Aryl Quinazolinone Derivatives as Novel Therapeutic Agents against Brain-Eating Amoebae

Article abstract of DOI:10.1021/acschemneuro.9b00596

Naegleria fowleri and Balamuthia mandrillaris are protist pathogens that infect the central nervous system, causing primary amoebic meningoencephalitis and granulomatous amoebic encephalitis with mortality rates of over 95%. Quinazolinones and their derivatives possess a wide spectrum of biological properties, but their antiamoebic effects against brain-eating amoebae have never been tested before. In this study, we synthesized a variety of 34 novel arylquinazolinones derivatives (Q1-Q34) by altering both quinazolinone core and aryl substituents. To study the antiamoebic activity of these synthetic arylquinazolinones, amoebicidal and amoebistatic assays were performed against N. fowleri and B. mandrillaris. Moreover, amoebae-mediated host cells cytotopathogenicity and cytotoxicity assays were performed against human keratinocytes cells in vitro. The results revealed that selected arylquinazolinones derivatives decreased the viability of B. mandrillaris and N. fowleri significantly (P < 0.05) and reduced cytopathogenicity of both parasites. Furthermore, these compounds were also found to be least cytotoxic against HaCat cells. Considering that nanoparticle-based materials possess potent in vitro activity against brain-eating amoebae, we conjugated quinazolinones derivatives with silver nanoparticles and showed that activities of the drugs were enhanced successfully after conjugation. The current study suggests that quinazolinones alone as well as conjugated with silver nanoparticles may serve as potent therapeutics against brain-eating amoebae.

Full text of DOI:10.1021/acschemneuro.9b00596

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Research Article
Aryl Quinazolinone Derivatives as Novel Therapeutic Agents against
Brain-Eating Amoebae
Mohammad Ridwane Mungroo, Muhammad Saquib Shahbaz, Ayaz Anwar, Syed Muhammad Saad,
Khalid Mohammed Khan, Naveed Ahmed Khan,* and Ruqaiyyah Siddiqui
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ABSTRACT: Naegleria fowleri and Balamuthia mandrillaris are
protist pathogens that infect the central nervous system, causing
primary amoebic meningoencephalitis and granulomatous amoebic
encephalitis with mortality rates of over 95%. Quinazolinones and
their derivatives possess a wide spectrum of biological properties,
but their antiamoebic effects against brain-eating amoebae have
never been tested before. In this study, we synthesized a variety of
34 novel arylquinazolinones derivatives (Q1−Q34) by altering both
quinazolinone core and aryl substituents. To study the antiamoebic
activity of these synthetic arylquinazolinones, amoebicidal and
amoebistatic assays were performed against N. fowleri and B.
mandrillaris. Moreover, amoebae-mediated host cells cytotopatho-
genicity and cytotoxicity assays were performed against human
keratinocytes cells in vitro. The results revealed that selected arylquinazolinones derivatives decreased the viability of B. mandrillaris
and N. fowleri significantly (P < 0.05) and reduced cytopathogenicity of both parasites. Furthermore, these compounds were also
found to be least cytotoxic against HaCat cells. Considering that nanoparticle-based materials possess potent in vitro activity against
brain-eating amoebae, we conjugated quinazolinones derivatives with silver nanoparticles and showed that activities of the drugs
were enhanced successfully after conjugation. The current study suggests that quinazolinones alone as well as conjugated with silver
nanoparticles may serve as potent therapeutics against brain-eating amoebae.
