Ursolic acid derivatives as potential agents against acanthamoeba Spp

Article abstract of DOI:10.3390/pathogens8030130

The current chemotherapy of Acanthamoeba keratitis relies on few drugs with low potential and limited efficacy, for all this there is an urgent need to identify new classes of anti-Acanthamoeba agents. In this regard, natural products play an important role in overcoming the current need and medicinal chemistry of natural products represents an attractive approach for the discovery and development of new agents. Ursolic acid, a natural pentacyclic triterpenoid compound, possesses a broad spectrum of activities including anti-Acanthamoeba. Herein, we report on the development by chemical transformation of an ursolic acid-based series of seven compounds (2-8), one of them reported for the first time. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity revealed that acylation/ether formation or oxidation enhances their biological profile, suggesting that the hydrophobic moiety contributes to activity, presumably by increasing the affinity and/or cell membrane permeability. These ursolic acid derivatives highlight the potential of this source as a good base for the development of novel therapeutic agents against Acanthamoeba infections.

Full text of DOI:10.3390/pathogens8030130

pathogens
Article
Ursolic Acid Derivatives as Potential Agents Against
Acanthamoeba Spp.
Ines Sifaoui 1,2, Rubén L. Rodríguez-Expósito 1, María Reyes-Batlle 1, Aitor Rizo-Liendo 1,
José E. Piñero 1,
* *
, Isabel L. Bazzocchi 3 , Jacob Lorenzo-Morales 1 and Ignacio A. Jiménez 3,
1
Instituto Universitario de Enfermedades Tropicalesy Salud Pública de Canarias, Universidad de La Laguna,
Avda Francisco Sanchez s/n, Campus de Anchieta, 38271 La Laguna Tenerife, Islas Canarias, Espana
Laboratoire Matériaux-Molécules et Applications, University of Carthage, La Marsa Tunisia, Tunisia
Instituto Universitario de Bio-Orgánica Antonio González, Departamento de Química Orgánica, Universidad
2
3
de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Islas Canarias, Espana
*
Correspondence: jpinero@ull.edu.es (J.E.P.); ignadiaz@ull.edu.es (I.A.J.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 August 2019; Accepted: 21 August 2019; Published: 23 August 2019
Abstract: The current chemotherapy of Acanthamoeba keratitis relies on few drugs with low potential
and limited efficacy, for all this there is an urgent need to identify new classes of anti-Acanthamoeba
agents. In this regard, natural products play an important role in overcoming the current need and
medicinal chemistry of natural products represents an attractive approach for the discovery and
development of new agents. Ursolic acid, a natural pentacyclic triterpenoid compound, possesses a
broad spectrum of activities including anti-Acanthamoeba. Herein, we report on the development by
chemical transformation of an ursolic acid-based series of seven compounds (2–8), one of them reported
for the first time. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity
revealed that acylation/ether formation or oxidation enhances their biological profile, suggesting
that the hydrophobic moiety contributes to activity, presumably by increasing the affinity and/or cell
membrane permeability. These ursolic acid derivatives highlight the potential of this source as a good
base for the development of novel therapeutic agents against Acanthamoeba infections.
Keywords: ursolic acid derivatives; Acanthamoeba; chemotherapy; programmed cell death
1. Introduction
Ursolic acid (UA) is a natural pentacyclic triterpenoid compound derived from the berries,
leaves, flowers, and fruits of medicinal herbs, such as Rosemarinus officinalis, Oldenlandia diffusa, and
Radix actinidiae. It has been reported to possess various effects such as antiviral [
1], antibacterial [2],
anti-inflammatory [ ], and anticancer activities [ ]. Its antitumor effects have been investigated
3
4
in vitro and in vivo and as inducing tumor differentiation, inhibiting the proliferation of cancer cells,
inducing apoptosis, as well as promoting the autophagy of cancer cells. Furthermore, UA could inhibit
the proliferation of cervical cancer lines, including HeLa, CaSki, and SiHa, furthermore, inducing
apoptosis in HeLa cells [5]. Few reports have described the action mode of UA toward the Leishmania
parasite. In this field, UA isolated from Petiveria alliaceae induced programmed cell death (PCD) in the
promastigote form of Leishmania amazonensis and early alterations in the mitochondria physiology of
the parasite [6].
