Exploring the Chemical Space of Benzothiazole-Based DNA Gyrase B Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.0c00416

We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV, based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed: varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To understand the mechanism of action and binding mode, we have obtained a crystal structure of compound 16a, bearing a primary amino group, in complex with the N-terminal domain of E. coli gyrase B (24 kDa) (PDB: 6YD9). Compound 15a, with a low molecular weight of 383 Da, potent inhibitory activity on E. coli gyrase (IC50 = 9.5 nM), potent antibacterial activity on E. faecalis (MIC = 3.13 μM), and efflux impaired E. coli strain (MIC = 0.78 μM), is an important contribution for the development of novel gyrase and topoisomerase IV inhibitors in Gram-negative bacteria.

Full text of DOI:10.1021/acsmedchemlett.0c00416

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Letter
Exploring the Chemical Space of Benzothiazole-Based DNA Gyrase B
Inhibitors
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⊥
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Ziga Skok, Michaela Barancokova, Ondrej Benek, Cristina Durante Cruz, Paivi Tammela,
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Tihomir Tomasic, Nace Zidar, Lucija Peterlin Masic, Anamarija Zega, Clare E. M. Stevenson,
Julia E. A. Mundy, David M. Lawson, Anthony Maxwell, Danijel Kikelj, and Janez Ilas*
̌
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ABSTRACT: We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV,
based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and
retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed:
varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position
and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To
understand the mechanism of action and binding mode, we have obtained a crystal structure of compound 16a, bearing a primary
amino group, in complex with the N-terminal domain of E. coli gyrase B (24 kDa) (PDB: 6YD9). Compound 15a, with a low
molecular weight of 383 Da, potent inhibitory activity on E. coli gyrase (IC50 = 9.5 nM), potent antibacterial activity on E. faecalis
(MIC = 3.13 μM), and efflux impaired E. coli strain (MIC = 0.78 μM), is an important contribution for the development of novel
gyrase and topoisomerase IV inhibitors in Gram-negative bacteria.
KEYWORDS: DNA gyrase, topoisomerase IV, GyrB, ParE, antibacterial, benzothiazole
he treatment of infections has advanced beyond
recognition since the first discoveries of antimicrobial
both strands of the double-helix. Gyrase has the unique
function of introducing negative supercoiling ahead of the
replication fork, while topo IV is involved in chromosome
decatenation. The enzymes consist of two pairs of subunits
with different functions, forming heterotetrameric structures:
A2B2 (gyrase) or C2E2 (topo IV). The main role of the GyrA
and ParC subunits is to release torsional stress by breaking and
rejoining strands of the DNA molecule. GyrB and ParE contain
the ATP-binding site and provide the energy required for the
enzyme function by ATP hydrolysis.12,13
T
compounds. Widespread distribution, low price, and high
safety profile have established antimicrobial drugs as a
cornerstone of modern medicine. However, due to rising
resistance issues, the efficacy of existing infection treatments is
rapidly decreasing. The dearth of new anti-infectives
introduced to the market over recent years, and no discovery
of novel classes, has deepened the problem.1−4 Antimicrobial
resistance is now a serious global health concern; infections
that are currently readily treatable could become untreatable in
the near future.5
DNA topoisomerases are enzymes that alter DNA topology,
being involved in important biological processes in the cell
(replication, transcription, chromosome condensation,
etc.).6−11 Bacterial DNA gyrase and DNA topoisomerase IV
(topo IV) are attractive targets for antibacterial drug discovery
due to their well-described structure and mechanism, as well as
their absence from eukaryotes. Both enzymes are type II
topoisomerases, sharing high functional and structural
similarity. They change the topology of DNA by cleaving
DNA gyrase inhibitors can influence the enzyme action at
different levels.14 Inhibition of the GyrA subunit can cause
stabilization of the DNA−enzyme complex, and thus broken
Received: July 29, 2020
Accepted: October 13, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00416
© XXXX American Chemical Society
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strands of DNA cannot be rejoined. GyrA inhibitors, so-called
gyrase poisons, are represented by fluoroquinolones, which are
widely in clinical use, although struggling with side effects and
growing resistance concerns.6 These problems have encour-
aged further research on inhibitors with different mechanisms
of action. GyrB inhibitors are not currently in clinical use;
novobiocin was withdrawn from the market in the 1960s due
to significant side effects. Since then, no other GyrB inhibitor
has passed clinical trials.7 Low cross-resistance between
potential GyrB inhibitors and fluoroquinolones is a promising
and motivating factor for further research. Novel gyrase
inhibitors based on various chemical structures were
discovered in recent years.8
Our research group has recently published several articles
focusing on ATP-competitive inhibitors of the DNA gyrase B
protein (GyrB).15−17 The compounds share a common
pyrrole-2-carboxamide moiety, originating from marine alka-
loids such as oroidin.18 Compounds are potent inhibitors of
gyrase; however, they lack potent antimicrobial activity. The
mode of action for this series of compounds was confirmed by
solving the crystal structure of a complex of E. coli GyrB with a
benzothiazole-type inhibitor 1 (PDB 5L3J).15
sulfur (benzothiazole) with nitrogen to give a benzimidazole
central scaffold. Indeed, benzimidazole-ureas were presented as
potent dual inhibitors of bacterial topoisomerases in a recent
study by Vertex Pharmaceuticals.29 In this manner, crucial
cation−π interactions between the core benzene ring and
Arg76 and the Glu50-Arg76 salt bridge interactions were
preserved. Additional interactions might have been introduced
by enlarging the central scaffold, but we did not plan to
increase the scaffold MW.
