Coumaric acid analogues inhibit growth and melanin biosynthesis in Cryptococcus neoformans and potentialize amphotericin B antifungal activity

Article abstract of DOI:10.1016/j.ejps.2020.105473

Fungal infections are on the rise, since the imunocompromised population is increasing due to AIDS/HIV, organ transplant and chemotherapy. Many environmental and pathogenic fungi are able to accomplish melanin biosynthesis as a virulence factor to promote host invasion. Melanized cells are more resistant to radiation, oxidative and osmotic stresses; also melanin confers an advantage in vivo, since melanized cells are more resistant to phagocytic engulfment and oxidative stress caused by the host defense cells and by some antifungal drugs, such as fluconazole (FCZ) and amphotericin B (AmB). Brown, red or black melanin pigments can be produced by the polyketide pathway (DHN-melanin) or from dihydroxyphenols, such as L-DOPA (L-3,4-dihydroxyphenylalanine) and L-tyrosine by polyphenoloxidases. Among several pathogenic fungi, Cryptococcus neoformans is a melanized yeast that causes pneumonia and meningoencephalitis in immunocompromised patients. The knockout of the laccase genes or other interruptions on melanin biosynthetic pathway generates cryptococcal strains with attenuated virulence in an animal model. In this study 16 analogues of coumaric and cinnamic acid were evaluated as possible tyrosinase inhibitors. We have identified some valuable inhibitors of C. neoformans growth and melanin biosynthesis disruption agents. The results showed that coumaric acid derivatives (1a-c), the ketones (3a-b) and 2-allylphenol (7c) are significant inhibitors of tyrosinase and melanization of the fungus. Two analogues (1b and 3b) were selected as promising antimelanogenic agents to be combined with AmB, showing to promote 16-fold reduction in the AmB fungicidal concentration with no appreciable cytotoxicity to mammalian cells. The data suggest that inhibition of the melanin biosynthesis by these compounds may increase the susceptibility of the cells to the oxidative stress generated by AmB. In summary, our data show that C. neoformans can be a suitable model system to test novel inhibitors that target melanin biosynthesis, and novel compounds for adjunct therapy against C. neoformans were identified.

Full text of DOI:10.1016/j.ejps.2020.105473

Journal Pre-proof
Coumaric acid analogues inhibit growth and melanin biosynthesis in
Cryptococcus neoformans and potentialize amphotericin B antifungal
activity
Lidiane Oliveira , M a´ rcio Ferrarini , Alexandre Paes dos Santos ,
Marina Themoteo Varela , Isabela Teresa Santos Corr eˆ a ,
Andr e´ Gustavo Tempone , Marcia S.C. Melhem ,
Marcelo A. Vallim , Jo a˜ o Paulo S. Fernandes , Renata C. Pascon
PII:
S0928-0987(20)30262-1
DOI:
Reference:
https://doi.org/10.1016/j.ejps.2020.105473
PHASCI 105473
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European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
12 June 2020
10 July 2020
17 July 2020
Please cite this article as: Lidiane Oliveira , M a´ rcio Ferrarini , Alexandre Paes dos Santos ,
Marina Themoteo Varela ,
Isabela Teresa Santos Corr eˆ a , Andr e´ Gustavo Tempone ,
Marcia S.C. Melhem , Marcelo A. Vallim , Jo a˜ o Paulo S. Fernandes , Renata C. Pascon , Coumaric
acid analogues inhibit growth and melanin biosynthesis in Cryptococcus neoformans and potential-
ize amphotericin B antifungal activity, European Journal of Pharmaceutical Sciences (2020), doi:
https://doi.org/10.1016/j.ejps.2020.105473
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Highlights




Novel coumaric acid analogues with fungal tyrosinase inhibitory activity are
described
These compounds also cause growth arrest and melanization inhibition in the
opportunistic yeast C. neoformans
The coumaric analogues described in this article potentialize amphotericin B
fungicidal activity in C. neoformans
C. neoformans may be useful as model system to screen for novel and more
potent antimelanogenic activity
1

Coumaric acid analogues inhibit growth and melanin biosynthesis in Cryptococcus
neoformans and potentialize amphotericin B antifungal activity
1
#
2#
3
2
Lidiane Oliveira, Márcio Ferrarini, Alexandre Paes dos Santos, Marina Themoteo
1
1
1,4
Varela, Isabela Teresa Santos Corrêa, André Gustavo Tempone, Marcia S. C.
3
2*
3*
Melhem, Marcelo A. Vallim, João Paulo S. Fernandes, Renata C. Pascon
#
These authors contributed equally to this work
1
Centre for Parasitology and Mycology, Instituto Adolfo Lutz, Avenida Dr. Arnaldo,
3
55, 01246-000 São Paulo, Brazil
2
Department of Pharmaceutical Sciences, Universidade Federal de São Paulo, Rua São
Nicolau 210, 09913-030 Diadema-SP, Brazil
3
Biological Sciences Department, Universidade Federal de São Paulo, Rua São Nicolau
2
10, 09913-030 Diadema-SP, Brazil
4
Federal University of Mato Grosso do Sul, School of Medicine, MS, Brazil
*
corresponding authors: renata.pascon@gmail.com and joao.fernandes@unifesp.br
Keywords: Cryptococcosis, coumaric acid analogues, melanin, amphotericin B,
antifungal agent, tyrosinase inhibitor
2