Acanthamoeba is one of the pathogen genera of free-living amoeba, widely distributed in the
environment, it has been isolated from different habitats namely soil, water, air, etc. Acanthamoeba exists
as two stages in their life cycle: A motile, trophic, and replicating trophozoite and a resistant cyst stage.
It could directly infect animal and humans causing several diseases like Acanthamoeba keratitis (AK)
and granulomatous amebic encephalitis (GAE) or by acting as a 'Trojan horse' for infective bacteria
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namely Legionella pneumophila [
7
]. Recently, Acanthamoeba keratitis cases are constantly increasing
due to the higher number of contact lens wearers [8]. The existence of a resistance stage; cyst in the
Acanthamoeba genus is the main concern in the development of effective therapeutic agents. Currently,
the first line treatments against Acanthamoeba keratitis are inefficient, due to the appearance of resistance
strains, and long-term use of eye drops are highly toxic for the patients [9,10]. Moreover, lately and
due to the lack of efficacy of the present treatment, the research for new effective drugs against this
parasite has grown exponentially.
In a previous report, we have pointed out the amoebicidal activity of UA. In fact, we observed
that this triterpenic acid could induce apoptosis in Acanthamoeba castellanii Neff by decreasing the
mitochondrial membrane potential and the ATP levels produced in cells [11].
Due to its low solubility in water, the use of ursolic acid in the pharmaceutics industry is still
limited [12]. To overcome these problems as well as to improve its selectivity effects towards parasites,
several derivatives from UA have been synthetized and proved to exhibit higher biological activity than
the origin compound [12]. In this context, the aim of this study was to evaluate the amoebicidal and the
toxic effects of ursolic acid derivatives and to elucidate the in vitro effect of the most effective molecules.
2. Results
2.1. Chemistry
The synthesis of seven analogues was carried out by alkylation, acylation, or oxidation of the
hydroxyl group of the C-3 position or carboxylic acid of the C-28 position of UA ‘1’ as presented in
Figure 1. Among the former synthetic derivatives, one out of seven was reported for the first time:
Derivative 7. The structure of the new compound was elucidated by high-resolution mass spectrometry
and NMR analysis. The known derivatives 2–6 and 8 were elucidated by comparison of their spectral
data with those reported in the literature.
Figure 1. Synthesis of ursolic acid analogues 2–8. Reagents and conditions: (i) Ac2O, Et3N, CH2Cl2;
(ii) PCC, acetone; (iii) CH3SO4, K2CO3, acetone; and (iv) Ac2O/p-NO2BzCl/BzCl, Et3N, CH2Cl2.

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2.2. Biological Study
The corresponding in vitro amoebicidal activities results are summarized in Table 1. The 50%
inhibitory concentration (IC50) was chosen as the appropriate and comparable data to give as previously
described [13]. The chemical modifications of UA enhanced the anti-Acanthamoeba activities with an
IC50 ranging from 40.0 for 1 to 21.4 for the 7 and from 15.3 for 1 to 6.7 for 7, respectively for A. Neff and
A. griffini.
Table 1. The 50% inhibitory concentrations (IC50s), the cytotoxic concentrations 50 (CC50) in µM and
the selectivity index (SI) values for the compounds used in this study against Acanthamoeba spp. and
murine macrophages measured using the Alamar blue assay. Mean concentration ± SD.
A. Castellanii Neff
A. Griffini
Murine Macrophages
CC50
SI for
A. Neff
SI for
A. Griffini
Compounds
IC50
IC50
1
2
3
4
5
6
7
8
40.0 ± 2.7
24.2 ± 0.3
34.9 ± 1.5
21.9 ± 0.9
22.7 ± 1.4
37.1 ± 1.3
21.4 ± 1.3
33.5 ± 2.1
3.02 ± 0.89
15.2 ± 0.3
11.1 ± 0.8
10.2 ± 0.5
8.7 ± 0.4
6.9 ± 1.5
9.1 ± 1.0
6.7 ± 1.8
11.5 ± 0.8
3.73 ± 0.98
17.7 ± 3.3
93.2 ± 7.2
>100
0.4
3.9
>2.9
1.2
>4.4
>2.7
>4.7
>3.0
2.5
1.2
8.4
>9.7
3.0
>14.5
>11.0
>14.9
>8.7
2.0
26.3 ± 1.7
>100
>100
>100
>100
Chlorhexidine*
7.40 ± 0.39
* Reference compound.
