. Many force field-based MD simulations of
biological systems containing Mg2+ have already been reported in literature [34-37]. In the
present study we used the particle mesh Ewald (PME) compatible Lennard-Jones
parameters for divalent metal ions in explicit solvent reported by Merz and co-workers [38].
An explicit solvent model for water, TIP3P, was used, and the complexes were solvated
with a 20 Å water cap. Sodium ions were added as counterions to neutralize the system.
Prior to MD simulations, two steps of minimization were carried out; in the first stage, we
kept the protein fixed with a position restraint of 500 kcal/mol•Å2 and we solely minimized
the positions of the water molecules. In the second stage, we minimized the entire system
through 5000 steps of steepest descent followed by conjugate gradient (CG) until a
convergence of 0.05 kcal/Å•mol, using the same procedure described above. PME
electrostatics and periodic boundary conditions were used in the simulation [39]. The MD
trajectory was run using the minimized structure as the starting conformation. The time
step of the simulations was 2.0 fs with a cutoff of 10 Å for the non-bonded interaction, and
17

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SHAKE was employed to keep all bonds involving hydrogen atoms rigid. Constant-volume
periodic boundary MD was carried out for 0.5 ns, during which the temperature was raised
from 0 to 300 K. Then 19.5 ns of constant pressure periodic boundary MD was performed
at 300 K using the Langevin thermostat to maintain constant the temperature of our
system. All the α carbons of the protein were blocked with a harmonic force constant of 10
kcal/mol•Å2 for the first 3.5 ns while in the last 16.5 ns no constraints were applied.
General Amber force field (GAFF) parameters were assigned to the ligands, while partial
charges were calculated using the AM1-BCC method as implemented in the Antechamber
suite of AMBER 14.
General Synthetic Procedures and Materials. Commercial products (1a,e and 4),
chemicals and solvents were of reagent grade; they were purchased from suppliers
(Sigma-Aldrich) and used as received. Anhydrous solvents were utilized without further
drying. Aluminum backed Silica Gel 60 plates (0.2 mm, Merck) were used for analytical
TLC, to follow the course of the reaction. Silica gel 60 (Merck 40–63 µm) was used for the
purification of intermediates and final compounds, through flash column chromatography.
Melting points were determined in open capillary tubes with a Büchi Melting Point 510. All
tested compounds were characterized by the means of FT-IR, 1H NMR, 13C NMR, MS and
1
HPLC to check their purity. H and 13C NMR spectra were acquired at ambient
1
temperature with a Varian-Oxford 300 MHz instrument, operating at 300 MHz for H and
75 MHz for 13C. Chemical shifts are expressed in ppm (δ) from tetramethylsilane
resonance in the indicated solvent (TMS: δ = 0.0 ppm), while J-couplings are given in
Hertz. The APT sequence was used when deemed necessary. IR spectra were acquired
with a Perkin Elmer Spectrum One FT-IR in a spectral region between 4000 and 450 cm-1
and analyzed by transmittance technique with 32 scansions and 4 cm-1 resolution. Solid
samples were mixed in a mortar with KBr (1:100) and pressed, using a hydraulic press (14
tons), to small tablets. MS analyses were carried out with a Thermo Finnigan (MA, USA)
LCQ Advantage system, equipped with a quaternary pump, a Diode Array Detector
(working wavelength: 254 nm) and a MS spectrometer, with an Electrospray ionization
source and an Ion Trap mass analyzer (ionization: ESI positive or ESI negative; capillary
temperature: 250°C; source voltage: 5.50 kV; source current: 4.00 µA; multipole 1 and 2
offset: -5.50 V and -7.50 V, respectively; intermultipole lens voltage: -16.00 V; trap DC
offset voltage: -10.00 V). The purity of the compounds was assessed by means of HPLC
(λ = 220 nm) and was ≥ 95% unless otherwise stated. Column: EVO C18 Phenomenex.
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Eluent system: aqueous formic acid (0.05%) / acetonitrile 50:50. Total flow: 1.2 mL/min.
Injection volume: 30 µL.
Compounds I-V were purchased from Enamine at the highest purity level available (≥ 95
%).
Details about specific synthetic procedures and analytical data (Table S2) of compounds
1a-o, 2a, 3 and 4 are reported in Supplementary material.
Procedure A for the synthesis of compounds 2b-d,f-j,m and 7.
Methyl 5-bromofuran-2-carboxylate (5, 1 mmol), the appropriate phenylboronic acid (1.3
mmol) and bis(triphenylphosphine)palladium(II) dichloride (5% mol) were dissolved in dry
1,4-dioxane (10 mL), under nitrogen atmosphere. A 2M sodium carbonate solution (2
mmol) was then added and the resulting mixture was stirred overnight at 90°C. After
completion, the solution was cooled to room temperature and then filtered on a celite pad.
The filtrate was diluted with water and extracted with ethyl acetate (3 x 4 mL). The organic
layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.