ABSTRACT
Fungal infections are on the rise, since the imunocompromised population is increasing
due to AIDS/HIV, organ transplant and chemotherapy. Many environmental and
pathogenic fungi are able to accomplish melanin biosynthesis as a virulence factor to
promote host invasion. Melanized cells are more resistant to radiation, oxidative and
osmotic stresses; also melanin confers an advantage in vivo, since melanized cells are
more resistant to phagocytic engulfment and oxidative stress caused by the host defense
cells and by some antifungal drugs, such as fluconazole (FCZ) and amphotericin B
(AmB). Brown, red or black melanin pigments can be produced by the polyketide
pathway (DHN-melanin) or from dihydroxyphenols, such as L-DOPA (L-3,4-
dihydroxyphenylalanine) and L-tyrosine by polyphenoloxidases. Among several
pathogenic fungi, Cryptococcus neoformans is a melanized yeast that causes pneumonia
and meningoencephalitis in immunocompromised patients. The knockout of the laccase
genes or other interruptions on melanin biosynthetic pathway generates cryptococcal
strains with attenuated virulence in an animal model. In this study 16 analogues of
coumaric and cinnamic acid were evaluated as possible tyrosinase inhibitors. We have
identified some valuable inhibitors of C. neoformans growth and melanin biosynthesis
disruption agents. The results showed that coumaric acid derivatives (1a-c), the ketones
(3a-b) and 2-allylphenol (7c) are significant inhibitors of tyrosinase and melanization of
the fungus. Two analogues (1b and 3b) were selected as promising antimelanogenic
agents to be combined with AmB, showing to promote 16-fold reduction in the AmB
fungicidal concentration with no appreciable cytotoxicity to mammalian cells. The data
suggest that inhibition of the melanin biosynthesis by these compounds may increase
the susceptibility of the cells to the oxidative stress generated by AmB. In summary, our
data show that C. neoformans can be a suitable model system to test novel inhibitors
3

that target melanin biosynthesis, and novel compounds for adjunct therapy against C.
neoformans were identified.
INTRODUCTION
Melanin is a negatively charged hydrophobic pigment produced by organisms in all
kingdoms of life (Hill, 1992). Especially in pathogenic fungi, such as Aspergillus
fumigatus, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrososi, Sporothrix
complex species, Cryptococcus neoformans and Cryptococcus gattii species complexes,
melanin plays multifunctional protective roles. Melanin protects from ultraviolet (UV)
radiation, ionizing radiation, oxidizing agents in the environment promoting survival
and confers resistance to phagocytic engulfment during oxidative immune system attack
in the human and animal host (Gomez and Nosanchuk, 2003). Therefore, the ability to
synthesize melanin has been considered an important virulence factor that allows host
colonization and pathogenesis establishment (Eisenman et al., 2020; Zaragoza, 2019).
There are two major pathways for melanin biosynthesis in fungi: the polyketide
pathway from 1,8-dihydroxynaphthalene (DHN), which is known as DHN-melanin; or
through oxidation of dihydroxyphenols starting from L-tyrosine and L-3,4-
dihydroxyphenylalanine (L-DOPA), which is a oxidation product of L-tyrosine, by the
action of a polyphenoloxidase (laccase), which catalyzes one-step reaction to quinone
intermediates that auto-oxidizes to melanin. Fungi may have any of these pathways, or
even both (Eisenman and Casadevall, 2012).
C. neoformans is a melanized fungus which may cause pneumonia and
meningoencephalitis, the leading fungal cause of death in immunocompromised
population, especially AIDS/HIV patients, those under chemotherapy and organ
4

transplanted patients (Zaragoza, 2019). The treatment against invasive fungal infections
IFIs) is in general difficult, often because the drugs used in the therapeutics have low
(
selective toxicity leading to hepato- and nephrotoxicity (Odds et al., 2003; Skipper et
al., 2019). Moreover, available antifungal drugs are scarce, since few selective targets
for antifungal development are druggable due to fungal and animal cells similarities. In
the case of cryptococosis the treatment is even more problematic due to the main site of
the infection, the central nervous system. Fluconazole (FCZ) and amphotericin B
(AmB) are the drugs of choice for cryptococosis treatment, which is required for long
periods of time, leading to the arising of resistant strains that are refractory to canonical
treatment (Skipper et al., 2019). AmB acts by binding to ergosterol, destabilizing the
plasma membrane, leading to ion loss and also kills fungal cells by generating high
levels of oxidative stress and accumulation of reactive oxygen species (ROS). One
important factor in order to evade oxidative stress caused by AmB in C. neoformans is
melanin, which acts as ROS scavenger (Mesa-Arango et al., 2014).
C. neoformans has only the L-DOPA pathway, which also depends on an exogenous
substrate for melanin production, either L-tyrosine or L-DOPA. The dependence of the
C. neoformans virulence on melanin production has been extensively reported in the
literature (Leopold Wager et al., 2016; Salas et al., 1996; Wang et al., 1995) and makes
this virulence factor an attractive one for inhibitory drugs development. Thus, by
targeting the melanin biosynthesis pathway, it is possible to find new drug candidates
that could reduce the virulence of C. neoformans, making it more susceptible to the
immune system and antifungal drugs, without directly harming the fungi cells and thus
avoiding the development of resistance.
A literature search revealed that p-coumaric acid (compound 1, Figure 1), a natural
product, possess inhibitory activity on human and fungal tyrosinases, which are
5