The analysis of variance by a two-factor ANOVA, illustrated that the biological activity was strain
dependent with p < 0.0001. In fact, the A. griffini was more sensitive to the derivatives than A. Neff.
The anti-trophozoite effect of the molecule was statistically significant with p < 0.0001. According to
the Figure 2A, for Acanthamoeba castellanii Neff, all the derivatives except 6 were statistically more
effective than the UA.
20
50
40
15
10
5
30
20
10
0
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Compounds
Compounds
A
B
Figure 2. Distribution of the IC50 of the trophocidal activities for Acanthamoeba castellanii Neff (
Acanthamoeba griffini (B).
A
) and
As for the Acanthamoeba griffini species, all the derivatives were statistically more effective than
UA as could be observed in the Figure 2B.
Considering the cytotoxicity towards the murine macrophage, if the inhibition could not reach
50% at the highest concentration, then >100
µM was given. Our data showed that the UA was the most
toxic followed by 4 and 2. Meanwhile, the other six derivatives possessed low toxicity with a CC50

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higher than 100
µM. In fact, as could be observed in Figure 3, at 50
µM 5 did not affect the morphology
of the murine macrophages, compared to 1.
Figure 3. Effect of an ursolic acid derivative at 50 µM in murine macrophages (J774A.1) observed by
inverted microscopy (20×) . Negative control (A), 1 (B) and 5 (C).
Based on their activity against Acanthamoeba and the toxicity towards the macrophages, compounds
5 and 7 were chosen for further studies. Both derivatives were observed to cause a dose-dependent
cysticidal effect. As shown in the histogram of Figure 4 For both strains no significant difference was
observed in the effect of both molecules.
25
20
15
10
5
0
5
7
A.castellanii Neff
A. griffini
Figure 4. Histogram plot of the cysticidal effects of both derivatives 5 and 7 showing the viability of
Acanthamoeba spp. cysts.
Derivatives 5 and 7 treated cells stained positive in the double-stain assay. When double staining
was performed, the tested drug at a concentration of IC90 86 µM and 75 µM respectively for 5 and
7could induced chromatin condensation proved by the bright-blue nuclei stain (Figure 5), although the
Propidium Iodide (PI) did not stain (data not shown).

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Figure 5. Acanthamoeba castellanii Neff trophozoite incubated with IC90 of 5 (B, E) and 7 (C, F) for 24 h.
Negative control (A, D). Hoechst stain is different in control cells, where uniformly faint-blue nuclei are
observed, and in treated cells, where the nuclei are bright blue. Images (20 ) are showing chromatin
) Hoechst channel (Magnification
×
condensation (blue) in treated cells. (A to C). Overlay images: (D to F
of 20× ).
Compounds 5 and 7 induced mitochondrial damages. As illustrated in the Figure 6, both
derivatives could depolarize the mitochondrial membrane potential by inhibition of JC-1 agglomeration
and emission of green fluorescence.
A
D
G
B
C
F
I
E
H
Figure 6. The effect of IC90 of 5 (B, E, H) and IC90 of 8 (C, F, I) on the mitochondrial potential, JC-1 dye
accumulates in the mitochondria of healthy cells as aggregates (red fluorescence) in cells treated with
the urolic acids derivatives for 24 h, due to the collapse of the mitochondrial potential, the JC-1 dye
remained in the cytoplasm in its monomeric form, green fluorescence. Images are representative of the
population of treated amoeba (20 ). (A to C) Overlay images, (D to F) JC-1 aggregates channel, and (G
×
to I) JC-1 monomers channel.