KEYWORDS: Brain-eating amoeba, antiamoebic, medicinal chemistry, quinazolinone, nanoparticles
high dosage of a mixture of drugs is recommended as
treatment against PAM and GAE.7,8 These drugs generally
lack specificity and exhibit high host cell cytotoxicity; therefore,
there is an urgent need for the development of efficient and
safer treatment options against these devastating infections.2
Quinazolinones and their derivatives are among one of the
most important heterocycles in medicinal chemistry as they
possess a wide spectrum of biological properties.9 It has been
reported that quinazolinones demonstrate antifungal activities
against organisms such as Aspergillus niger, Candida albicans,
and Aspergillus clavatus.10−12 Moreover, quinazolinones also
possess antibacterial activity against several bacteria, including
Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Listeria
monocytogenes, Klebsiella pneumoniae, Escherichia coli, Enter-
ococcus faecalis, Pseudomonas aeruginosa, and Salmonella
typhimurium.10,12−16 Furthermore, the anticancer properties
INTRODUCTION
■
Naegleria fowleri and Balamuthia mandrillaris are known as
brain-eating amoebae that infect the central nervous system
(CNS) and cause primary amoebic meningoencephalitis
(PAM) and granulomatous amoebic encephalitis (GAE),
respectively.1,2 Infection of the CNS with free-living amoebae
almost always proves to be fatal with a mortality rate of over
95%.3−6 Despite the high mortality rate, currently there is no
single effective drug available against these infections. Drugs
such as amphotericin B, miconazole, rifampin, chlorampheni-
col, ornidazole, dexamethasone, ceftriaxone, fluconazole,
miltefosine, azithromycin, 5-fluorocytosine, pentamidine,
sulfadiazine, clarithromycin, trifluoperazine, ketoconazole,
flucytosine, itraconazole, metronidazole, thioridazine, and
artesunate have shown efficacy against N. fowleri and B.
mandrillaris.5 Specifically, a combination of amphotericin B,
fluconazole, rifampin, azithromycin, dexamethasone, and
miltefosine is effective against PAM due to N. fowleri, while
a combination of pentamidine, sulfadiazine, flucytosine,
fluconazole, azithromycin, and miltefosine is effective against
GAE due to B. mandrillaris.5−8 However, the optimal regimen
against these pathogens has not been established and still a
Special Issue: CNS Pathogens
Received: November 7, 2019
Accepted: December 23, 2019
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Figure 1. (A) Amoebicidal activities of quinazolinones were determined. Briefly, B. mandrillaris was incubated with various quinazolinones for 24 h
and viability was then determined using Trypan blue as described in the Methods section. The results revealed that quinazolinones caused a
reduction in the number of viable B. mandrillaris cells. The results are representative of at least three independent experiments performed in
duplicate. The data are presented as the mean standard error (*P < 0.05, **P < 0.01, ***P < 0.001 using two sample t test; two tailed
distribution). (B) Amoebicidal activities of quinazolinones were determined. Briefly, N. fowleri was incubated with various quinazolinones for 24 h
and viability was then determined using Trypan blue as described in Methods. The results revealed that quinazolinones caused a reduction in the
number of viable N. fowleri cells. The results are representative of at least three independent experiments performed in duplicate. The data are
presented as the mean standard error (*P < 0.05, **P < 0.01, ***P < 0.001 using two sample t test; two tailed distribution).
of quinazolinones have been evaluated, and it was reported
that the compounds exhibit potent anticancer activity at low
concentrations.17−19 Quinazolinones also exhibit noteworthy
effects against viruses, such as vaccinia virus, Pox viruses,
influenza A virus, and human immunodeficiency virus.16,20,21
Several quinazoline and quinazolinone derivatives have also
been reported to exhibit antiparasitic activities such as
antileishmanial,22 antiplasmodial,23 antiacanthamoebic,24 anti-
trypanosoma,25 and antitoxoplasma activities.26 However,
despite their broad range of biological properties, quinazoli-
nones have not been tested against N. fowleri and B.
mandrillaris.
