A search for dual action HIV-1 reverse transcriptase, bacterial RNA polymerase inhibitors

Article abstract of DOI:10.3390/molecules22111808

Using molecular modeling approach, potential antibacterial agents with triazole core were proposed. A moderate to weak level of antibacterial activity in most of the compounds have been observed, with best minimal inhibitory concentration (MIC) value of 0

Full text of DOI:10.3390/molecules22111808

molecules
Article
A Search for Dual Action HIV-1 Reverse
Transcriptase, Bacterial RNA Polymerase Inhibitors
Agata Paneth 1,*, Tomasz Fra˛czek 2, Agnieszka Grzegorczyk 3 ID , Dominika Janowska 1,
Anna Malm 3 and Piotr Paneth 2,
*
ID
1
Department of Organic Chemistry, Medical University of Lublin, 20-093 Lublin, Poland;
dominika.hagel@gmail.com
Institute of Applied Radiation Chemistry, Lodz University of Technology, 90-924 Lodz, Poland;
tomasz.fraczek@p.lodz.pl
Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland;
agnieszka.grzegorczyk@umlub.pl (A.G.); anna.malm@umlub.pl (A.M.)
Correspondence: agata.paneth@umlub.pl (A.P.); piotr.paneth@p.lodz.pl (P.P.);
Tel.: +48-81-448-7243 (A.P.); +42-631-3199 (P.P.)
2
3
*
Received: 4 October 2017; Accepted: 22 October 2017; Published: 25 October 2017
Abstract: Using molecular modeling approach, potential antibacterial agents with triazole core were
proposed. A moderate to weak level of antibacterial activity in most of the compounds have been
observed, with best minimal inhibitory concentration (MIC) value of 0.003 mg/mL, as shown by the
15 against S. epidermidis. Studied compounds were also submitted to the antifungal assay. The best
antifungal activity was detected for 16 with MIC at 0.125 and 0.25 mg/mL against C. albicans and
C. parapsilosis, respectively.
Keywords: triazoles; antibacterial activity; molecular modeling
1. Introduction
Human immunodeficiency virus infection is associated with the progressive loss of CD4+ T cells
and massive dysregulation of the immune system [1]. Such conditions, commonly referred to as
acquired immunodeficiency syndrome (AIDS), or AIDS related complex (ARC), make HIV patients
vulnerable to opportunistic infections such as bacterial, fungal, viral, protozoal, and neoplastic diseases,
which ultimately lead to death. Indeed, according to global HIV and AIDS statistics, since the start of the
epidemic, an estimated 78 million people have become infected with HIV and 35 million people have died
of AIDS-related illnesses [2]. Despite the recent success of highly active antiretroviral therapy (HAART)
in improving the prognosis for HIV-infected individuals, challenges to effective use of these therapeutic
strategies remain, including issues of adherence, toxicities, detrimental side effects, drug resistance,
and persistent viral replication in latent reservoirs [
even more challenging due to potential drug-drug interactions and the ongoing prevalence of resistant
strains in HIV-1 populations [ ]. Therefore, there is an urgent need to develop anti-HIV agents with dual
3]. Treatment of bacterial/HIV-1 co-infection is
4
antiviral and antibacterial potency. The simplification of antiretroviral therapy to single-pill combination
would not only offer the convenience to address the above mentioned issues but it would also contribute
to improving patient satisfaction, their quality of life, medication adherence, and lowering the health care
costs [5]. In this scenario, our recently reported triazole non-nucleoside reverse-transcriptase inhibitors
(NNRTIs) [
that the most potent of the studied compounds had IC50 in the range of 3.3–18.5
wild-type, making them a good starting point for developing antiretroviral drugs. In turn, bacterial RNA
polymerase (RNAP), a proven target for broad-spectrum antibacterial therapy [ ], is structurally and
functionally similar to reverse transcriptase (RT) [10]. Consequently, it is reasonable to suppose that
6] (Figure 1) have emerged as very suitable pharmacophore structures. Results revealed
µM against the HIV-1
7–9
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www.mdpi.com/journal/molecules