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Histograms of the fluorescence (Figure 7) demonstrated that the treatment with 5 and 7 decreased
the Acanthamoeba castellanii Neff mitochondrial membrane potential (∆Ψm) by 49.0% and 35.0% when
treated with IC90, respectively.
1.0
0.8
0.6
0.4
0.2
0.0
Negative control
5
7
Figure 7. Mitochondrial membrane potential (∆ψm) showing a change in the ratio of fluorescence
intensity at 590/530 nm after the 24 hours of treatment with both IC90 of 5 and 7.
The mitochondrial damage was also checked by measuring the ATP level generated in 24 h.
We found out that the treated cells with the IC90 produced half of the ATP produced by the untreated
cells (Figure 8).
2.5×107
2×107
1.5×107
1×107
5×106
0
Negative control
5
7
Figure 8. The effect of both molecules on the ATP content, using a CellTiter-Glo®Luminescent Cell
Viability Assay. Results represent the percentage relative to the negative control.
Derivatives 5 and 7 increased reactive oxygen species (ROS) levels in Acanthamoeba. Treated
amoebae with the IC90 for 24 hours generated higher level of ROS than the negative control demonstrated
by the red fluorescence emitted (Figure 9).

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A
B
C
Figure 9. Increased levels of ROS in Acanthamoeba Neff incubated with compounds 5 (
24 h. Negative control ( ): Cells exposed to CellROX Deep Red in the absence of triterpenic acids.
Images (63 ) are representative of the cell population observed in the performed experiments. Cells
were observed in a Leica TSC SPE- confocal microscope.
B) and 7 (C) for
A
®
×
3. Discussion
Organic chemistry plays an important role in the pharmaceutical industry and drug discovery
process [14]. Even though, the natural products serve as a source of raw material for innovative drug
research, a chemical modification remains imperative to enhance the efficacy/safety profile of a new
drug [15–18]. Latterly, many attempts on structural modifications of UA have been made to improve
its efficacy and specificity against cancer cells [19], HIV virus [20], and Plasmodium falciparum [21,22],
among others.
The hit compound (UA, 1) and their derivatives (2–8) were evaluated in vitro for their growth
inhibition capacity against the trophozoite stage of Acanthamoeba spp: A. castellanii Neff and A. griffini,
and against murine macrophages, wherein the latter used to calculate the selectivity index. All
derivatives made in this study demonstrated in general higher anti-Acanthamoeba activity than the
origin product, with lower toxicity towards murine macrophages.
Moreover, it is noteworthy that analogues 2, 4, 5, and 7 were the favorable trend for optimal
trophocidal activity against A. castellanii Neff, with an activity two-fold higher than the original product.
Taking into consideration the promising results obtained for UA derivatives on A. castellanii, this series
of compounds was evaluated against A. griffini. The results indicated that all analogues exhibited a
higher effect than the hit compound, UA. Moreover, compounds 5 and 7 displayed a growth inhibition
of A. griffini two-fold higher than the UA and with an IC50 of 6.9 and 6.7 µM, respectively.
After confirming that the analogues showed potent activity against both strains, the cytotoxicity
against murine macrophages was tested to determine the selectivity index as shown in Table 1.
According to Suffnes et al. (1999) the selectivity index (SI) is considerately good when its value is higher
than 2 [23]. Among the evaluated compounds, five of them showed a selectivity index higher than 10
for A. griffini than the murine cell line, including the most active analogues on this strain, compound 5
and 7. Furthermore, five compounds had a SI higher than the reference drug chlorhexidine.

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The influence of the substitution pattern of the uran-12-ene skeleton on the amoebicidal activity
was examined, revealing the following trends on the preliminary structure-activity relationship.