polyphenoloxidases (Boo, 2019). The evident similarity between compound 1 and the
substrate for melanin biosynthesis, the amino acid L-tyrosine (highlighted in Figure 1),
suggests that the carboxylic acid and the phenol motifs are important to the interaction
with the enzyme, explaining why compound 1 acts as inhibitor (Pei et al., 2016).
In our hypothesis, antimelanogenic compounds could inhibit the melanization process in
fungi, making them more susceptible to the host immune system, oxidative stress and
indeed to death by some antifungal agents. The hydrophilic nature of p-coumaric acid
impairs its penetration through cell membranes, and thus a set of analogues were
designed to increase the lipophilicity and reduce the ionization profile of the prototype,
but maintaining their efficacy as melanization inhibitors. Considering this, the present
work aimed to evaluate analogues of p-coumaric acid to assess their potential as
adjuctive antimelanogenic agents against C. neoformans.
The results obtained show that these analogues led to C. neoformans growth arrest,
reduced melanin biosynthesis and further, acted in combination with AmB which causes
oxidative stress in the cell (Baginski and Czub, 2009). These results unraveled a novel
prototype for adjunct treatment against C. neoformans and possibly other
microorganisms that use L-DOPA/L-tyrosine for melanin biosynthesis. Moreover, our
results also suggest that C. neoformans may be a biological model for screening the
antimelanogenic activity of novel compounds, as exemplified with the inhibitors herein.
MATERIALS AND METHODS
Chemicals
The p-coumaric (1), cinnamic (2), 4-methoxycinnamic (4) and caffeic (5) acids, vanillic
acid (6a) and alcohol (6b), eugenol (7a), isoeugenol (7b) and 2-allylphenol (7c) (Figure
6

1
) were commercially acquired with adequate purity for synthesis and testing in the
biological assays (Sigma-Aldrich, USA). All other chemicals such as salts, solvents,
acids and bases were purchased (LabSynth, Brazil) in adequate purity grade to be used
without further preparation. The synthesized compounds were characterized through
1
hydrogen nuclear magnetic resonance ( H NMR) and compared to former literature
reports for these compounds. Reported melting points (mp) were recorded in a capillary
melting point apparatus (Gehaka, PF1500), when applicable.
Design of the compounds
Considering that compound 1 (Figure 1) is a known antimelanogenic agent, several
analogues were designed to assess structural features that could play a role in this
activity. For this purpose, a set of analogues were designed as potential latent forms
(prodrugs) of 1 with improved penetration on fungal cells (compounds 1a-c). Moreover,
since phenols may act as antiradical agents, the roles of the phenol group in melanin
formation process and in antifungal activity were checked by testing the cinnamic acid
analogues (2a-c), p-methoxycinnamic acid (4) caffeic acid (5), which present structural
variations on the phenolic moiety. Other phenolic compounds (6a-b) were also inserted
in the set for this purpose. Finally, considering the similarity with the amino acid
substrates, the ketone analogues 3a-b and the eugenol analogues 7a-c were included in
the set to check if the carboxylic acid group is essential to the antimelanogenic and
antifungal activities. In fact, there are previous reports from the antimelanogenic
activity of eugenol, which raised these questions (Ali et al., 2016).
General procedure for the synthesis of the esters
7

The ester derivatives (p-coumarates 1a-c and cinnamates 2a-c) were synthesized
following classic Fischer esterification. In a flask, 1 mmol of the corresponding acid (1
or cinnamic acid 2) and 0.1 mL of concentrated sulfuric acid were added to an excess
(10 mL) of corresponding alcohol (methanol, ethanol or isopropanol) as solvent. The
reaction mixture was stirred under reflux for 3-24 hours until no more starting acid was
present (monitored by thin layer chromatography, TLC). The acid was neutralized with
saturated sodium bicarbonate solution, and the excess of alcohol was evaporated under
reduced pressure. The residue was taken up in 15 mL ethyl acetate and washed three
times with 15 mL saturated sodium bicarbonate solution and dried over anhydrous
sodium sulfate. The solvent was removed and the crude product was purified through
open silica gel column chromatography using hexane:ethyl acetate as eluent. The yields
are given after chromatographic purification.
General procedure for the synthesis of the compounds 3a and 3b
The α,β-unsaturated compounds were obtained following classic aldol condensation
reaction under basic conditions, as previously described by our group (Varela and
Fernandes, 2018). Equimolar amount of ketone (acetone for 3a and 2-pentanone for 3b)
and p-hydroxybenzaldehyde (2 mmol each) were dissolved in ethanol, and afterwards 2
mL of 40% sodium hydroxide solution was added dropwise. The reaction proceeded
under reflux for 24 hours. After neutralization, ethanol was evaporated and the crude
product was precipitated. The solid material was purified through open silica gel
column chromatography, using hexane:ethyl acetate as eluent. The yields are given after
chromatographic purification.
8