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our NNRTIs should also inhibit RNAP. We have therefore submitted compounds to molecular docking
studies in the RNAP “switch region” with a mechanistic function close to that of the NNRTI binding site
that is followed by antimicrobial assay. The results of these combined in silico and experimental studies
are presented in this paper.
2. Results and Discussion
2.1. Rationale
As mentioned in the Introduction, the “switch region” has been discovered as an interesting new
binding domain of bacterial RNAP. A natural antibiotic myxopyronin B and its synthetic derivative
desmethyl myxopyronin B have been demonstrated to target this region [11]. Although myxopyronin
B is highly active in vitro, its clinical use is hampered by insufficient physicochemical properties [12,13]
.
Therefore, novel RNAP “switch region” inhibitors are being developed; due to their different binding
mode as compared to rifamycin antibiotics, such inhibitors are expected to overcome existent resistance [9].
Based on the functional and structural similarities between bacterial RNAP “switch region”
and the NNRTI allosteric binding site, following a structure-based design strategy, Hartmann et al.
identified the first synthetic 2-ureidothiophene-3-carboxylic acids as dual RNAP/RT inhibitors [14].
The compounds were characterized by a potent antibacterial activity against S. aureus and in cellulo
antiretroviral activity against NNRTI-resistant strains. Recently, we have identified novel triazole
NNRTIs also following the molecular approach. In vitro activity determination using HIV-1 RT wild
type inhibition assay led to the identification of the compounds 1–11 (Figure 1) showing inhibitory
activity with an IC50 values in the range of 3.3 to 76 µM.
R1
R1
R2
R2
1–4 (series I)
5–7 (series II)
R2
H
N
N
N
N
R2
R1
S
Cl
N
R1
O
O
S
Cl
R2
O
R2
H
8–10 (series III)
11
12
Figure 1. General structures of our recently reported triazole non-nucleoside reverse-transcriptase
inhibitors (NNRTIs) [ ] and thiophene-2-carboxylic acid derivative (12) with inhibitory activity against
6
RNA polymerase (RNAP) [14].
To find out whether our previously reported triazole NNRTIs are also good candidates for
bacterial RNAP inhibitors, the compounds 1–11 were docked to the “switch region” of the RNAP
using a cocrystal structure of Thermus thermophilus RNAP complex (PDB code 3DXJ) and their bonding
mode was analysed.
According to the docking scores presented in Figure 2, with the exception of 11 (docking score
−
13.30)
,
all of the triazoles series I–III are predicted to bind with comparable or even a higher affinity
21.29) and previously reported RNAP inhibitor 12
20.15). Out of these, the best binding affinity is predicted for triazoles series I with
31.22 ( ), followed by 28.39 ( ), 27.17 ( ), and 26.72 ( ) due to their ability to
than both native myxopyronin (docking score
−
(docking score
docking score
−
−
2
−
4
−
1
−
3
form intermolecular hydrogen bonds in the nitrophenyl-amide core. Indeed, as depicted in Figure 3,
for the compound with the best docking score, its position in the binding pocket is stabilized by three
H-bond interactions of its nitro group with the amine groups of Arg1031 and two H-bond interactions
2

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(as donor and acceptor) of its amide group with the C=O group of Leu618 and the H-N group of
Gln1019. Its binding is further strengthened through close interactions of the thiophene ring with
Gly1033, Glu1034, Val1466, Gly620, Val1037, Lys621, Ile1467, Leu619, and Leu1053.
1 (docking score: −27.17)
9 (docking score: −23.40)
2 (docking score: −31.22)
10 (docking score: −20.82)
3 (docking score: −26.72)
11 (docking score: −13.30)
4 (docking score: −28.39)
12 (docking score: −20.15)
5 (docking score: −26.95)
13 (docking score: −29.41)
Figure 2. Cont.

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6 (docking score: −22.63)
14 (docking score: −30.92)
7 (docking score: −21.27)
15 (docking score: −26.89)
8 (docking score: −25.62)
16 (docking score: −26.41)
11) [ ], known bacterial RNA
Figure 2. Docking scores for previously reported triazole NNRTIs (
1
–
6
polymerase inhibitor (12) [14], and designed triazoles with the N-(2-nitrophenyl)acetamide core (13–16)
within bacterial RNAP “switch region”.
Figure 3. Binding mode of 2 within bacterial RNAP “switch region”.