As shown in Table 1, compound 1 that contained two oxygenated positions, a hydroxyl group at
C-3, and a carboxylic acid at C-28, showed lower potency than those analogues (2–8). These results
indicated that a substituent at C-3 or C-28 on the core skeleton is relevant for the activity. Moreover,
the substituent at C-3 is crucial for the inhibition of the parasite, with the presence of a hydroxyl group
being unfavorable with respect to an ester or ketone (1 versus 2 and 3). In addition, esterification of
carboxylic acid has a favorable effect on the activity, as revealed by comparison of potency of the hit
compounds 1 with its methyl ester derivative 4. Our results were accordingly similar to the anti-tumor
activity of UA reported by several authors. In general, the acetylation of the C-3 of UA revealed to
be beneficial for obtaining high anti-tumor activity [24,25]. Our findings support that lipophilicity
plays an important role in the trophocidal capacity of these series of compounds especially on the
cytotoxicity profile.
In the last decades, programmed cell death (PCD) or apoptosis-like has been described in unicellular
protists, including the Plasmodium, Trypanosoma, and Leishmania genera, and Acanthamoeba [11,26].
As originally defined in multicellular organisms, apoptosis refers to certain morphological modifications
that occur during genetically controlled cell death. This process shows a common event in both
multicellular and unicellular including chromatin condensation, loss of mitochondrial membrane
potential, nuclear DNA fragmentation, cell shrinkage, and the formation of apoptotic bodies [26,27].
Hoechst 33342 is a selective DNA fluorochrome used to investigate the chromatin condensation [28].
Our results indicated that 5 and 7 induced chromatin condensation in treated cells, and resulted in the
induction of apoptotic cell death. In the same idea, Chen et al. (2019) have reported that ursolic acid
DNA condensation and fragmentation (ladder) resulted in the induction of apoptotic cell death in
NCI-H292 cells [29].
Depolarization of the mitochondrial membrane potential is a substantial evidence of apoptosis
induction. The (∆Ψm) was measured by spectrofluorometric analysis using the mitochondrial
membrane potential sensitive cationic dye JC-1 [30]. In healthy cells, the dye accumulates in the
mitochondria as aggregates and gives a red fluorescence signal. In apoptotic cells, the mitochondrial
potential collapses, and the JC-1 does not accumulate in the mitochondria and remains in the cytoplasm
giving a green fluoresce signal. Both molecules 5 and 7 induced mitochondrial malfunction. Several
reports, confirmed the mitochondria transmembrane potential collapse in different cancer cells treated
with ursolic acid [31]. In our previous work, we have proved the effect of UA activating the PCD in
Acanthamoeba by affecting the mitochondrial membrane potential [11]. Additionally, other authors
showed the apoptotic effect of this triterpenic acid in cancer cell lines. Li et al. (2014) observed
that ursolic acid was able to inhibit the proliferation of HeLa cells by inducing apoptosis via the
mitochondrial intrinsic pathway [32]. Furthermore, the effect of ursolic acid (UA) on the proliferation
of the human breast cancer cell line, MDA-MB-231 was investigated by Kim et al. (2011). They found
that UA inhibited the proliferation of the cancer cells through both a mitochondrial death pathway
and extrinsic death receptor dependent pathway [33].
The mitochondrial membrane potential is crucial for ATP generation in the respiratory chain [34].
Furthermore, affecting the mitochondria function will provoke a decrease in intracellular ATP
produced [34,35]. Herein, the ATP level was affected as a result of the mitochondria collapse.
Another aspect of the mitochondrial malfunction is the generation of oxidative stress in the cells.
In fact, the oxidative stress is known as imbalance of the ROS production over antioxidant defenses [36].
Our results show a higher production of ROS in the treated cells comparing to the negative control,
which will confirm again the mitochondria damage. Isopropyl 3β-hudroxyurs-12-en-28-oat a derivative
from ursolic acid to induce apoptosis in NTUB1 cells (human bladder cancer cells) via the excessive
production of ROS [37].
In this study it was demonstrated that both compounds 5 and 7 possessed a selective and
interesting in vitro activity against Acanthamoeba strains. Additionally, the solubility of both analogues

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was higher than the origin compound 1 (ursolic acid), which would ease their application in drug
formulation as eye drops to treat AK. To enhance the amoebicidal activity of the actual treatment a
formulation based on one of the actual derivatives and a currently used therapeutic agent such as
chlorhexidine or biguanide could be a good option. Nevertheless, further studies using in vivo models,
which would allow us to obtain data such as tissue penetration and drug half-life among others should
be developed in the near future.