Compound 7 was obtained from methyl 3-bromobenzoate (6, 1 mmol) and 4-
nitrophenylboronic acid (1.3 mmol) following the same procedure.
Procedure B for the synthesis of compounds 2k,l,o.
Methyl 5-boronofuran-2-carboxylate (8, 1.3 mmol), the appropriate bromo derivatives
(10, or 12, or 13, 1.0 mmol) and bis(triphenylphosphine)palladium(II) dichloride (5% mol)
were dissolved in dry 1,4-dioxane (10 mL), under nitrogen atmosphere. A 2M sodium
carbonate solution (2 mmol) was then added and the resulting mixture was stirred in a
microwave synthesizer (Biotage Initiator Classic) for 1 h at 60°C. After completion, the
solution was cooled to room temperature and then filtered on a celite pad. The filtrate was
diluted with water and extracted with ethyl acetate (3 x 4 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated in vacuo.
Procedure C for the synthesis of compounds 1b-d,f-o and 3.
The appropriate methyl ester derivative (2b-d, f-m, o, 7, 1 mmol) was dissolved in a
mixture of tetrahydrofuran – ethanol 1:1 (15 mL) and a 1M solution of sodium hydroxide
(2.5 mmol) was added dropwise while stirring. The reaction mixture was heated at reflux
for 5 h. After completion, the solvent was evaporated under reduced pressure; the
aqueous phase was washed with chloroform (1 x 5 mL), acidified with hydrochloric acid
19

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(3M) and then extracted with ethyl acetate (3 x 7 mL). The organic layers were washed
with brine, dried over anhydrous sodium sulfate and then concentrated in vacuo. The
resulting solid was finally washed with cool hexane (3 mL). Variations to this general
procedure were applied to the synthesis of 1d,l (see Supplementary material for details).
Compound 1m (1 mmol) was further treated with potassium hydroxide (4.5 mmol) in
water (12 mL) at reflux for 6 h. After completion, the solution was acidified with
hydrochloric acid (10M) and then extracted with ethyl acetate (3 x 7 mL). The organic
layers were washed with brine, dried over anhydrous sodium sulfate and then
concentrated in vacuo to obtain 1n as a light yellow solid.
1
Characterization of 1a-o, 2a, 3, 4. All compounds were characterized using H
NMR,13C NMR, MS, IR spectroscopy, and RP-HPLC. The purity was ≥ 95% according to
analytical RP-HPLC. 1H NMR and 13C NMR spectra, together with RP-HPLC
chromatograms are provided in Supplementary material.
Production and purification of chorismic acid. Chorismic acid was produced using
the Escherichia coli KA12 strain, according to Grisostomi et al. [40] and purified by a
modified protocol described in Supplementary material.
MbtI Expression, Purification and Inhibition assays. Rv2386c gene, encoding the
MbtI was cloned into pET28b expression vector, and the protein was expressed in BL21
(DE3) E. coli cells and purified as reported in Supplementary Information. Enzymatic
activity was determined at 37 °C by
The standard reaction mixture contained 50 mM HEPES pH 8.0, 0.2 mM NADH, 10 mM
MgCl2, 3 units of -lactic dehydrogenase (Sigma Aldrich), 10-20 µM MbtI, and the
L-lactic coupled assay, as previously described [14].
L
reactions were started by the addition of the substrate (CHA). Steady-state kinetic
parameters were determined by assaying the enzyme at variable concentrations of
chorismic acid (10-500 µM). The experiments were performed in triplicate, and the kinetic
constants Km and kcat determined by fitting the data to the Michaelis-Menten equation
using Origin 8 software. Initially, MbtI inhibition was screened for all compounds at 100 µM
(dissolved in a solution containing 10% DMSO) and 50 µM chorismic acid. The compound
solubility in the medium assay was verified up to 100 µM concentration. For compounds
that significantly inhibited the enzyme activity, IC50 and Ki values were determined. For
IC50 determinations, the enzyme activities were measured in the presence of compound
and values were estimated according to the Equation 1, where A[I] is the enzyme activity at
inhibitor concentration [I] and A[0] is the enzyme activity without inhibitor.
20

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[I]
A[I]=A[0]×ꢀ1-ꢁ ꢂ
ꢃ
Equation 1
I +IC
50
The inhibition constant (Ki) values were determined by assaying the MbtI enzymatic
activity at different substrate and compound concentrations, using an adapted equation for
competitive inhibition (Equation 2) with Origin 8 software.
V
[S]
max
v=
Equation 2
ꢁ ꢂ
I
ꢁ ꢂ
S + K ꢄ1+
ꢅ
m
K
i
Pan Assay Interference Compounds (PAINS) analysis. As reported by Baell and
Holloway, there is a number of substructural features which could help to identify
compounds that appear as PAINS in many biochemical high-throughput screens [41,42].