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Since the final recognition in binding site is likely to be dominated by electrostatic forces [15],
geometry optimizations of and previously reported RNAP inhibitor 12 were carried out and molecular
2
electrostatic potentials (MEP) were visualized (Figure 4). Neutral fragments are characterized by green
color, the negative charge is represented by red, while the positive charge is marked in blue. As can be
seen, both of the compounds seem to be electrostatically similar enough to be recognized by the enzyme.
(2)
(12)
Figure 4. Molecular electrostatic potential (MEP) of 2 and 12.
To prove the favoured binding mode in the “switch region” of the bacterial RNAP proposed
for triazoles with the N-(2-nitrophenyl)acetamide core four potential inhibitors 13–16 with higher
affinity than native myxopyronin were designed (see Figure 2), and their antibacterial potency
against gram-positive and gram-negative bacterial strains was tested in vitro. For antibacterial assay,
two previously reported triazoles series I with the best docking scores, i.e.,
2 and 4, were also selected.
2.2. Chemistry
Synthesis of triazoles series A (
starting with appropriate carboxylic acid hydrazide
followed by basic cyclization of the produced thiosemicarbazides
in a good yield. Reaction of triazoles with 2-bromo-N-(2-nitrophenyl)acetamide in the presence of
2,
4
,
13
–
16) was accomplished through a three-step procedure
(Scheme 1). Reaction with isothiocyanate
delivered the intermediate triazoles
A
B
C
C
potassium iodide or tetrabutylammonium iodide and potassium carbonate yielded the final triazoles
(2, 4, 13–16) in a one-pot reaction.
H
O
H
N
O
H
N
H
N
N
N
2NCS
R
NaOH
R1
R1
R1
N
H
A
H
S
N
H
R2
N
S
R2
B
C
O
Br
N
H
N
O
O
O
O
N
H
N
N
N
R1
S
N
O
R2
,
,
--
2 4 13 16
Scheme 1. Synthetic route for triazoles (2, 4, 13–16).

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2.3. Antimicrobial Assay
The triazoles (
2,
4,
13–16) were then submitted for antibacterial assay against a panel of Gram-positive
and Gram-negative reference bacterial strains using broth microdilution assay. The ciprofloxacin was
included as a control. Results revealed that with the exception of 15 with MIC at 0.003 mg/mL
against S. epidermidis, all of the tested compounds possessed limited antibacterial activity. Among
them, the best inhibitory activity against Gram-positive bacterial strains was observed for 13 (MIC range
0.06–0.5 mg/mL), followed by 15 (MIC range 0.003–1 mg/mL), 16 (MIC range 0.25–1 mg/mL) and
almost inactive compounds 2, 4, 14. In all of the cases, MICs for Gram-negative bacteria were higher in
comparison to MICs for Gram-positive bacteria, which suggests their lower sensitivity to tested triazoles.
Indeed, with the exception of B. bronchiseptica, all of the Gram-negative strains were able to grow at high
concentration (MICs of 1 mg/mL) or even were insensitive to compounds tested.
Finally, we completed our antibacterial assay by evaluating the antifungal potency of 2, 4, 13–16
against the yeast Candida albicans and Candida parapsilosis. The fluconazole was included as a control.
No appreciable antifungal activity was observed. As indicated by results collected in Table 1, the best
inhibitory activity was observed for 16 with MICs at 0.125 and 0.25 mg/mL against Candida albicans
and Candida parapsilosis, respectively, while no marked difference was observed in the activity profiles
for remaining compounds.
Table 1. Antimicrobial data (minimal inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC), mg/mL) for 2, 4, 13–16.
2
4
13
14
15
16
Control *
Gram-Positive Bacteria
MIC; MBC (mg/mL)
S. aureus ATCC 6538
S. aureus ATCC 43300
S. aureus ATCC 25923
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 10876
M. luteus ATCC 10240
1; >1
n.a.
n.a.
1; >1
1; >1
1; >1
1; 2
n.a.
1; >1
n.a.
1; >1
0.125; 1
1; >1
1; >1
0.5; 2
0.25; 1
0.25; >1
0.06; 0.125
0.125; 1
0.5; >1
n.a.
n.a.
0.5; >1
1; >1
0.5; >1
0.003; 1
1; >1
1; >1
0.25; >1
0.5; >1
0.5; >1
0.25; >1
1; >1
0.24; 0.24
0.24; 0.24
0.49; 0.49
0.49; 0.49
0.03; 012
0.12; 0.12
0.98; 1.95
0.5; >1
0.5; 2
1; >1
1; >1
1; >1
0.5; >1
1; >1
0.125; 0.5
0.5; >1
Gram-negative bacteria
MIC; MBC (mg/mL)
E. coli ATCC 35218
E. coli ATCC 25922
S. typhimurium ATCC 14028
K. pneumoniae ATCC 13883
P. mirabilis ATCC 12453
P. aeruginosa ATCC 9027
B. bronchiseptica ATCC 4617
1; >1
1; >1
1; >1
n.a.
n.a.
1; >1
1; >1
n.a.
n.a.
n.a.
n.a.
n.a.
1; >1
1; >1
1; >1
1; >1
1; >1
1; >1
0.5; >1
1; >1
1; >1
1; >1
n.a.
n.a.
1; >1
0.5; >1
n.a.
n.a.
n.a.
n.a.
n.a.
1; >1
1; >1
1; >1
1; >1
n.a.
0.015; 0.015
0.008; 0.008
0.008; 0.008
0.015; 0.015
0.015; 0.015
0.12; 0.24
1; >1
0.5; >1
1; >1
0.5; >1
1; >1
0.5; >1
0.12; 0.12
Yeasts
MIC; MBC (mg/mL)
C. parapsilosis ATCC 22019
C. albicans ATCC 2091
C. albicans ATCC 10231
0.5; 1
0.5; 1
1; 1
0.5; 1
0.5; 0.5
1; 1
0.5; 1
0.5; 0.5
0.5; 1
0.5; 1
0.5; 1
1; 1
0.5; 1
0.5; 0.5
0.5; 1
0.25; 1
0.125; 0.25
1; 1
1.95; 1.95
0.98; 0.98
0.98; 1.95
* MICs (×10−3) for ciprofloxacin (antibacterial assay) and fluconazole (antifungal assay).
3. Materials and Methods
3.1. Chemistry
All of the reagents and solvents of analytical grade or higher were purchased from commercial sources
and were used without purification unless otherwise stated. NMR spectra were recorded using Bruker
Avance spectrometers (250, 300, and 700 MHz) (Bruker BioSpin GmbH, Rheinstetten, Germany). Analytical
thin layer chromatography was performed with Merck60F254 silica gel plates (Merck, Darmstadt, Germany)
and visualized by UV irradiation (254 nm). Melting points were determined on a Fisher–Johns block
(Thermo Fisher Scientific, Schwerte, Germany), the reported values are uncorrected. Elemental analyses
were determined by an AMZ-CHX elemental analyzer (PG, Gdan´sk, Poland) (all results were within