The focus of the optimization strategy was the replacement
of the oxalyl moiety, which is likely too acidic and might impair
permeation into bacteria and thus diminish antimicrobial
activity. As confirmed by the crystal structure of 1, the oxalyl
moiety extending out of the binding pocket and making only a
single direct hydrogen bond to the protein, via the carbonyl
group to Arg136, can be replaced without loss of inhibitory
activity. We systematically changed the acidic oxalyl moiety to
(i) a neutral small aliphatic acyl moiety (acetamide group), (ii)
a neutral aromatic moiety (isonicotinamide moiety), (iii) a
neutral polar group with H-bond donor/acceptor potential
(urea derivative), and (iv) an aliphatic primary amino group
(derivatives of glycine and beta alanine).
Our goal was to explore the chemical space of pyrrole-
benzothiazole GyrB inhibitors by selecting those that retain
potent enzyme inhibition while optimizing their physicochem-
ical properties. Several strategies of modification illustrated in
Figure 1 were followed: alteration of the pyrrole carboxamide
Synthesis of benzimidazole compounds, illustrated in
Scheme 1, starts with coupling of 6-nitro-1H-benzo[d]-
a
Scheme 1
Figure 1. Structure of previously discovered compound 1 (PDB
5L3J) with highlighted positions selected for structure modifications.
moiety, alteration of the central scaffold, including variation of
the substitution position, and most importantly modification of
the oxalyl moiety.
a
Reagents and conditions: (a) 2,2,2-trichloro-1-(4,5-dibromo-1H-
The 4,5-dibromo-1H-pyrrole-2-carboxamide interacting with
the protein through hydrogen bonding interactions of the
pyrrole nitrogen and adjacent carbonyl with Asp73, either
directly or via a network of hydrogen bonds involving
conserved water molecule, respectively does not permit
substantial changes. Modification to 3,4-dichloro-5-methyl-
1H-pyrrole-2-carboxamide has several potential beneficial
properties. The key role of the pyrrole nitrogen and
nonsubstituted carboxamide moiety remains unchanged.
Smaller chlorine atoms (compared to bromine) are likely to
fit better into the E. coli topo IV hydrophobic pocket and the
Staphylococcus aureus gyrase hydrophobic pocket, which has a
smaller volume than the E. coli gyrase hydrophobic pocket.19
The 3,4-dichloro-5-methyl-1H-pyrrole-carboxamide moiety is
present in the natural antibiotics kibdelomycin20−22 and
amycolamicin23,24 and was used in the Astra Zeneca
pyrrolamide series of gyrase B inhibitors.25−27
To explore the influence of the central scaffold we
interchanged the substituents on positions 2 and 6 of
benzothiazole, resulting in interchanging the position of the
benzene ring and thiazole ring of the benzothiazole moiety28 in
the binding site and its π−cation interactions with Arg76, as
well as influencing the acidity of the pyrrole amide proton. An
obvious bioisosteric approach was the replacement of the ring
pyrrol-2-yl)ethan-1-one, Na2CO3, DMF, 80 °C, 16 h; (b) SnCl2·
2H2O, EtOH, reflux, 12 h; (c) ethyl oxalyl chloride (for 4a) or methyl
malonyl chloride (for 4b), Et3N, 1,4-dioxane, rt, 12 h; (d) 1 M
NaOH, 1,4-dioxane, rt, 16 h.