Considering the potent activity of quinazolinones against
eukaryotic fungi and parasites, we hypothesize that quinazo-
linones possess antiamoebic activity against brain-eating
amoebae N. fowleri and B. mandrillaris. A library of 34 aryl
quinazolin-4(3H)-one derivatives was synthesized by multi-
component reactions, and these compounds were thoroughly
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Figure 2. (A) Representative effects of quinazolinones on B. mandrillaris-mediated HeLa cell cytotoxicity. Briefly, amoebae (5 × 105 amoebae/0.5
mL) were incubated with 100 μg/mL of quinazolinones and then added to HeLa cells monolayers for 48 h at 37 °C in a 5% CO2 incubator as
described in the Methods section. The results showed that some quinazolinones inhibited parasite-mediated host cell damage. The data are
presented as the mean standard error (*P < 0.05, **P < 0.01 using two sample t test; two tailed distribution). (B) Representative effects of
quinazolinones on N. fowleri mediated HeLa cell cytotoxicity. Briefly, amoebae (5 × 105 amoebae/0.5 mL) were incubated with 100 μg/mL of
quinazolinones and then added to HeLa cells monolayers for 48 h at 37 °C in a 5% CO2 incubator as described in the Methods section. The results
showed that some quinazolinones inhibited parasite-mediated host cell damage. The data are presented as the mean standard error (*P < 0.05
using two sample t test; two tailed distribution).
characterized by standard spectroscopic techniques including
nuclear magnetic resonance spectroscopy and mass spectrom-
etry as reported previously.24 These compounds were tested
against N. fowleri and B. mandrillaris for their amoebicidal
properties, their amoebistatic properties, and their ability to
inhibit amoebae-mediated host cell damage. Moreover, some
of these compounds were further conjugated with silver
nanoparticles as representatives to determine if the anti-
amoebic activity is enhanced by nanoparticles conjugation. For
the first time, we showed that nanoparticle-coated quinazoli-
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Figure 3. (a) Amoebistatic activities of quinazolinones were determined. Briefly, B. mandrillaris was incubated with various quinazolinones onto a
monolayer of feeder cells for 24 h and viability was then determined using Trypan blue as described in the Methods section. The results revealed
that quinazolinones caused a reduction in the growth of B. mandrillaris cells. The results are representative of at least three independent
experiments performed in duplicates. The data are presented as the mean standard error. (b) Amoebistatic activities of quinazolinones were
determined. Briefly, N. fowleri was incubated with various quinazolinones onto a monolayer of feeder cells for 24 h and viability was then
determined using Trypan blue as described in the Methods section. The results revealed that quinazolinones caused a reduction in the growth of N.
fowleri cells. The results are representative of at least three independent experiments performed in duplicate. The data are presented as the mean
standard error (*P < 0.05, **P < 0.01 using two sample t test; two tailed distribution).
nones exhibited potent in vitro activity against brain-eating
amoebae and conjugation of drugs with silver nanoparticles
significantly enhanced activity of several derivatives against N.
fowleri and B. mandrillaris. Furthermore, nanoparticle-coated
quinazolinones inhibited parasite-mediated human cell cyto-
toxicity. Based on the results presented in the present study,
quinazolinones can be considered as potential candidate for
future drug development studies against brain-eating amoebae.
(DNA) synthesis and inhibition of cell wall synthesis in
bacteria, while inducing apoptosis, inhibition of DNA repair
and attachment to DNA in cancer cells.14−26 Therefore, we
hypothesized that these compound classes may possess
antiamoebic activities and tested them against N. fowleri and
B. mandrillaris for their amoebicidal properties, amoebistatic
properties and their ability to inhibit amoebae-mediated host
cell damage.
Several Quinazolinones Showed Amoebicidal Activ-
ity against B. mandrillaris and N. fowleri. Amoebicidal
assays were performed to determine the ability of quinazoli-
nones to kill the amoebae. The use of 100 μg/mL
quinazolinones caused a reduction in the number of viable B.
mandrillaris as shown in Figure 1A. Q5, Q7, Q10, Q11, Q13−
Q15, Q18, Q19, Q25, Q26, and Q30−Q32 showed statistically
significant effects against B. mandrillaris when compared to the
solvent control (P < 0.05 using two sample t test; two tailed
distribution). The compound that resulted in the lowest
RESULTS AND DISCUSSION
■
Primary amoebic meningoencephalitis and granulomatous
amoebic encephalitis are fatal brain infections caused by N.