The corresponding filters have been included in the Filter-itTM software and compound 1a
was thus filtered by using this program. The result highlighted that this compound did not
possess any of the substructural features shared by the most common PAINS. Moreover,
to verify the possible formation of aggregates, the ability of compound 1a to inhibit MbtI
activity was also tested in the presence of 0.1 mg/mL of bovine serum albumin (BSA) or in
the presence of 0.01% (v/v) Triton X-100 as detergent [43]. As reported in Figure S93, the
IC50 value in the presence of BSA (7.28 ± 1.41 µM) or in the presence of Triton X-100
(8.00 ± 1.28 µM) are not significantly different, supporting the notion that the ligand did not
act as an aggregate. Finally, to exclude a promiscuous enzyme inhibition due to covalent
reaction with cysteines [44]. MbtI inhibition activity of compound 1a was also tested in the
presence of 100 µM of 1,4-dithio-DL-threitol (DTT). As depicted in Figure S93, the IC50 was
not influenced by DTT (7.84 ± 1.07 µM), thus excluding an interaction of 1a with the MbtI
cysteines.
Minimal inhibitory concentration determinations. The MIC99 of 1a and 2a, against M.
tuberculosis H37Rv, was determined in solid medium. A single colony was inoculated in
complete Middlebrook 7H9 supplemented with 10% OADC Middlebrook Enrichment, and
grown at 37 °C until exponential growth phase (~108 CFU/mL). Dilutions to the final
concentration of ~106 CFU/mL were performed and about 1 µL of cell culture was streaked
onto plates containing two-fold serial dilutions of appropriate compound. MIC values were
assigned as the lowest drug concentrations inhibiting bacterial cell growth. The
experiments were performed in triplicate. Additionally, the compound was assayed against
M. bovis BCG in liquid medium, in high and low iron media, by determining the MIC
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against cells grown both in 7H9/OADC medium and in low-iron Chelated Sauton’s medium
[45].
Siderophore production assay. Siderophore activity present in the culture was tested
in M. bovis BCG through the isolation of mycobactins [46] or using the Universal CAS
liquid assay [28]. To this purpose, M. bovis was grown in 7H9 medium, subcultured in
chelated Sauton’s medium, then diluted 1:1000 in chelated Sauton’s containing different
concentrations of compound 1a. After 15 days of incubation at 37°C, cells were harvested
by centrifugation. For the isolation of mycobactins, cell pellets were extracted in ethanol
overnight. 0.1 M FeCl3 in ethanol was added until no further change in colour was
observed, and the mixture incubated at room temperature for 1 h. Mycobactins were then
extracted in chloroform, washed with water 3 times to remove the excess of iron, and
evaporated. Residue was dissolved in methanol. The concentration of mycobactins was
determined by measuring the absorbance at 450 nm (1% solution of mycobactins gives an
absorbance of 42.8) [46]. For Universal CAS assay 100 µl of cell cultures supernatant was
mixed with 100 µl of CAS assay liquid solution in 96 well plate, incubated 10 min at room
temperature, then the absorbance was measured at 630 nm. The siderophore units were
calculated using the following equation
A -A
r
s × 100 Equation 3
A
r
where Ar is the absorbance at 630 nm of the blank medium with CAS assay solution and
As is the absorbance of the culture supernatants with CAS assay solution.
AUTHOR INFORMATION
Corresponding Authors
*stefania.villa@unimi.it. ++390250319368, ORCID: 0000-0002-0636-7589;
*tiziano.tuccinardi@unipi.it. ++390502219595, ORCID: 0000-0002-6205-4069.
Author Contributions.
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
Acknowledgments
22

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We acknowledge the University of Milan (Linea B) and the University of Pavia for the
financial support. We thank Prof. Peter Kast, ETH Zurich, for kindly providing the E. coli
KA12 strain.
ASSOCIATED CONTENT
Supplementary material. Virtual screening evaluation, NMR spectra, IR and MS data,
HPLC chromatograms, chemical and biological experimental procedures and
supplementary figures, PDB file of the I-MbtI complex. This material is available free of
charge at website.
ABBREVIATIONS USED
Mtb, Mycobacterium tuberculosis; MbtI, Mycobacterium tuberculosis salicylate synthase;
TB, Tuberculosis; VS, virtual screening; DUD, Directory of Useful Decoys; CHA, chorismic
acid; CG, conjugate gradient; PME, Particle mesh Ewald; GAFF, general Amber force
field; BSA, bovine serum albumin; CAS, chrome azurol S.
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ACCEPTED MANUSCRIPT
Highlights
A VS study identified new MbtI inhibitors bearing the furan ring as potential antitubercular
agents.
1a exerts a slightly better in vitro inhibitory activity on MbtI, compared to the best inhibitor
reported to date.
1a is the first potent MbtI inhibitor endowed with a promising activity against
Mycobacterium tuberculosis.
Siderophore production assays suggest a correlation between the antimycobacterial
effects of 1a and the iron-uptake inhibition.