Tyrosinase inhibition assay
The compounds were screened for the tyrosinase inhibition assay in two different
concentrations (1 mM and 10 µM). Briefly, in a 96-well plate, 20 µL of each compound
in a 10% DMSO stock solution (10 mM and 1 mM, respectively), 80 µL of phosphate
buffer (pH 6.5) and 50 µL of mushroom tyrosinase (Sigma-Aldrich, USA) solution in
the same phosphate buffer (400 U/well) were added to each well. The plate was
incubated for 15 minutes at 37 ºC and afterwards, 50 µL of freshly prepared L-tyrosine
solution (1.5 mM in phosphate buffer) was added and the plate was monitored in a plate
reader (490 nm) at a 37 ºC for every 3 minutes during 30 minutes. The DMSO 1%
solution in phosphate buffer was used as negative control. The acquired data were
normalized as percentage of tyrosinase activity, and statistically evaluated in the
GraphPad Prism 5.0 software.
Strains, media and microbiological reagents
The strain type C. neoformans var. grubii serotype A H99 (ATCC 208821) was used in
the study. Also, H86 mutant, which has an interruption on the laccase gene
(CNAG_03465) was included in the study. The strains were routinely grown on rich
medium YEPD (1% yeast extract, 2% bacto-peptone, 2% glucose) at 30°C on plates or
in liquid with 150 rpm in a rotatory shaker.
-
Lac mutant construction
H86 strain was isolated in a mutant collection made by Agrobacterium tumefaciens
random insertional mutagenesis described previously by our group (de Gontijo et al.,
9

2
014). Briefly, C. neoformans mutant was created by Agrobacterium mediated-
transformation according to the method described by Idnurm et al. (Idnurm et al., 2004).
Briefly, H99 derived C. neoformans strain KN99α (Nielsen et al., 2003) was co-cultured
with A. tumefaciens LBA4404 strain harboring the plasmid pPZP-Hyg2 (kindly donated
by Dr. J. Heitman) for 48 hours, then aliquots were platted onto YEPD medium
supplemented with 200 μg/mL hygromycin, and cefotaxime (100 μg/mL) to eliminate
Agrobacterium strain (de Gontijo et al., 2014; Walton et al., 2006). After 48 to 72 hours,
antibiotic resistant colonies were transferred to Guizzotia abssynica agar plates (75g/L
bird seed, 0.1% dextrose, 2% agar), and incubated at 30°C for up to 96 hours when
melanin production was evaluated. Sequencing revealed that the hygromicin cassette
integrated and disrupted the LAC1 locus (CNAG_03465).
Disk diffusion method
Stock solutions of the 16 inhibitors and positive controls (timol and eugenol) were
prepared in 50% dimethylsulfoxide (DMSO) at 500 mM; AmB at 1600 µg/mL in
DMSO and FCZ at 5120 µg/mL in H O. The disk diffusion method was used as
2
described by the Clinical and Laboratory Standards Institute (CLSI, 2009) with small
modifications. YEPD plates were inoculated with a sterile swab dipped into a cell
suspension (MacFarland´s scale 1) made from a saturated overnight cultured (YEPD)
inoculated with a single colony. After plates were dried, the sterilized disks impregnated
with 5 µL of each inhibitor and then placed on the inoculated plates. The cultures were
grown at 30 °C for 48 hours before the results were documented. To calculate the
activity of compounds, an inhibition index was calculated: the diameter of the inhibition
zone (millimeters) was divided by 6 mm, which is the diameter of the disk.
1
0

Minimal inhibitory concentration determination
Microdilution tests were conducted according to document 7.3 of European Committee
on Antimicrobial Susceptibility Testing (EUCAST) for yeasts (Arendrup et al., 2016)
with some modifications. Briefly, the minimum inhibitory concentration (MIC) values
were determined using 96 wells microtiter plates. Sabouraud agar plates containing
yeast colonies were cultured overnight at 30 °C. Five colonies were picked and diluted
6
in 0.9% of saline and were adjusted to 1 to 5 x 10 CFU/mL using the McFarland´s
3
scale. This cell suspension was serially diluted to 0.5-2.5 x 10 CFU/mL in RPMI-1640
medium (Sigma-Aldrich) added with MOPS and 2% glucose, pH 7.0 and added to the
microtiter plate. The colonies formation units (CFU) counts and viability were
confirmed on YEPD (100 µL of diluted cells). Inhibitor compounds and reference
standards compounds were serially diluted (two-fold) and added to each well.
Sterilization negative control containing liquid medium and a positive growth control
containing fungal suspension, amended with DMSO control (10 µL) were included. The
microtiter plates were incubated at 30°C for 48 hours. The MIC was considered the
lowest concentration at which at least 90% of growth was inhibited. All tests were
performed in triplicate. Candida krusei (ATCC 6258) and C. parapsilosis (ATCC
2
2019) were used as quality control strains.
Checkerboard assays
Antifungal interaction was evaluated by the microdilution broth checkerboard method
(Nooney et al., 2005). The following combinations were tested: AmB (from 0.03 to 0.5
mg/L), FCZ (from 2 to 32 mg/L) along with compounds 1b and 3b (from 62.5 to 1000
1
1