fowleri and B. mandrillaris respectively.7,8 The high mortality
rates despite the existing treatment options suggest the need
for novel drugs. Quinazolinones have previously been shown to
demonstrate a wide spectrum of biological properties.9
Quinazolinones have been shown to act using various
mechanisms, including inhibition of deoxyribonucleic acid
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Figure 4. (Top) FE-SEM images of B. mandrillaris treated with quinazolinones. Amoebae treated with quinazolinones were fixed on glass coverslips
by using glutaraldehyde. Following fixation, images were recorded using a field-emission scanning electron microscope (Hitachi SU8010). (A)
Untreated (B) treated with 100 μg/mL Q15. (Bottom) FE-SEM images of N. fowleri treated with quinazolinones. Amoebae treated with
quinazolinones were fixed on glass coverslips by using glutaraldehyde. Following fixation, images were recorded using a field-emission scanning
electron microscope (Hitachi SU8010). (A) Untreated (B) treated with 100 μg/mL Q5.
number of viable B. mandrillaris is Q30, resulting in 86% cell
death. Moreover, selected quinazolinones resulted in a
reduction of the number of viable N. fowleri cells as shown
in Figure 1B. These include Q1, Q2, Q4, Q5, Q7, Q8, Q10−
Q12, Q14−Q21, Q23, Q24, and Q27−Q33. The drugs with
the highest amoebicidal activities against N. fowleri were Q23
with a reduction of viable cells by 85%.
of Q11, Q14, Q29, Q30, and Q33 inhibited the growth of B.
mandrillaris but was not statistically significant when compared
to the solvent control which inhibited the growth of the
amoebae by 47.5%. As shown in Figure 3A, Q14 and Q30
inhibited the growth of the amoeba by 60% and 58%,
respectively, when compared to untreated B. mandrillaris. A
decrease in the growth was also observed in N. fowleri treated
with 100 μg/mL of some quinazolinones as shown in Figure
3B. Q14, Q20, Q29, and Q33 are the compounds that were
able to inhibit the growth of the amoebae. Q20 inhibited the
growth of N. fowleri by 51%, while Q14 inhibited the growth of
the amoebae by 42% when compared with untreated amoebae.
Scanning Electron Microscopy. The effects of the
quinazolinones on the morphology of the amoebae were
observed using field-emission scanning electron microscopy
(FE-SEM) at 5×, 103×, and 2 × 104×. A difference in
morphology was observed between the untreated B.
mandrillaris cells and the amoebae treated with Q15 as
shown in Figure 4, top. The untreated amoebae appeared as a
circular mass with an uneven surface. While the treated cells
were also circular in shape, but they appeared wrinkled and the
formation of craters (white arrows) surrounded by a thick film
(black arrows) was observed. A difference in morphology was
also observed between the untreated and treated N. fowleri as
shown in Figure 4, bottom. The untreated N. fowleri appeared
as a circular mass with an uneven surface, while the amoebae
treated with Q5 appeared as a shrunken circular mass with the
formation of craters with a comparatively smooth surface
(white arrows) surrounded by a thick border (black arrows).
Several Quinazolinones Reduced the Cytopathoge-
nicity of B. mandrillaris and N. fowleri against Human
Cells. Assays were performed to assess the effect of the drugs
on the cytopathogenicity of the amoebae against human cells.
A concentration of 100 μg/mL of Q1, Q2, Q4, Q5, Q8, Q16−
Q18, Q22, Q23, Q29, Q33, and Q34 resulted in a statistically
significant decrease in the cytopathogenicity of B. mandrillaris
against HeLa cells when compared to untreated B. mandrillaris
as shown in Figure 2A (P < 0.05). As compared to
cytopathogenicity of 69% caused by untreated amoebae, B.
mandrillaris treated with Q23 and Q8 had a cytopathogenicity
of 8% and 16%, respectively. A decrease was also observed in
the cytopathogenicity of N. fowleri treated with 100 μg/mL of
selected quinazolinones as shown in Figure 2B. Q8, Q17, Q18,
and Q28 reduced the cytopathogenicity of the amoeba when
compared to the untreated N. fowleri which exhibited a
cytopathogenicity of 66%. N. fowleri treated with Q28 had a
cytopathogenicity of 14% as compared to 66% for the
untreated amoebae.