4. Materials and Methods
4.1. Molecules/Chemicals
Ursolic acid was purchased from Extrasynthese (Cymit quimica, Barcelona, Spain). The
compounds tested in this study are shown in Figure 1.
The known derivatives were prepared according to the literature: 2 [24,38], 3 [24,38], 4 [24,38],
5 [38,39], 6 [40,41], and 8 [42,43].
Derivative 7 was prepared as follows: A solution of compound 4 (6 mg), two drops of dry
trimethylamine, and 8 mg of p-nitrobenzoyl chloride in dry CH2Cl2 (1 mL) were stirred under
argon atmosphere at room temperature. The progress of the reaction was monitored by Thin Layer
Chromatography (TLC) using hexane/ethyl acetate (9:1). After the mixture was concentrated to dryness
under reduced pressure, the residue was purified by preparative-TLC using hexane/ethyl acetate (9:1)
to afford the corresponding compound 7 (5.4 mg).
3β-(p-nitrobenzoyloxy)-urs-12-en-28-oic acid methyl ester (7).
20
Light white lacquer; [
α
]
D = +33.3 (c 0.24, CHCl3); for 1H NMR (CDCl3, 600 MHz),
δ 0.80 (3H, s,
H-24), 0.90 (3H, d, J = 6.4 Hz, H-29), 0.95 (1H, m, H-5), 0.97 (3H, s, H-23), 0.98 (3H, d, J = 6.4 Hz, H-30),
1.03 (3H, s, H-25), 1.05 (3H, s, H-26), 1.11 (1H, m, H-15), 1.12 (3H, s, H-27), 1.19 (1H, m, H-1), 1.33 (1H,
m, H-21), 1.37 (1H, m, H-7), 1.44 (1H, m, H-6), 1.52 (1H, m, H-21), 1.55 (1H, m, H-7), 1.59 (1H, m, H-6),
1.60 (1H, m, H-19), 1.61 (1H, m, H-9), 1.62 (1H, m, H-22), 1.70 (1H, m, H-22), 1.71 (1H, m, H-16), 1.75
(1H, m, H-1), 1.81 (2H, m, H-2), 1.82 (1H, m, H-15), 1.95 (H, m, H-20), 1.96 (2H, m, H-11), 2.04 (1H, m,
H-16), 2.67 (1H, d, J = 11.6 Hz, H-18), 4.81 (1H, dd, J = 6.4, 10.1 Hz, H-3), 5.28 (1H, t, J = 3.4 Hz, H-12),
3.64 (3H, s, OCH3), p-NO2Bz [8.22 (2H, d, J = 8.9 Hz), 8.31 (2H, d, J = 8.9 Hz)]. 13C NMR (CDCl3, 125
MHz), δ 16.0 (q, C-25), 17.4 (q, C-24), 17.5 (2 x q, C-26, C-29), 18.7 (t, C-6), 21.6 (q, C-30), 23.8 (t, C-11),
26.5 (t, C-16), 28.5 (t, C-2), 28.7 (t, C-15), 28.8 (q, C-23), 31.0 (t, C-21), 33.3 (t, C-7), 37.2 (t, C-22), 37.3 (s,
C-10), 38.8 (t, C-1), 39.4 (d, C-20), 39.6 (d, C-19), 39.9 (s, C-4), 40.3 (s, C-8), 42.4 (s, C-14), 48.0 (d, C-9),
48.1 (s, C-17), 53.2 (d, C-18), 55.7 (d, C-5), 83.3 (d, C-3), 125.5 (d, C-12), 140.1 (s, C-13), 171.3 (s, C-28),
51.7 (q, OMe), p-NO2Bz [123.9 (2
×
d), 131.0 (2
×
d), 134.9 (s), 150.8 (s), 163.8 (s)]. ESIMS m/z 642 [M +
Na]+ (100). HRESIMS m/z 642.3779 [M + Na]+ (calculated for C38H53NO6, 642.3771).
