C.R. Polaquini et al.
Bioorganic Chemistry 109 (2021) 104668
(
m-OH), which are regioisomers of MAC 5 (m-OMe) and MAC 6 (p-OH),
the effects on prokaryotic and eukaryotic cells, the selectivity indexes
(SI) of selected compounds were determined as the ratio between the
IC50 and MIC values against M. tuberculosis and A. baumannii. MAC 4 was
more selective than MACs 6 and 8, with SI values ranging from 5.4 to
15.6. Furthermore, the comparison between the SI values of MAC 4
suggests it is more promising as an antitubercular agent, since it has
higher SI values for this species (SI values of 15.6 and 14.2 for MRC-5
and A549 cells, respectively). According to Orme and collaborators,
candidates for new antitubercular agents must have a SI value ≥10 [59].
respectively. MAC 7 was inactive (MIC > 125 µg/mL) against Gram-
negative and Gram-positive species, and it displayed less activity (MIC
7
.8 µg/mL) than its regioisomer MAC 5 (MIC 3.9 µg/mL) against
M. tuberculosis. On the other hand, MAC 8 (MIC 0.9 µg/mL) demon-
strated similar antitubercular activity to the regioisomer MAC 6 (MIC
0
.9 µg/mL). Regarding Gram-negative and Gram-positive species, MAC
displayed higher activity than MAC 6 against A. baumannii, MSSA,
8
MRSA and S. epidermidis. In general, these results indicated that the
position of OMe and OH substituents affects the bioactivity, suggesting
that the meta position was more relevant for antibacterial and antitu-
bercular activities than para.
2.4. Genotoxic evaluation of MAC 4
In order to determine whether the active MACs displayed bacteri-
cidal or bacteriostatic activity against Gram-negative and Gram-positive
species, the ratios between the MBC and MIC values were calculated.
Antibacterial agents are generally regarded as bactericidal if MBC/MIC
ratio <4 [51,52]. All active MACs exhibited MBC/MIC ratios between 1
and 2, indicating that the compounds have bactericidal activity against
these species.
In order to further investigate the safety of MAC 4, we evaluated its
genotoxic effects on human cells by the single cell gel electrophoresis
assay (alkaline comet assay). This method is able to measure the damage
induced by oxidizing, alkylating or intercalating agents in the DNA
[60,61]. A549 cells were exposed to MAC 4 at ¼IC50 and ½IC50 (19.8
and 39.7 µmol/L, respectively) for 24 h and their nuclei were submitted
to electrophoresis. The A549 line was selected because it was the most
sensitive to MAC 4, and subinhibitory concentrations were used in order
to guarantee high percentages of metabolically viable cells. Hydrogen
peroxide (1 mmol/L) was used as a reference genotoxic compound.
Fig. 3 presents the genotoxic parameters of MAC 4, including the tail
moment and the percentage of DNA in tail, which are directly propor-
tional to the DNA break [62]. A549 cells exposed to MAC 4 at ¼IC50 and
½IC50 did not show significant genotoxic effects compared to the
negative control (untreated cells). In addition, the comparison with the
MACs 4, 6 and 8 showed a broad spectrum and potent activity,
mainly against M. tuberculosis and A. baumannii, with MIC values
ranging from 0.9 to 7.8 µg/mL. M. tuberculosis is the main etiological
agent of tuberculosis, a communicable lung disease that is one of the top
1
0 causes of death worldwide and the leading cause of death from a
single infectious agent [37]. A. baumannii belongs to the ESKAPE path-
ogens, a bacterial group that also includes E. faecium, S. aureus, Klebsiella
pneumoniae, P. aeruginosa and Enterobacter spp. [53]. These microor-
ganisms are recognized as the leading cause of nosocomial infections
worldwide [54]. In addition, the carbapenem resistant A. baumannii
belongs to the critical category of World Health Organization’s priority
pathogens list for the development of new antibiotics [55], which re-
inforces the importance of the search for agents against A. baumannii.
genotoxic parameters of cells treated with H O2 (positive control)
2
corroborated the weak genotoxic effect of MAC 4. Furthermore, MAC 4
genotoxic parameters were similar to the CUR ones, and CUR is
considered safe for humans [63].