Various Quinazolinones Inhibited the Growth of B.
mandrillaris and N. fowleri. Selected quinazolinones were
tested for amoebistatic activity. A concentration of 100 μg/mL
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Figure 5. Cytotoxic effects of quinazolinones against host cells. Briefly, 100 μg/mL of quinazolinones were incubated with HaCaT cells monolayers
for 24 h at 37 °C in a 5% CO2 incubator as described in Materials and Methods. The results showed that quinazolinones have limited host cell
damage. The data are presented as the mean standard error.
Quinazolinones Exhibited Limited Cytotoxicity
against Human Cells. The cytotoxic effects of the
quinazolinones on human epithelial cells (HaCaT) was
determined by lactate dehydrogenase assay. The results
indicate that most of the quinazolinones had a cytotoxicity
lower than 25% at a concentration of 100 μg/mL as shown in
Figure 5. Q34 had no cytotoxic activity against HaCaT while
Q7 and Q33 only expressed limited (less than 5%) cytotoxicity
against the cell line.
Quinazolinones were Successfully Conjugated with
Silver Nanoparticles. Formation of a characteristic band at
around 420 nm, which suggests the formation of the silver
nanoparticles was observed for six of the quinazolinone
conjugated with silver nanoparticles (Supporting Information).
Peaks can be observed at below 400 nm, suggesting formation
of nanoparticles, while the zeta potential of quinazolinones
after conjugation with silver nanoparticles showed peaks at low
millivolts suggesting stability of nanoparticles (Supporting
Information).
All the quinazolinones conjugated silver nanoparticles gave
an average size of less than 50 nm. Notably, Ag-Q13 has an
average size of 6 nm and Ag-Q15 has an average size of 5 nm.
The zeta potential of quinazolinones conjugated with silver
nanoparticles was found to be averaged in the range of −30 to
−22 mV, indicating stability of the small particles.
Conjugation of Quinazolinones with Silver Nano-
particles Enhanced the Activity of Q24 against B.
mandrillaris against N. fowleri. The activity of quinazoli-
nones before and after conjugation was investigated. Q13
activity was enhanced from no visible amoebicidal activity
before conjugation to causing 40% amoebicidal effect against
B. mandrillaris while activity of Q24 was increased from 14% to
an amoebicidal effect of 42% as shown in Figure 6A. A 41%
decrease in viability of B. mandrillaris was observed for Q34
conjugated with silver nanoparticles, while 25% activity was
observed for the drug before conjugation. The activity of Q12,
Q13, Q15, Q18, Q24, and Q34 was investigated before and
after conjugation with silver nanoparticles against N. fowleri.
The activity of Q24 was enhanced from 37% to 53% against N.
fowleri after conjugation with silver nanoparticles (Figure 6B).
Structure−Activity Relationship. Q8 and Q18 showed
amoebicidal activities against both amoebae and also reduced
their cytopathogenicity. Q30 and Q23 showed the most
amoebicidal and Q14 and Q20 amoebistatic activities against
B. mandrillaris and N. fowleri, respectively. Q23 and Q28 are
the quinazolinones that were able to reduce the cytopathoge-
nicity of B. mandrillaris and N. fowleri, respectively. Q8
possesses an iodine, while Q18 possesses a methyl group at
position 2 on the benzene ring attached to the quinazoline.