imidazol-2-amine with 2,2,2-trichloro-1-(4,5-dibromo-1H-pyr-
rol-2-yl)ethan-1-one and a weak base in DMF. The nitro group
of intermediate 2 is reduced using tin(II) chloride dihydrate to
give amino-derivative 3, further acylated with respective acyl
chloride to obtain esters 4a−b. Corresponding carboxylic acids
5a−b were obtained by alkaline hydrolysis.
Synthesis of benzothiazole-based compounds is illustrated in
Scheme 2. Acylation of 6-nitro-benzo[d]thiazol-2-amine (6)
with ethyl oxalyl chloride/methyl malonyl chloride/acetyl
chloride gives intermediates 7a, 7b, and 7c respectively.
The nitro group in position 6 of the benzothiazole was then
reduced using hydrogen and palladium catalyst to obtain
compounds 8a−c. Coupling with 3,4-dichloro-5-methyl-1H-
pyrrole-2-carboxylic acid (activated in situ with oxalyl
chloride) in DCM in the presence of pyridine as base gave
compounds 9a−c. The ethyl oxalyl derivative 9a was further
hydrolyzed to carboxylic acid 10. Synthesis of the reversed
isomers with the pyrrole moiety at position 2 is more
B
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a
reduced using tin(II) chloride dihydrate to give key amino
derivative 12, which was further derivatized. Acylation with
oxalyl chloride moiety gave ester 13. The corresponding
carboxylic acid 14 is isolated after hydrolysis with 1 M sodium
hydroxide. Reaction of amino derivative 12 with acetic
anhydride in the presence of triethylamine in DCM gave
target compound 15a. To obtain the urea derivative 15b,
aminobenzothiazole 12 was first stirred at RT with CDI in
DMF for 3 h and heated to 50 °C overnight after addition of
ammonia gas. Compounds 15c−e were prepared by coupling
of 12 with nicotinic acid (15c), Boc-β-alanine (15d), or Boc-
glycine (15e) in the presence of coupling agents EDC, HOBt,
or NMM, respectively, in DMF. The Boc protective group of
15d−e was finally removed with HCl in dioxane, to obtain
16a−b.
Scheme 2
Compounds were evaluated in E. coli and S. aureus gyrase
supercoiling assays as well as in E. coli and S. aureus topo IV
relaxation assays. The results are presented in Tables 1−3 as
IC50 values or residual activity (RA) of the enzyme in a
concentration of 10 μM of the inhibitor.
The benzimidazole-based series was shown to be only
weakly active (Table 1), with inhibitory activity against E. coli
gyrase in the micromolar range. Carboxylic acids 5a−b were
approximately 10-fold more active than the corresponding
esters 4a−b, due to possible ionic interactions additional to
hydrogen bonds with Arg136. The most potent compound 5a
(IC50 = 0.60 μM) showed weak activity against S. aureus gyrase
and E. coli/S. aureus topo IV). Direct comparison of
benzothiazole 1 with benzimidazole 5a shows that replacement
of sulfur with nitrogen resulted in 10-fold lower enzymatic
inhibition against E. coli gyrase. Poor activity results and poor
solubility of benzimidazoles pointed us back to the
benzothiazole central scaffold, and the benzimidazole series
was not further extended. Replacement of 4,5-dibromo-1H-
pyrrole with 3,4-dichloro-5-methyl-1H-pyrrole indeed resulted
in good inhibitory activity against E. coli gyrase, but even more
importantly it introduced good inhibitory activity against E. coli
topo IV (14: IC50 topo IV = 75 nM) and potent inhibitory
activity against S. aureus gyrase and topo IV, which was
completely absent in the case of the dibromo analogue 1.