4.2. In Vitro Drug Sensitivity Assay
4.2.1. Acanthamoeba strains
The anti-Acanthamoeba activity of molecules was evaluated against the Acanthamoeba castellanii
Neff (ATCC 30010) type strain from the American Type Culture Collection and Acanthamoeba griffini
obtained in previous studies [13,44]. Those strains were grown axenically in Peptone Yeast Glucose
(PYG) medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract, and 1.5% (w/v) glucose)
containing 40 µg of gentamicin ml−1 (Biochrom AG, Cultek, Granollers, Barcelona, Spain).
4.2.2. In Vitro Effect Against the Trophozoite Stage of Acanthamoeba spp.
The anti-Acanthamoeba activities of the compounds were determined by the Alamar Blue assay
as previously described [13
microtiter plate with 50
L from a stock solution of 104 cells mL−1. Amoebae were allowed to adhere
for 15 min and 50 L of serial dilution series of the eye drop solution was added. Finally, the Alamar
,45]. Briefly, Acanthamoeba strains were seeded in duplicate on a 96-well
µ
µ

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Blue assay reagent (Bioresource, Europe, Nivelles, Belgium) was added into each well at an amount
equal to 10% of the medium volume. The plates were then incubated for 96 h at 28 ◦C with a slight
agitation and the emitted fluorescence was measured with an Enspire microplate reader (PerkinElmer,
Massachusetts, USA) at 570/585 nm.
4.2.3. In Vitro Effect Against the Cyst Stage of Acanthamoeba spp.
The cysticidal activity was determined by the Alamar Blue assay at 144 h and confirmed visually
by inverted microscopy. Cysts of both strains were prepared as described by Lorenzo-Morales et al.
(2008) [46]. Briefly, trophozoite were transferred from PYG medium based cultures (trophozoite
medium) to Neff
´
s encystment medium (NEM; 0.1 M KCl, 8 mM MgSO4
·
7H2O, 0.4 mM CaCl2 2H2O,
·
1 mM NaHCO3, and 20 mM ammediol (2-amino-2-methyl-1,3-propanediol; Sigma Aldrich Chemistry
Ltd., Madrid, Spain), pH 8.8, at 25 ◦C) and were cultured in this medium with gentle shaking for a
week in order to obtain mature cysts. After that, mature cysts were harvested and washed twice using
the PYG medium.
A serial dilution of the tested molecules was made in PYG. The in vitro susceptibility assay was
performed in sterile 96-well microtiter plates (Corning
tested and 5
104 mature cysts of Acanthamoeba/ml were added. The final volume was 100
well. After 7 days of incubation with the drugs, the plate was centrifuged at 3000 rpm for 10 min. The
supernatant was removed and replaced with 100 L of fresh medium PYG in each well. Finally, 10 µL
™). To these wells the drug concentration to be
×
µL in each
µ
of the Alamar Blue assay reagent (Biosource, Europe, Nivelles, Belgium) was placed into each well,
and the plates were then incubated for 144 h at 28 ◦C with slight agitation and the emitted fluorescence
was periodically examined with an Enspire microplate reader (PerkinElmer, Waltham, MA, USA) at
570/585 nm.
4.2.4. Cytotoxicity Activity
Cytotoxicity of the selected compounds was evaluated after 24 h incubation of murine macrophage
J774.A1 cell line (ATCC # TIB-67) with different concentrations of the tested compounds at 37 ◦C in a
5% CO2 humidified incubator. The viability of the macrophages was determined with the Alamar Blue
assay as previously described [47].
4.2.5. Double-Stain Assay for Programmed Cell Death Determination
A double-stain apoptosis detection kit (Hoechst 33342/PI; GenScript, Piscataway, NJ, USA) and
an inverted confocal microscope (Leica DMI 4000B) were used. The experiment was carried out by
following the manufacturer’s recommendations, and 105 cells/well were incubated in a 24-well plate
for 24 h with the previously calculated IC90. The double-staining pattern allows the identification
of three groups in a cellular population: Live cells will show only a low level of fluorescence, cells
undergoing PCD will show a higher level of blue fluorescence (as chromatin condenses), and dead
cells will show low-blue and high-red fluorescence (as the propidium iodide stain enters the nucleus).