Q14 and Q28 contain a methoxy group, while Q23 possesses a
butyl group at position 4 on the benzene ring attached to the
quinazoline. Q30 possesses two methoxy groups, while Q20
possesses a methyl and a chlorine group at position 2 and 5 on
the benzene ring attached to the quinazoline. The results
suggest that quinazolinones that exhibited activity against B.
mandrillaris and N. fowleri possess chlorine, methyl, or
methoxy groups. Previous studies have shown that the
presence of halogen atoms and lipophilic electron withdrawing
groups on quinazolinones enhances their antimicrobial
activity.27−31 It has also been shown that the antimicrobial
activity of quinazolinones can be enhanced by the presence of
methyl groups.27 Moreover, the enhancement of the
antimicrobial activity of quinazolinones by the presence of
methoxy groups has also been reported.9,29 Furthermore, it has
been reported that the varying positions and types of
substituents on the quinazolinones may alter the biological
properties of the drugs.32
Small sizes of nanoparticles make them efficient carriers
which enhances both pharmacokinetics and pharmacodynam-
ics of drugs.33 Furthermore, it has been previously shown that
conjugation of drugs with silver nanomaterials may enhance
activity of drugs against brain-eating amoebae.30 Selected
quinazolinones were therefore conjugated with silver nano-
particles and tested for amoebicidal activities against N. fowleri
and B. mandrillaris. It has been previously reported that
quinazolinones conjugated with silver possess potent anti-
microbial activities.34 This is consistent with our results which
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Figure 6. (A) Amoebicidal activities of quinazolinones were determined. Briefly, B. mandrillaris was incubated with various quinazolinones for 24 h,
and viability was then determined using Trypan blue as described in the Methods section. The results show the effect of quinazolinones before and
after conjugation on B. mandrillaris cells. The results are representative of at three independent experiments performed in duplicates. The data are
presented as the mean standard error (*P < 0.05 using two sample t test; two tailed distribution). (B) Amoebicidal activities of quinazolinones
were determined. Briefly, N. fowleri was incubated with various quinazolinones for 24 h, and viability was then determined using Trypan blue as
described in the Methods section. The results show effect of quinazolinones before and after conjugation on N. fowleri cells. The data are presented
as the mean standard error (*P < 0.05 using two sample t test; two tailed distribution).
suggest that the conjugation of quinazolinones with silver
nanoparticles enhances their in vitro activity against both B.
mandrillaris and N. fowleri.
In conclusion, several quinazolinones, mainly those possess-
ing halogen, methoxy, methyl, and butyl substituents, exhibited
potent antiamoebic activity. The antiamoebic effects of
METHODS
■
Synthesis of Aryl Quinazolines. A total of thirty four aryl
quinazolinones were synthesized via one pot reaction of 2-amino-3-
methylbenzoic acid or 2-amino-4,5-dimethoxybenzoic acid (1 mmol)
with triethoxymethane (3 mmol) and varying substituted anilines (1
mmol) refluxed in acidic medium using acetic acid for 8−12 h to
afford 15 3-aryl-8-methylquinazolin-4(3H)-ones and 19 3-aryl-6,7-
dimethoxyquinazolin-4(3H)-ones as described previously.22 After the
complete consumption of starting materials monitored by thin layer
chromatography analysis, the reaction mixture was poured in water,
precipitates formed were filtered, and the mixture was thoroughly
washed with ultrapure water and dried under vacuum. The crude solid
obtained was crystallized by ethanol. The structural elucidation and
purity were determined by nuclear magnetic resonance spectroscopy
(1H NMR), electron impact mass spectrometry (EI-MS), and
elemental analysis. The structure and molecular details of all 34
quinazolinones are also shown to be enhanced after
conjugation with silver nanoparticles. This suggest that
quinazolinones hold promise for drug development against B.
mandrillaris and N. fowleri. However, the mechanism of action
of the drugs, as well as the activity of the drugs in vivo, is not
clear and will be investigated in future studies.
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a
Table 1. Table Shows the Different Quinazolinones That Were Tested against the Amoebae
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Table 1. continued
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Table 1. continued
a
Name, molecular formula, structure, molecular weight, and code name of the quinazolinones are shown.
quinazolinone derivatives are depicted in Table 1. The compounds
were numbered Q1−Q34 based on their arrangement in Table 1.