When comparing compounds with the pyrrole attached to
position 2 (compound 14) to a regioisomer with the pyrrole
attached to position 6 (compound 10), the inhibitory activity
on E. coli gyrase is favorable for compound 10 and even more
favorable regarding E. coli topo IV inhibitory activity as well as
S. aureus gyrase and topo IV. Overall, compound 14 has
a
Reagents and conditions: (a) corresponding acyl chloride, Et3N, 1,4-
dioxane, rt, 4 h; (b) H2, 10% Pd/C, EtOH (for 8a and 8c) or MeOH
(for 8b), rt, 24 h; (c) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl
chloride, pyridine, DCM; (d) 1 M NaOH, MeOH, rt, 24 h,; (e) 3,4-
dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, reflux, 16
h; (f) SnCl2·2H2O, EtOH, reflux, 12 h; (g) ethyl oxalyl chloride, Et3N,
1,4-dioxane, rt, 8 h (for the synthesis of 13); (g) Ac2O, Et3N, DCM,
rt, 2 h (for the synthesis of 15a); or CDI, DMF, rt, 3 h; NH3, rt, 16 h
(for the synthesis of 15b); or nicotinic acid, EDC, NMM, HOBt,
DMF, rt, 12 h (for the synthesis of 15c); or corresponding Boc-amino
acid, EDC, NMM, HOBt, DMF, rt, 12 h (for the synthesis of 15d−e);
(h) 4 M HCl, 1,4-dioxane, rt, 5 h.
demanding. The coupling of benzothiazole 6 with the pyrrole-
2-carbonyl chloride moiety was done in refluxing toluene
without any base, to obtain intermediate 11. The nitro group is
Table 1. Inhibitory Activity of Series I of Compounds with Benzimidazole Central Scaffold
a
IC50 (μM) or RA (%)
Cmpd
n
R
E. coli gyrase
E. coli topo IV
S. aureus gyrase
S. aureus topo IV
4a
4b
5a
5b
1
0
1
0
1
Et
Me
H
4.0 1.6 μM
7.0 3.4 μM
0.60 0.32 μM
1.5 0.2 μM
0.058 μM
100%
100%
12 2 μM
100%
13 μM
11 μM
100%
100%
80 23 μM
100%
>100 μM
0.041 μM
100%
100%
31 1 μM
100%
10 μM
27 μM
H
novobiocin
0.17 μM
a
Residual activity of the enzyme at 10 μM concentration of the compound.
C
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Table 2. Inhibitory Activity of Benzothiazole Compounds with Acidic Terminal Functional Groups
a
IC50 (nM) or RA (%)
Cmpd
n
R
E. coli gyrase
E. coli topo IV
100%
100%
6 400 3 000 nM
100%
75 28 nM
13 000 nM
11 000 nM
S. aureus gyrase
S. aureus topo IV
9a
9b
10
13
14
1
0
1
0
0
0
Et
Me
H
Et
H
290 170 nM
200 180 nM
29 16 nM
48 12 nM
4.8 2.1 nM
58 nM
69%
59%
100%
48%
250 130 nM
100%
38 16 nM
>100 μM
41 nM
910 340 nM
100%
290 180 nM
10 000 nM
27 000 nM
novobiocin
168 nM
a
Residual activity of the enzyme at 10 μM concentration of the compound.
Table 3. Inhibitory Activity of Benzothiazole Compounds with Neutral or Basic Terminal Functional Groups
a
IC50 (nM) or RA (%)
Cmpd
R
E. coli gyrase
66 8 nM
16 000 4 000 nM
9.5 2.5 nM
26 8 nM
2 500 1 500 nM
110 20 nM
29 15 nM
110 50 nM
280 10 nM
E. coli topo IV
100%
100%
4 600 100 nM
5 200 2 700 nM
100%
100%
60%
10 000 2 000 nM
100%
S. aureus gyrase
S. aureus topo IV
9c
12
35 400 nM
60%
400 120 nM
1 300 600 nM
100%
100%
100%
1 600 300 nM
5 800 3 100 nM
100%
100%
100%
290 180 nM
100%
15a
15b
15c
15d
15e
16a
16b
-CH3
-NH2
-pyridine-4-yl
-CH2CH2NHBoc
-CH2NHBoc
780 nM
260 120 nM
1 500 600 nM
380 nM
-CH2CH2NH3 Cl−
+
-CH2NH3 Cl−
+
a
Residual activity of the enzyme at 10 μM concentration of the compound.
superior enzymatic activity against all four tested enzymes
compared to novobiocin. Benzothiazole with 3,4-dichloro-5-
methyl-1H-pyrrole on position 2 was thus selected to explore
the possible replacements of the unfavorable oxalyl moiety.