mg/L). For test, AmB and FCZ (Sigma-Aldrich) pure powder drugs were dissolved
according to the manufacturer’s instructions and diluted in RPMI-1640 (Sigma-
3
Aldrich). The final inoculums were 0.5 to 2.5 x 10 CFU/mL. Plates were incubated at
3
5 °C for 72 hours. The interaction coefficient between each antifungal drug and
inhibitor compounds (named A and B) was quantitatively evaluated as means of the
fractional inhibitory concentration index (FICI), which was calculated by using the
following formula: FICI = FIC A (MIC A in combination/MIC A) + FIC B (MIC B in
combination/MIC B). The interaction was defined as synergistic if FICI ≤ 0.5;
indifferent if FICI > 0.5 to ≤ 4; and antagonistic if FICI was > 4.0 (Odds, 2003).
Mammalian Cytotoxicity assay
®
Murine fibroblasts NCTC cells (ATCC clone 929) cultured in RPMI-1640 medium
with 10% fetal bovine serum, were counted in a hemocytometer chamber, seeded at 6 x
4
1
5
0 /well and incubated in with the compounds (200 - 1.56 µM) for 48 h at 37 °C in a
% CO -humidified incubator. The cell viability was determined using the 3-(4,5-
2
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Pinto et al.,
013). The cytotoxicity was determined through non-linear regression using the
GraphPad Prism 5.0 software, to obtain the 50% cytotoxic concentration (CC ).
2
50
L-DOPA melanin assay
Melanin was assayed according to previously published protocol at 30 °C for 48 hours
Paliwal and Randhawa, 1978; Price et al., 1982). In brief, the medium was prepared
with glucose (15 mM), magnesium sulfate (10 mM), potassium phosphate (KH PO4
(
2
2
1.4 mM), glycine (13 mM), thiamine (3 µM), bacto-agar (1.5%) and L-DOPA (1 mM)
at pH 5.5. The compounds were added at lethal and sub lethal concentrations to the
6
medium, mixed and poured on plates. Cell suspensions of H99 and H86 strains (2 x 10
1
2

CFU/mL in saline) made from cell growing on a fresh YEPD plate were used to
inoculate (5 µL) the melanization medium after solidification.
RESULTS AND DISCUSSION
Characterization of the compounds
1
Methyl coumarate (1a). White solid, 56% yield (mp_141-144 °C). H NMR (300
MHz, CDCl ): δ 7.65 (d, J = 16.0 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz,
3
2
H), 6.30 (d, J = 16.0 Hz, 1H), 6.11 (s, 1H), 3.81 (s, 3H). Spectral data are in
accordance with previous literature (Huang et al., 2010; Lopes et al., 2019).
1
Ethyl coumarate (1b). White solid, 53% yield (mp 78-81 °C). H NMR (300 MHz,
CDCl ): δ 7.56 (d, J = 16.0 Hz, 1H), 7.32 (d, J = 8.5 Hz, 2H), 6.99 (s, 1H), 6.80 (d, J =
3
8
3
.5 Hz, 2H), 6.21 (d, J = 16.0 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz,
H). Spectral data are in accordance with previous literature (Lu, 2011).
1
Isopropyl coumarate (1c). Yellowish solid, 65% yield (mp 72-75 °C). H NMR (300
MHz, CDCl ): δ 7.62 (d, J = 15.9 Hz, 1H), 7.49–7.31 (m, 2H), 6.95–6.82 (m, 2H), 6.28
3
(d, J = 15.9 Hz, 1H), 5.14 (quint, J = 6.2 Hz, 1H), 1.32 (d, J = 6.2 Hz, 6H). Spectral
data are in accordance with previous literature (Soda et al., 2012).
1
Methyl cinnamate (2a). Yellowish oil, 59% yield. H NMR (300 MHz, CDCl ): δ 7.69
3
(
d, J = 16.0 Hz, 1H), 7.50 (dd, J = 3.6, 1.9 Hz, 2H), 7.43–7.30 (m, 3H), 6.44 (d, J = 16.0
Hz, 1H), 3.79 (s, 3H). Spectral data are in accordance with previous literature (Omar,
014).
2
1
3

1
Ethyl cinnamate (2b). Yellowish oil, 58% yield. H NMR (300 MHz, CDCl ): δ 7.69
3
(d, J = 16.0 Hz, 1H), 7.51 (dd, J = 6.6, 2.9 Hz, 2H), 7.46–7.30 (m, 3H), 6.44 (d, J = 16.0
Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). Spectral data are in
accordance with previous literature (Omar, 2014).
1
Isopropyl cinnamate (2c). Yellow oil, 48% yield. H NMR (300 MHz, CDCl ): δ 7.67
3
(
d, J = 16.0 Hz, 1H), 7.50 (d, J = 4.3 Hz, 2H), 7.39–7.32 (m, 3H), 6.41 (d, J = 16.0 Hz,
H), 5.14 (quint, J = 6.2 Hz, 1H), 1.31 (d, J = 6.2 Hz, 6H). Spectral data are in
accordance with previous literature (Jia, 2006).
1
(1E)-1-(4-Hydroxyphenyl)but-1-en-3-one (3a). Yellowish solid, 62% yield (mp 109-
1
1
6
11 °C). H NMR (300 MHz, CDCl ): δ = 7.55-7.36 (m, 3H), 6.87 (d, J = 8.7 Hz, 2H),
3
.61 (d, J = 16.1 Hz, 1H), 5.55 (br. s, 1H), 2.37 (s, 3H). Spectral data are in accordance
with previous literature (Varela et al., 2019).
(1E)-1-(4-Hydroxyphenyl)hex-1-en-3-one (3b). Yellowishsolid, 57% yield (mp 117-
1
1
6
0
2
20 °C). H NMR (300 MHz, CDCl ): δ = 7.58-7.40 (m, 3H), 6.86 (d, J = 8.62 Hz, 2H),
3
.63 (d, J = 16.1 Hz, 1H), 5.26 (br. s, 1H), 2.63 (t, J = 7.3 Hz, 2H), 1.81-1.61 (m, 2H),
.98 (m, 3H). Spectral data are in accordance with previous literature (Varela et al.,
019).
Inhibition assay on fungal tyrosinase
The compounds with similarities to the coumaric and cinnamic acid (all the set with
exception of the simple phenols 6a and 6b) were tested for their inhibitory effect on
fungal tyrosinase using the classical protocol from literature (Ali et al., 2016) in two
different concentrations (1 mM and 10 µM) to check for a concentration-dependent
1
4