2
.5. Evaluation of MAC 4 chemical stability
2
.3. Evaluation of MACs 4, 6 and 8 toxicity on human cells and
One of the main factors that hinder the clinical use of CUR is its poor
determination of selectivity indexes
stability under physiological conditions [11]. In order to assess whether
the new monocarbonyl analog has overcome this limitation, we inves-
The evaluation of the effects against human cells is a relevant step to
investigate the selectivity and safety of new antibacterial agents. We
selected the compounds MACs 4, 6 and 8, which demonstrated a broad
spectrum and potent activity, in order to evaluate their toxicity on
human cells. Since M. tuberculosis and A. baumannii were the most sen-
sitive species to the selected compounds and cause pulmonary infection
◦
tigated MAC 4 stability at pH 7.4 and 37 C by monitoring the decrease
in its UV–Vis absorption for 24 h (Fig. 4). The initial concentration of
MAC 4 was reduced at 28% after 24 h, while CUR initial concentration
reduced at 57%. These data indicate MAC 4 is more stable than its
parent compound. Our findings corroborated other investigations about
the increase in the chemical stability of monocarbonyl analogs when
compared with CUR [17,64], supporting the hypothesis β-diketone
moiety of curcumin is correlated with the poor chemical stability [12].
[
56,57], we investigated the toxicity against human lung cells, including
fibroblast (MCR-5) and epithelial (A549) cell lines. MACs 4, 6 and 8
toxic effects were expressed according to the concentration required in
order to reduce cell viability at 50% (IC50) (Table 2). Doxorubicin was
used as a reference cytotoxic agent [58].
2
.6. Effects of MAC 4 on the divisome of B. Subtilis
MAC 4 was less toxic on MRC-5 and A549 cells (IC50 87.6 and 79.4
µmol/L, respectively) than MACs 6 and 8 (17.5 µmol/L ≤ IC50 ≤ 52.4
µmol/L). In addition, MAC 4 was about 120 and 50 times less toxic than
doxorubicin on MRC-5 and A549 cells, respectively. In order to compare
In order to investigate whether cell division is a target for the new
curcumin analog, we evaluated the effects of MAC 4 on the divisome
using B. subtilis strain expressing FtsZ-GFP, which labels the bacterial
divisome. B. subtilis was selected because this species is commonly used
in studies on the antibacterial action mode of new agents [65,66] and its
division process has been extensively explored [6]. B. subtilis was treated
with MAC 4 at ½MIC (73.6 µmol/L) for up to 30 min and analyzed under
the microscope. Hexyl gallate was used as a reference divisome dis-
ruptor [65].
Table 2
Toxicity on human lung cells and selectivity indexes of selected compounds.
Compounds
MRC-5
IC50
SI
A549
IC50
SI
Mt
Ab
Mt
Ab
Fig. 5 depicts phase contrast and fluorescence microscopy repre-
sentative images of the negative and positive controls, and treatments
with MAC 4 after 15 and 30 min. Untreated B. subtilis cells (negative
control) showed normal pattern of septation in which the divisome
(marked by the fluorescence of FtsZ-GFP) was located in the middle of
the cells as bars perpendicular to the long axis of the rods (Fig. 5A). The
same pattern was observed for B. subtilis exposed to 1% DMSO (vehicle
control) (data not shown). When cells were treated with hexyl gallate
(positive control), there was a complete divisome disruption, and the
MAC 4
MAC 6
MAC 8
CUR
87.6 ± 2.1
52.4 ± 1.2
17.5 ± 0.9
42.1 ± 0.7
0.7 ± 0.04
15.6
11.9
3.8
ꢀ
6.0
1.7
1.1
ꢀ
79.4 ± 1.3
50.5 ± 2.0
23.3 ± 1.8
87.6 ± 0.2
1.6 ± 0.2
14.2
11.5
5.1
ꢀ
5.4
1.6
1.5
ꢀ
Doxorubicin
ꢀ
ꢀ
ꢀ
ꢀ
IC50 (in µmol/L) = concentration required in order to reduce cell viability at
5
0%; SI (IC50/MIC) = selectivity index; MRC-5 = lung fibroblast cells; A549 =
lung epithelial cells; Mt = Mycobacterium tuberculosis; Ab = Acinetobacter bau-
mannii; ꢀ not determined.
4
Polaquini, Carlos R.