The results of the E. coli gyrase inhibitory activity assays reveal
that the anionic center is not required for potent inhibitory
activity. Compounds with the acetyl moiety, the urea
derivative, and the glycine derivative with a free primary
amino group all possess E. coli gyrase inhibitory activity in the
low nanomolar range (10−25 nM). Having an aromatic moiety
(15c) pointed to the water environment (and possibly having
π−cationic interactions with Arg136) is clearly not optimal for
this series of compounds, although such an approach was
successful in tricyclic inhibitors of GyrB (PDB: 4KFG).30
Investigation of the Boc-protected amino acid derivatives 15d
and 16a reveals that the bulky lipophilic moiety can have
favorable binding to E. coli GyrB. Although this might seem
contradictory, it is known from thermodynamic evaluations
that the binding of compound with “unfavorable” lipophilic
moieties extending into a water environment can be beneficial
as more polar/ionized groups can pay a high desolvation
penalty, which contributes to net unfavorable binding.31 The
amino compound 12, lacking the carbonyl group, is a very
weak binder, which indicates that a carbonyl moiety is a
prerequisite for potent enzyme binding. Acetyl derivative 15a
with lowest molecular weight in the series and single digit
nanomolar binding with IC50 = 9.5 nM seemed very
interesting; therefore, regioisomer 9c with a pyrrole attached
to position 6 and acetyl to position 2 of benzothiazole was
prepared. The trend observed already from previous
compounds was the same in this series: the isomer 15a with
pyrrole attached to position 2 of benzothiazole was 7-fold
more potent than regioisomer with pyrrole attached to position
6.
To confirm binding to the ATP-pocket of GyrB and to gain
insight into molecular interactions, crystallization of selected
ligands with the 24 kDa fragment (N-terminal subdomain) of
GyrB was attempted. Among crystallization attempts with 15a,
16a, and 9c, cocrystals of 16a with E. coli GyrB24 were
obtained and the structure was solved to a resolution of 1.60 Å
(Figure 2). The 3,4-dichloro-5-methyl-1H-pyrrole moiety of
16a is bound to the adenine-binding pocket, making a
hydrogen bond between the pyrrole NH group and Asp73
side chain, while interaction of the Asp73 side chain and
D
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through a H-bond (2.87 Å), while the primary amino group of
β-alanine is pointed toward the water environment, making no
interactions with the protein. While interactions of the oxalyl
moiety of 1 with Arg136 were not apparent (PDB code 5L3J,
Resolution: 2.83 Å), improved resolution now offers clear
insight into the binding mode. Hydrogen bonding of Arg136
with the carbonyl oxygen of β-alanine is evident, thus
demonstrating that a carboxylic acid moiety is not prerequisite
for binding, and explains strong enzymatic inhibitory activity of
compounds having a neutral side chain.
Compounds were assayed on 4 wild type bacterial strains to
determine their antibacterial activity (E. coli, S. aureus,
Enterococcus faecalis, Pseudomonas aeruginosa) (Table 4).
Additionally, we included two other E. coli strains, an E. coli
strain with impaired outer membrane (lpxC deletion mutant)
and an E. coli strain with a defective efflux pump (tolC deletion
mutant), to evaluate if weak antibacterial activity on wild type
could be due to weak permeability and/or strong efflux. Both
benzimidazole and benzothiazole inhibitors with the oxalyl
moiety (5a, 10, and 14) were inactive in all bacterial strains
(MIC > 50 μM). Ester derivative 9b which had good
enzymatic inhibitory activity (IC50 = 200 nM) showed also a
good MIC (1.56 μM) in the E. coli strain with a defective efflux
pump, indicating that the ester moiety is beneficial for entry
into bacteria; however, such compounds are often the
substrates for efflux pumps. Ester derivative 13 which had
very good enzymatic inhibitory activity (IC50 = 48 nM)
showed only weak MIC (50 μM) in the E. coli strain with a
defective efflux pump.