Stock concentrations of 10 mg/mL were prepared in methanol and
stored at room temperature. Solvent controls contained the same
volume of methanol as the volume used from the stock solutions.
Henrietta Lacks (HeLa) and HaCaT Cell Culture. HeLa and
HaCaT cells were used as a food source for N. fowleri and B.
mandrillaris cells and for determination of cytotoxicity. Human
cervical adenocarcinoma cells (HeLa) (ATCC CCL-2) were obtained
from American Type Culture Collection, while human keratinized
skin cells (HaCaT) (CLS:300493) were acquired from CLS Cell
Lines. Cells were cultivated in Roswell Park Memorial Institute
(RPMI) 1640 medium, supplemented with 10% fetal bovine serum
(FBS), 1% minimum essential medium amino acids, 1% L-glutamine,
and 1% antibiotics (supplemented RPMI-1640) at 37 °C and 5%
CO2. When fully confluent, the cells were detached from the flasks
using trypsin, transferred to a tube, spun at 1258g for 5 min,
resuspended in supplemented RPMI-1640, and transferred into tissue
culture flasks or tissue culture plates.35,36
mandrillaris cells from the brain of a mandrill baboon (ATCC 50209)
were utilized in this study. Amoebae were cultured as previously
described.37,38 Briefly, the amoebae were cultured in RPMI-1640
medium supplemented with 1% antibiotics with HeLa cell monolayers
were used as food source at 37 °C in a 5% CO2 incubator.
Amoebicidal Assays. Amoebicidal assays were performed as
previously described.38 Briefly, 5 × 105 amoebae cells were incubated
with 100 μg/mL of drugs and/or solvents (methanol) in 24-well
plates in RPMI-1640. Amoebae were incubated with RPMI-1640 for
negative control. For positive controls, antiamoebic drugs, amphoter-
icin B and ketoconazole were utilized at 100 μg/mL. Plates were
incubated at 37 °C and 5% CO2 for 24 h. The viability of amoebae
was assessed by counting live (unstained) cells, after addition of 0.1%
Trypan blue to each well, using a hemocytometer. The data are
representative of the mean standard error of several independent
experiments performed in duplicate.
Cytopathogenicity Assays. Cytopathogenicity assays were
performed as previously described.38 Briefly, 5 × 105 amoebae cells
were treated with drugs for 2 h. Amoebae were then collected by
centrifugation at 1258g for 5 min. The pellet was resuspended in
RPMI-1640. The amoebae were incubated in RPMI-1640 in 24-well
Culture of Amoebae. N. fowleri cells isolated from the
cerebrospinal fluid of a patient (ATCC 30174) as well as B.
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plates containing HeLa monolayers at 37 °C and 5% CO2 and were
observed until 50% damage was noted for untreated amoebae. The
supernatant was then collected and tested for the presence of lactate
dehydrogenase (LDH) enzyme using a cytotoxicity detection kit
(Roche Diagnostics). The percentage LDH release was determined as
follows: [(absorbance of sample − absorbance of control)/
(absorbance for total LDH release − absorbance of control)] ×
100 = percentage cytotoxicity. The data are representative of the
mean standard error of several independent experiments performed
in duplicate.
AUTHOR INFORMATION
Corresponding Author
■
Naveed Ahmed Khan − American University of Sharjah,
Sharjah, United Arab Emirates; orcid.org/0000-
0001-7667-8553; Phone: +971-6515-5555;
Email: naveed5438@gmail.com
Other Authors
Mohammad Ridwane Mungroo − Sunway University,
Bandar Sunway, Malaysia
Muhammad Saquib Shahbaz − University of Karachi,
Karachi, Pakistan
Ayaz Anwar − University of Karachi, Karachi, Pakistan
Syed Muhammad Saad − University of Karachi, Karachi,
Pakistan
Amoebistatic Assays. For amoebistatic assays, 5 ×105 amoebae
were incubated with 100 μg/mL drugs and/or solvent (methanol) in
24-well plates containing RPMI-1640 on HeLa monolayers at 37 °C
and 5% CO2 for 24 h. RPMI-1640 alone was used as a negative
control, while 100 μg/mL amphotericin B and ketoconazole were
used as positive controls. Live amoebae were then quantified as
described for amoebicidal assay using Trypan blue exclusion assay.