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±
0.5% of the theoretical values). Mass spectra (ESI TOF) were recorded using Waters LCT Premier XE
mass spectrometer (Waters, Milford, MA, USA).
3.1.1. General Procedure for Synthesis of Thiosemicarbazides B
A solution of corresponding carboxylic acid hydrazide
A
(0.01 mol) and equimolar amount of
appropriate isothiocyanate in 25 mL of anhydrous ethanol was heated under reflux for 2 h. Next,
the solution was cooled and the solid formed was filtered off, washed with diethyl ether, dried,
and crystallized from ethanol.
3.1.2. General Procedure for Synthesis of Triazoles C
A solution of the corresponding thiosemicarbazide derivative
B (0.01 mol) in 2% sodium
hydroxide (10 mL) was refluxed and the progress of the reaction was monitored by thin layer
chromatography. After 2 h, the reaction was completed and the reaction mixture was cooled and then
acidified with 3 M hydrochloric acid. The precipitate was filtered, washed with water, and crystallized
from ethanol.
3.1.3. Synthesis of 2-Bromo-N-(2-nitrophenyl)acetamide
◦
o-Nitroaniline (0.01 mol) was dissolved in methylene chloride and cooled down to 0 C. Potassium
carbonate (0.03 mol) was added and then freshly distilled bromoacetyl bromide (0.015 mol) was added
dropwise for 1–3 min. After the reaction was completed (controlled by TLC, chloroform–methanol
29:1), the precipitate was centrifuged, washed with methylene chloride and discarded. Combined
extracts were concentrated under reduced pressure and the product was precipitated with heptane,
centrifuged, washed twice with pentane, and dried.
3.1.4. General Procedure for Synthesis of the Triazoles (2, 4, 13–16)
One equivalent of the corresponding triazole
C and 1.7 mg of potassium iodide (or tetrabutylammonium
iodide, 0.3 eq.) were dissolved/suspended in 2 mL of methanol. 5.2 mg of K2CO3 (1.1 eq.) was added
and the mixture was heated to 60 ◦C under reflux for 20 min. After cooling to <30 ◦C, 1.2 equivalent of the
2-bromo-N-(2-nitrophenyl)acetamide was added and the reaction mixture was stirred for 1–2 h, monitored by
TLC. Subsequently, the reaction mixture was filtered, evaporated, dissolved in methylene chloride, filtered,
concentrated, and layered with heptane. The product precipitated or crystallized within 24 h was washed
with heptane and dried. If the product separated as an oil, addition of 1–2 drops of methylene chloride often
caused solidification.
Physicochemical characterization of all compounds was reported elsewhere [
obtained are in agreement with those previously reported.
6,16,17]. The data
3.2. Antimicrobial Assay
The in vitro antimicrobial activity of (2, 4, 13–16) was tested against a panel of microorganisms from
American Type Culture Collection (ATCC): Gram-positive bacteria (Staphylococcus aureus ATCC 6538,
Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876),
Gram-negative bacteria (Escherichia coli ATCC 3521, Escherichia coli ATCC 25922, Salmonella typhimurium
ATCC 14028, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027, Proteus mirabilis
ATCC 12453), Bordetella brochiseptica ATCC 4617, and yeasts (Candida parapsilosis ATCC 22019,
Candida albicans ATCC 2091, Candida albica◦ns ATCC 10231). Microorganisms were stored in the broth
media containing 17% (v/v) glicerol at
onto Tripticase Soy Agar (in case of bacteria) and Sabouraud agar (in case of yeasts) and incubated in
ambient air for 24 h at 35 ◦C or 48 h at 30 ◦C for bacteria and yeasts, respectively.
−
70 C. Before the experiments, all of the strains were transferred