A significant improvement in MIC value was expected with
the compound lacking the acidic oxalyl moiety. Compound
16b, having a glycine moiety with a free primary amino group,
was the only compound showing some antibacterial activity on
S. aureus wild type (MIC = 25 μM), confirming that good
enzymatic inhibitory activity (S. aureus gyrase IC50 = 380 nM)
with suitable physicochemical properties is needed for
antibacterial activity. When looking at Gram-positive E. faecalis,
the results were more encouraging, as four compounds showed
good antibacterial activity. Both compounds with a free amino
group (16a MIC = 12.5 μM and 16b MIC = 6.25 μM) had
good antibacterial activity. Boc-protected analogue 15e and
compound 15a with an acetyl moiety had even better
antibacterial activity with MIC = 3.13 μM, which also
correlated with enzymatic inhibitory potency. When looking
at more challenging Gram-negative E. coli and P. aeruginosa,
Figure 2. Crystal structure of the complex formed between 16a and E.
coli GyrB 24 kDa fragment (PDB: 6YD9). The protein is depicted in
cartoon representation covered by a semitransparent molecular
surface. Omit mFobs-DFcalc positive difference electron density for
the ligand at 1.6 Å resolution is depicted at two contour levels: 3σ
(magenta mesh) and 12σ (black mesh), with the latter highlighting
the locations of the electron-dense chlorine and sulfur atoms. Shown
below are the interactions between amino acid residues with ligand
(red line: hydrogen bond, blue line: π−cation interactions).
pyrrolamide carbonyl oxygen is bridged by a coordinated water
molecule (NHCO−H2O H-bond distance is 2.61 Å).
Comparison with the GyrB crystal structure of kibdelomycin
(PDB: 4URM)32 and Astra Zeneca’s pyrrolamide (PDB:
3TTZ)33 reveals identical interactions of the 3,4-dichloro-5-
methyl-1H-pyrrole moiety. There is a strong π−cation
interaction (3.28 Å) between the aromatic ring and Arg76.
The carbonyl moiety of β-alanine interacts with Arg136
Table 4. Antibacterial Activity of Selected Compounds
a
MIC (μM)
E. coli (ATCC
S. aureus (ATCC
E. faecalis (ATCC
P. aeruginosa (ATCC
E. coli
(JD17464)
E. coli
(JW5503)
b
c
Cmpd
25922)
29213)
29212)
27853)
4a, 4b, 5a, 5b, 9a, 9c, 10, 14, 15c,
15d
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
9b
13
15a
15b
15e
16a
16b
ciprofloxacin
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
0.05 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
25 μM
>50 μM
>50 μM
3.13 μM
>50 μM
3.13 μM
12.5 μM.
6.25 μM
3.02 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
3.02 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
1.56 μM
50 μM
0.78 μM
1.56 μM
0.78 μM
12.5 μM
3.13 μM
1.51 μM
0.121 μM
0.015 μM
a
b
MIC (minimum inhibitory concentration that inhibits the growth of bacteria by ≥90%) values against E. coli and S. aureus. E. coli strain with
c
impaired outer membrane, lpxC deletion mutant. E. coli strain with defective efflux pump, tolC deletion mutant.
E
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̌
́
the results were less encouraging, as none of the compounds
showed antibacterial activity.
Michaela Barancokova − University of Ljubljana, Faculty of
Pharmacy, 1000 Ljubljana, Slovenia
̌
́ ́
Ondrej Benek − University of Hradec Kralove, Faculty of
Testing on the E. coli strain with impaired outer membrane
(lpxC deletion mutant), revealed that permeability is not a
main issue as tested compounds did not show any antibacterial
activity. On the contrary, the compounds were very active
when tested on the E. coli strain with a defective efflux pump.
The most potent compounds were 15e and 15a with
submicromolar MIC values (MIC = 0.78 μM). Both are
neutral molecules with lipophilic moieties (acetyl and Boc).
Compounds with free amino group were also potent
antibacterial compounds (16a and 16b) but were not superior
to the neutral compounds (15a and 15e). This is somehow
contrary to the recent proposal that the introduction of a
primary amino group improves entry and causes accumulation
in Gram-negative bacteria.34 To confirm the mechanism of
uptake for the reported compounds (porin pathway or direct
diffusion),35 additional studies should be performed in order to
guide optimization.36,37 Urea derivative 15b was also a potent
antibacterial compound on the efflux impaired E. coli strain
(MIC = 1.56 μM), while it was inactive on all other strains.