effect. The results from tyrosinase inhibition are shown in Table 1. As can be noted, the
coumaric acid esters 1a-c were the most effective inhibitors of tyrosinase activity,
followed by the ketone analogues 3a and 3b. This result suggest that the phenol group
in 4-position of the aromatic ring is essential to adequate tyrosinase inhibition, since the
non-phenolic cinnamic acid analogues 2a-c and the methoxy analogue 4 showed poor
inhibitory activity on the enzyme. Moreover, the presence of the acidic group seems not
essential, as suggested by the activities from the ketones 3a-b. The presence of other
substituents on the aromatic ring seems detrimental to the inhibitory activity, as
exampled by the results from caffeic acid 5 and eugenols 7a-b. However, compound 7c
showed considerable inhibitory activity, suggesting that switching the hydroxyl to the 2-
position increased the inhibitory activity on tyrosinase.
Tyrosinase inhibitors have been widely explored in cosmetic, medical and food
applications. Many tyrosinase inhibitors have been described over the years, and most
of them have been screened based on fungal tyrosinase inhibition assays, because
human tyrosinase is not readily available from commercial sources (Zolghadri et al.,
2
019). Recently, human and fungal tyrosinases have been reported as requiring different
molecular motifs for inhibition, implying this enzyme may be a drug target with high
selective toxicity against melanotic fungi (Mann et al., 2018).
Inhibition of growth and melanization in C. neoformans
The possibility that tyrosinase may be a selective target for antifungals, and the fact that
these compounds inhibit fungal tyrosinase, led to the question of whether they would
inhibit the growth of C. neoformans wild type (H99) strain by disk diffusion assay. As
presented in Table 2, compounds 1b and 3b were able to inhibit fungal growth at higher
1
5

disk diffusion index levels (2.92 and 2.58, respectively) compared to the positive
control (timol). All ester analogues (1a-c) presented higher growth inhibition than
coumaric acid (1), suggesting that these substitutions would improve the antimicrobial
activity in the fungal cell. The remaining ketone analogue 3a also presented
considerable inhibitory activity. On the contrary, cinnamic acid (2), its analogues (2a-c),
methoxycinnamic acid (4) and caffeic acid (5) had no important activity against fungal
growth.
MIC test were done to confirm and complement information given by disk diffusion
assay. To evaluate the structural motifs involved in the antifungal activity that may
interfere in the melanization assays, we also added the vanillin-derived compounds, 6a
and 6b, as well as previously known analogues derived from eugenol (7a-c). These
compounds present some similarities to the coumarates and, also, earlier our group
demonstrated that 7a has antimicrobial activity (Martho et al., 2019; Martho et al.,
2016). The MIC results are described in Table 3 and are consistent to those obtained
from the disk diffusion assay. The compounds 1b and 3b presented the lowest MIC
values among the inhibitors tested, which is in line with the disc diffusion assays. These
results suggest that coumaric acid (1a-c) and ketone analogues (3a-b), not only promote
high inhibitory effect on tyrosinase activity (Table 1), but also affect fungal growth
(Table 2 and 3). Also, phenol moiety as radical scavenger seems to be important for
tyrosinase and growth inhibition, since compounds 2a-c, 4 and 5 promoted the lowest
tyrosinase inhibitory activity and had no effect on growth.
Since coumaric acid and ketone analogues inhibited both tyrosinase activity and fungal
growth, it is possible that these compounds also interfere with melanin biosynthesis in
the fungus. Since melanization is an important virulence factor in C. neoformans, it was
worth checking this hypothesis. Therefore, we tested the effects of MIC and sub-lethal
1
6