4.2.6. Intracellular ROS Production Using CellROX®Deep Red Staining
The generation of intracellular ROS was detected using the CellROX
(Invitrogen). The cells were treated with IC90 of both 5 and 7 for 24 h and exposed to CellROX
Red (5
M, 30 min) at 26 ◦C in the dark. Cells were observed in a Leica TSC SPE-confocal microscope
®Deep Red fluorescent probe
®Deep
µ
equipped with inverted optics at λexc = 633 and λem = 519 nm.
4.2.7. Analysis of Mitochondrial Membrane Potential
The collapse of an electrochemical gradient across the mitochondrial membrane during apoptosis
was detected with the JC-1 mitochondrial membrane potential detection kit (Cell Technology). After
being treated with IC90 of the test solution for 24 h, the cells were centrifuged (1000 r.p.m.
×
10 min)

Pathogens 2019, 8, 130
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and resuspended in JC-1 buffer. After that, 100
µ
L of each treated culture was added to a black 96
well plate (PerkinElmer) then 10 µ
L of JC-1 was added and incubated at 26 ◦C for 30 min. Analysis
for the mean green and red fluorescence intensity was done using an Enspire microplate reader
(PerkinElmer, Waltham, MA, USA) after 30 minutes. As well as images were taken on an EVOS
fluorescence microscope from the Advanced Microscopy Group (AMG). The staining pattern allows
the identification of two groups in a cellular population: Live cells will show only red fluorescence;
and cells with low mitochondrial potential, (undergoing PCD) will show a higher level of green and
red fluorescence.
4.2.8. Measurement of ATP
The ATP level was measured using a CellTiter-Glo Luminescent Cell Viability Assay. The effect
of the drug on the ATP production was evaluated by incubating (105) of cells/ml with the previously
calculated IC90 of the selected derivatives.
4.2.9. Statistical Analysis
All data are expressed as mean
±
SD. For statistical comparisons between two groups, multiple
comparisons were performed by a two factor analysis of variance (ANOVA), and a p-value (p) < 0.05
denoted the presence of a statistically significant difference. Statistical analyses were carried out using
the GraphPad Prism8.0.2 software program (GraphPad Software, San Diego, CA, USA).
5. Conclusions
All tested derivatives demonstrated activity against Acanthamoeba spp. Based on the activity and
cytotoxicity results compounds 5 and 7 were the most potent drug; higher activity versus lower toxicity.
The structure-activity relationship (SAR) analysis showed that a substituent at C-3 or C-28 on the core
skeleton of ursolic acid was relevant for the activity. In addition, both derivatives possessed good
cysticidal activity against both tested strains. Those products could induce PCD in the parasite by the
mitochondrial pathway. Chemical modification of triterpenoids as ursolic acid could be considered as
a potent route to design new and effective amoebicidal drugs.
Author Contributions: Data curation, I.A.J.; J.L.-M.; I.S. and M.R.-B.; Formal analysis, I.S., I.A.J.; J.E.P.; Funding
acquisition, J.E.P., I.A.J., J.L.-M. and I.L.B.; Investigation, all authors. Methodology, M.R.-B., I.S., I.A.J., J.E.P.
and J.L.-M; Project administration, J.E.P.; Resources, All authors.; Supervision, J.E.P., I.A.J., I.L.B. and J.L.-M.;
Writing—original draft, I.S.; Writing—All authors. Review & editing, all authors.
Funding: This research was funded by the grants PI18/01380, Fondo Europeo de Desarollo Regional (FEDER) and
RICET (project no. RD16/0027/0001 of the program of Redes Temáticas de Investigación Cooperativa, FIS), Spanish
Ministry of Science, Innovation and Universities, Madrid, Spain. MRB was also funded by RICET. I.S. was funded
by Agustin de Betancourt program. D.S.N.-H. was funded by a grant from the Agencia Canaria de Investigaci n,
n cofunded by FEDER. O.C. was funded by a grant from Vicerrectorado de
ó
Innovación y Sociedad de la Informació
Internacionalización, Universidad de La Laguna.
Conflicts of Interest: The authors declare no conflict of interest.
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