In summary, three series of GyrB/ParE inhibitors were
designed, synthesized, and evaluated in enzymatic and
antibacterial assays. Chemical space was thoroughly explored;
compounds with acidic, basic, and neutral properties were
synthesized, possessing very potent inhibitory activity on E. coli
and/or S. aureus gyrase and/or topo IV. The best compounds
were active on Gram-positive bacterium E. faecalis with the
best compound having MIC= 3.13 μM. The compounds were
inactive on Gram-negative bacteria because they are good
substrates for bacterial efflux pumps, but 15a and 15e showed
potent antibacterial activity on the efflux impaired E. coli strain
(MIC = 0.78 μM). Compound 15a with low molecular weight
383 Da displayed potent inhibitory activity on E. coli gyrase
(IC50 = 9.5 nM) and potent antibacterial activity on E. faecalis
(MIC = 3.13 μM) and on the efflux impaired E. coli strain
(MIC = 0.78 μM) and thus makes an important contribution
for the development of novel gyrase and topoisomerase
inhibitors in Gram-negative bacteria.
́
́
Science, Department of Chemistry, 500 03 Hradec Kralove,
Czech Republic; orcid.org/0000-0001-6840-1933
Cristina Durante Cruz − Centre for Drug Research, Division of
Pharmaceutical Biosciences, Faculty of Pharmacy, University of
Helsinki, Helsinki FI-00014, Finland
̈
Paivi Tammela − Centre for Drug Research, Division of
Pharmaceutical Biosciences, Faculty of Pharmacy, University of
Helsinki, Helsinki FI-00014, Finland; orcid.org/0000-
0003-4697-8066
̌
̌
Tihomir Tomasic − University of Ljubljana, Faculty of
Pharmacy, 1000 Ljubljana, Slovenia; orcid.org/0000-0001-
5534-209X
Nace Zidar − University of Ljubljana, Faculty of Pharmacy,
1000 Ljubljana, Slovenia; orcid.org/0000-0003-1905-
0158
̌
̌
Lucija Peterlin Masic − University of Ljubljana, Faculty of
Pharmacy, 1000 Ljubljana, Slovenia
Anamarija Zega − University of Ljubljana, Faculty of Pharmacy,
1000 Ljubljana, Slovenia; orcid.org/0000-0003-4065-
0019
Clare E. M. Stevenson − Department of Biological Chemistry,
John Innes Centre, Norwich NR4 7UH, U.K.
Julia E. A. Mundy − Department of Biological Chemistry, John
Innes Centre, Norwich NR4 7UH, U.K.
David M. Lawson − Department of Biological Chemistry, John
Innes Centre, Norwich NR4 7UH, U.K.
Anthony Maxwell − Department of Biological Chemistry, John
Innes Centre, Norwich NR4 7UH, U.K.; orcid.org/0000-
0002-5756-6430
Danijel Kikelj − University of Ljubljana, Faculty of Pharmacy,
1000 Ljubljana, Slovenia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00416
Author Contributions
⊥
̌
Z.S. and M.B. contributed equally. The manuscript was
written with contributions from all of the authors.
ASSOCIATED CONTENT
■
Funding
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00416.
The work was funded by the EU H2020 ITN-ETN Project
INTEGRATE (Project Reference: 642620), EU FP7 Inte-
grated Project MAREX (Project No. FP7-KBBE-2009-3-
245137), Academy of Finland (Grant No. 277001), and
Slovenian Research Agency (Grant No. P1-0208). Work in
A.M.’s lab was supported by the Biotechnology and
Biosciences Research Council (UK) Institute Strategic
Programme Grant BB/P012523/1, and the Wellcome Trust
(Investigator Award 110072/Z/15/Z).
(i) Experimental conditions for biochemical and micro-
biological assays, (ii) experimental conditions for
cocrystallization, collection, and refinement statistics
for compound 16a, (iii) experimental procedures for the
synthesis of all the compounds described in this
manuscript, and (iv) representative analytical data
(PDF)
Notes
The authors declare no competing financial interest.
AUTHOR INFORMATION
■
ACKNOWLEDGMENTS
Corresponding Author
■
̌
Janez Ilas − University of Ljubljana, Faculty of Pharmacy, 1000
We acknowledge Diamond Light Source for access to beamline
I04 under proposal MX18565 and Sara Henderson for
providing the protein sample.
Ljubljana, Slovenia; orcid.org/0000-0002-0124-0474;
Phone: +386-1-4769576; Email: janez.ilas@ffa.uni-lj.si
Authors
ABBREVIATIONS
̌
■
Ziga Skok − University of Ljubljana, Faculty of Pharmacy, 1000
Ljubljana, Slovenia
MIC, minimal inhibitory concentration; RA, residual activity
F
https://dx.doi.org/10.1021/acsmedchemlett.0c00416
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