MIC (half of MIC) concentration of these inhibitors on melanin production by the wild
type H99 strain. Also, the H86 mutant which has an interruption in the laccase 1 gene
(CNAG_03465) was added to this experiment as a control to set the visual parameter
for a strain that produces low levels of melanin. An A. tumefaciens insertion
mutagenesis collection comprising 980 stable transformants was screened on bird seed
agar (Guizzotia abssynica agar plates) and a single lac- mutant (H86) was uncovered.
The confirmation that insertion occurred on the laccase 1 gene (CNAG_03465) was
obtained by inverted PCR followed by DNA sequence (data not shown). C. neoformans
genome encodes two laccase genes (CNAG_03464 and CNAG_03465) that are
arranged side by side on chromosome 8; however only CNLAC1 gene was interrupted
leaving a second laccase gene intact. Due to this second functional laccase gene (Lac2),
H86 strain still produces a light brown pigment, which represents low levels of melanin
biosynthesis, however it is much lower compared to the wild type H99 in the presence
of L-DOPA (Figure 2A, right panel). Both strains produce opaque white colonies on
medium lacking L-DOPA (Figure 2A, left panel).
The compounds that did not show activity at MIC assay (MIC > 1000 µg/mL, except 6a
and 6b) were excluded from this analysis (1c, 2a-c, 4 and 5). In addition to growth
reduction, these compounds caused a massive salt precipitation in the L-DOPA
medium, impairing any growth, even at very low concentrations, imposing a technical
problem to this experiment with these compounds. Therefore, we were unable to work
with these inhibitors regarding melanization. Figure 2B (1, 1a and 1b), 2C (3a and 3b)
and 2D (6a-b and 7a and 7c) show that all substances tested are able to inhibit
melanization of C. neoformans at different degrees depending on the tested compound
and dose (lethal and sub lethal doses). It is worth to mention that compound 1b (Figure
2
B) caused visual, large growth reduction at both MIC and sub MIC concentrations.
1
7

This set of coumarate analogues allowed the investigation on the molecular moieties
that are important for the antimelanogenic activity and C. neoformans growth arrest
(
disk diffusion and MIC assays). The data have shown that the 4-phenol motif present
90
in the compounds 1 and 3 is important to achieve adequate enzyme inhibition associated
to growth inhibition. Accordingly, compounds lacking this group such as cinnamic acid
analogues (2a-c) and methoxycinnamic acid (4), and also with additional substitution
such as caffeic acid (5) showed poor antimelanogenic and fungistatic activities,
suggesting that the coumarate framework (3-[4-phenol]propyl-2-ene 1-carbonyl motif)
present in compounds 1 and 3 is important to achieve antifungal effect associated with
tyrosinase inhibition. As can be observed in Tables 1, 2 and 3, the coumarate analogues
1
a-c and 3a-b had better inhibitory activity on tyrosinase and were also more efficient
in inhibiting fungal growth when compared to coumaric acid (1) and analogues,
suggesting that the inhibitory activity on fungal growth is correlated to the ability of
inhibiting melanization. The ethyl coumarate 1b and the ketone analogue 3b are
noteworthy due to the best results against both tyrosinase and C. neoformans growth
and melanization. The esterification of 1 as exemplified by 1b, and the bioisosteric
replacement of the carboxylic acid to the ketone (3b) improve the lipophilicity of these
molecules, which may allow better penetration into the fungal cells and increase these
effects.
Interestingly, we checked that several of the tested compounds blocked melanization in
C. neoformans in variable degrees, which is in line with the inhibitory activity over
tyrosinase. For instance, compounds 1b and 3b are inhibitors of the C. neoformans
growth (Tables 2 and 3 and Figure 2), showing comparable fungistatic effect to eugenol
(7a). Eugenol is a known antifungal agent, and due to its structural similarity to
phenylalanine, it apparently depends on targeting amino acid permeases (Darvishi et al.,
1
8

2
013; Martho et al., 2019; Martho et al., 2016). Curiously, several compounds showed
to inhibit the L-DOPA induced melanization of the C. neoformans in a concentration-
dependent manner. As can be noted in Figure 2, the best tyrosinase inhibitors also
inhibited the melanization of the fungi, suggesting that this mechanism is involved in
the antimelanogenic effect of the compounds.
Eugenol and analogues were already reported as antimelanogenic agents (Ali et al.,
2
016; Zolghadri et al., 2019), although their inhibitory activity on tyrosinase is variable.
Our data showed that 7a and 7b are poor inhibitors of the enzyme, while compound 7c
showed increased inhibitory activity in relation to other allylphenols. However, these
compounds showed to inhibit the C. neoformans melanization induced by L-DOPA
(
Figure 2). To investigate if this happened due to radical scavenging effect of the phenol
group, other phenolic analogues (i.e. 6a and 6b) were also tested. The compounds 6a,
b, 7a and 7c showed to inhibit the melanization in both wild type and mutant C.
6
neoformans (Figure 2), suggesting that these compounds may act through different
mechanism of action rather than tyrosinase inhibition. Further studies are required to
unveil this mechanism.
The most promising compounds 1a-c and 3a-b were assessed for their cytotoxicity to
mammalian cells. For this assessment, NCTC clone L929 cells were used to compare if
the compounds would cause unspecific damage to mammalian cells and evaluate if the
compounds may exert selective fungal toxicity. The results showed that no important
cytotoxicity was observed at the maximum concentration tested (200 µM) for the tested
compounds (Table 3), which is in line with other reports from literature (Lopes et al.,
2
019).
1
9