The data are representative of the mean standard error of several
independent experiments performed in duplicate.
Khalid Mohammed Khan − University of Karachi,
Karachi, Pakistan
Scanning Electron Microscopy. Amoebae were fixed with 2.5%
glutaraldehyde for scanning electron microscopy.39 Amoebae were
treated with drugs for 2 h and washed with phosphate buffered saline
(PBS), fixed in 2.5% glutaraldehyde for 2 h, and resuspended in 50 μL
of Milli-Q water. A volume of 10 μL of the suspension was then
placed on a coverslip and left for 10 min to allow adherence and were
dried overnight. Field-emission scanning electron microscopy (FE-
SEM) (Hitachi SU8010) was then conducted to capture high
magnification images of the treated and untreated amoebae.
Cytotoxicity Assays. Cytotoxic effects of the drugs were
determined as previously described.35,39 The cytotoxic activity of
the drugs was investigated against HaCaT cells. Drugs at 100 μg/mL
were incubated with HaCaT cells in RPMI-1640 for 24 h at 37 °C and
5% CO2. The RPMI-1640 was then collected and tested for the
presence of lactate dehydrogenase (LDH) enzyme by using a
cytotoxicity detection kit (Roche Applied Science). The percentage
LDH released was determined as follows: [(absorbance of sample −
absorbance of control)/(absorbance for total LDH release −
absorbance of control)] × 100 = percentage cytotoxicity.
Nanoparticle Conjugation. Silver nanoparticles (AgNPs) were
conjugated with selected representative aryl quinazolinones by one
phase reduction method as previously described.38 Briefly, 1 mM
quinazolinone was magnetically stirred with 1 mM silver nitrate
aqueous solution, followed by addition of sodium borohydride
aqueous solution as reducing agent. Addition of sodium borohydride
caused a change in the color of the solution to yellow-brown,
indicating formation of AgNPs. To maximize yield, the mixture was
further stirred for 1 h. The procedure was repeated by varying volume
ratios of aryl quinazolinones to silver nitrate solution, while still using
sodium borohydride as a reducing agent. The same procedure was
used to obtain unconjugated AgNPs for use in biological assays.
Characterization of Nanoparticle Conjugation Using UV−
Vis Spectroscopy and Dynamic Light Scattering. UV−vis
absorption spectra were used for measuring the characteristic surface
plasmon resonance band of AgNPs as described previously.40
Dynamic light scattering technique was used at 25 °C, and an angle
of 90° to determine the size distribution and zeta potential of AgNPs
particles as previously described.40
Ruqaiyyah Siddiqui − Sunway University, Bandar
Sunway, Malaysia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.9b00596
Author Contributions
M.R.M. and M.S.S. contributed equally. N.A.K. and R.S.
established the idea and supervised the study. M.S.S. and
S.M.S. synthesized molecules and characterized nanoparticles
under the supervision of K.M.K. AA analyzed the data related
to the synthesis and characterization. MRM conducted all the
bioassays and prepared the first draft of manuscript. N.K. and
R.S. corrected and finalized the manuscript for submission.
The manuscript was submitted with the endorsement of all
authors.
Funding
This work is supported by the American University of Sharjah,
UAE and HEJ, Pakistan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors are grateful to Sunway University, Malaysia for
support of this work.
■
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ASSOCIATED CONTENT
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sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.9b00596.
UV−vis spectra, particle sizes, and zeta potentials of
quinazolinones conjugated with silver nanoparticles
(PDF)
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