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The in vitro antimicrobial activity were performed using broth microdilution method in 96-well
microtitrate plates, allowing for estimation of MIC (minimum inhibitory concentration) and MBC
(minimum bactericidal concentration) or MFC (minimum fungicidal concentration). Mueller-Hinton
broth (MHB) and Mueller-Hinton broth with 2% glucose (MHB 2%) were used to determine antibacterial
and antifungal activity, respectively. The tested compounds were dissolved in dimethyl sulfoxide (DMSO)
to a concentration of 10 g/L. First, stock solution (1 g/L) of the tested compounds were prepared in
MHB and MHB 2%. Then, serial twofold dilution of these compounds in the same media were made in
order to obtained final concentrations of samples ranging from 0.008 × 10−3 to 1 mg mL−1
.
The colonies of each strain were resuspended in sterile physiological saline to get an optical
density equal to 0.5 McFarland. Then, 2 µL of the suspension was put into the wells to obtained final
concentration of inoculum approximately of 106 CFU mL−1 and 104 CFU mL−1 in case of bacteria
and yeasts, respectively. The last two wells were positive and negative controls. The positive control
was inoculated with bacteria and yeasts suspension only, while the negative control was left blank
without inoculation. The MICs of the compounds were recorded as the lowest◦concentration where
◦
no viability was observed after incubation at 35 C (in case bacteria) and 30 C (in case yeasts) in
ambient air for 24 h. The results of MICs were checked by the spectrophotometry of absorbance at
580 nm using Absorbance Microplate Reader EL×800 (BioTek Instruments, Inc., Winooski, VT, USA).
After determination of the MICs, MBCs, and MFCs were determined by spreading 5 µL suspension
from each well showing no growth onto Mueller-Hinton Agar (MHA) for bacteria, and Mueller-Hinton
Agar with 2% glucose (MHA 2%) for yeasts. The MBCs and MFCs of the compounds were recorded as
the lowest concentration that kills 99.9% of the bacteria (fungi) [18
performed in triplicate.
,19]. All of the experiments were
3.3. Computational Details
Conformational search was performed using the Amber force field as implemented in HyperChem
8.0.3. [20] and default convergence criteria. For the most stable conformers population analysis was
carried out using the Merz-Kollman scheme [21] at the HF/6-31G theory level with the use of the
Gaussian package [22].
3.4. Docking Studies
The docking simulations were performed using the FlexX docking module of the LeadIT
environment as implemented in the LeadIT 2.1.9 program (BioSolveIT GmbH, Augustin, Germany)
using cocrystal structure of Thermusthermophilus RNAP complex (PDB code 3DXJ). The active sites
were defined to include all of the atoms within 10 Å radius of the native ligand. To validate the docking
protocol, ligands co-crystallized with the proteins were initially docked into the crystal structure of the
appropriate enzymes; the best conformations obtained were practically identical with the experimental
ones. Subsequently, studied compounds were docked using the same docking parameters. The first
100 top ranked docking poses were saved for each docking run.
4. Conclusions
In this study, molecular docking approach combined with antimicrobial assay was used to find
new compounds with dual antiretroviral and antibacterial potency. No appreciable antibacterial
activity was found for all of the tested triazole NNRTIs. These results, though seemingly disappointing,
could be useful for future research.
Acknowledgments: The reported studies were supported by the grant 2011/02/A/ST4/00246 (2012–2017) from
the Polish National Science Centre (NCN).
Author Contributions: A.P.: synthesis, writing a manuscript; T.F.: synthesis; A.G., D.J., A.M.: antimicrobial assay;
P.P.: molecular modelling, writing a manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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