Combination of selected analogues with AmB and FCZ
Considering melanin biosynthesis as an important factor not only for intracellular
survival during macrophage engulfment but also to counteract oxidative stress caused
by antifungals as FCZ and AmB (Mesa-Arango et al., 2014), the combination of these
drugs with the antimelanogenic compounds was tested. We hypothesized that ROS
generated by antifungal drugs would be more difficult to counter act during
melanization inhibition, augmenting antifungal activity. One possible advantage of this
would be a synergistic/addictive effect between two drugs, which could lead to the
administration of lower levels of one of them or both. To test this hypothesis, the
selected compounds (1b and 3b) and AmB and FCZ were submitted to checkerboard
experiments. The results presented in Table 4 showed that none of the compounds
interfere with the MIC values of FCZ, suggesting no synergism among them or any
possibility of lower doses of FCZ. However, compound 1b had a 2-fold concentration
reduction in the presence of FCZ, however not enough to count as synergistic
interaction. The combination of AmB and 1b and 3b caused a reduction in AmB MIC
from 0.5 to 0.03 mg/L, which is a 16-fold reduction. Therefore, even though FIC was
indifferent for both combinations, the reduction in AmB was relevant. This result
suggests that melanin is involved in the neutralization mechanism of the reactive
oxygen species generated by AmB (Mesa-Arango et al., 2014) and FCZ (Peng et al.,
2
018), which consequently dependent of adequate tyrosinase activity.
The biosynthesis of melanin is a wide spread phenomenon in fungal biology. It is a
transversal feature that affects environmental and medical important fungi in several
ways: it protects the fungal cells from desiccation, osmotic and oxidative stresses and
radiation (Eisenman et al., 2020). Accordingly, melanization is very important for
fungal pathogens to evade phagocytosis. Additionally, melanin may also bind to other
2
0

molecules (covalently and non-covalently), which may explain its protective function in
fungal cells (Gomez and Nosanchuk, 2003; Hamilton and Gomez, 2002; Liu et al.,
1
999; Zaragoza, 2019).
For these reasons, the melanin biosynthetic pathway has been the target of many
attempts to develop novel antifungals. In C. neoformans melanin is considered a crucial
virulence factor; lack of melanin leads to virulence attenuation in animal model of
infection (Eisenman et al., 2020; Wang et al., 1995). The antipsychotic drug
trifluoperazine not only inhibits the growth of melanized cells, but also reduces
mortality in mice animal model (Wang and Casadevall, 1996). Melanin can also be
affected by the herbicide glyphosate, which has been shown to inhibit its biosynthesis
due to blocking the aromatic amino acid pathway. This herbicide was able to increase
survival in animal model infected with C. neoformans wild type strain (Nosanchuk et
al., 2001).
Coumaric acid analogues have already been reported as antiparasitic agents against
Trypanosoma cruzi, which also shown to accumulate reactive oxygen species leading to
oxidative stress (Lopes et al., 2019). Similar data have been reported for bacteria; Yuan
et al (2020) suggested the combinatory use of tyrosinase inhibitors and antibacterial
drugs leads to dose reduction, increase efficiency and avoidance of multi resistance
strain arise (Yuan et al., 2020). To the best of our knowledge, this is the first report in
literature regarding the positive combination between antimelanogenic agents and
classical antifungals.
Since AmB is an antifungal with high toxicity and high cost, the combination with other
compounds may allow dose reduction, a positive measure in the clinics. Besides, lower
doses of AmB may reduce treatment cost as well and may help to minimize the rise of
2
1

resistant strains. Our results suggest that antimelanogenic compounds such as 1b
present potential as adjunctive in antifungal therapy to help AmB and other classical
antifungals to exert their fungicidal effects by inhibiting the production of melanin as
protective factor.
CONCLUSIONS
In summary, coumaric acid analogues with inhibitory activity on tyrosinase were
identified as inhibitors of fungal growth with remarkable inhibitory activity on melanin
biosynthesis in C. neoformans. Interestingly, the combinatorial usage of melanin
inhibitors and AmB may increase the efficacy and lower the effective doses of this toxic
fungicidal drug largely used in the clinics. Compound 1b is noteworthy due to its potent
antimelanogenic effect and addictive effect with AmB against C. neoformans.
Additionally, we have shown that C. neoformans may represent a model organism for
screening studies in the search for novel melanin inhibitors. Further, this paper added
important concepts on future combination chemotherapy against C. neoformans
infections.
ACKNOWLEDGMENTS
This work was supported by São Paulo Research Foundation - FAPESP (grants nº
2
015/04400-9, 2016/14542-8, 2018/10279-6 and 2016/25028-3) to MAV, RCP, AGT
and JPSF. The authors are also grateful to FAPESP for the scholarship to MTV (grant nº
018/03918-2), and to Coordination of Superior Level Staff Improvement – CAPES
financial code 001) for the scholarship to MF. JPSF and AGT are grateful to the
2
(
2
2

National Council for Scientific and Technological Development – CNPq for the
fellowship awards (grant 306355/2018-3).
Author’s contributions
Lidiane Oliveira: Data curation, Formal analysis, Methodology, Writing - original draft
Márcio Ferrarini: Data curation, Formal analysis, Methodology, Writing - original draft
Alexandre Paes dos Santos: Data curation, Formal analysis, Methodology
Marina Themoteo Varela: Data curation, Formal analysis, Methodology, Writing -
original draft
Isabela Teresa Santos Corrêa: Data curation, Formal analysis, Methodology, Writing -
original draft
André Gustavo Tempone: Resources, Funding acquisition, Investigation, Writing -
original draft, Supervision, Writing - original draft
Marcia S. C. Melhem: Resources, Funding acquisition, Investigation, Writing - original
draft, Supervision, Writing - original draft
Marcelo A. Vallim: Resources, Funding acquisition, Investigation, Writing - original
draft, Supervision, Writing - original draft
João Paulo S. Fernandes: Resources, Funding acquisition, Investigation, Writing -
original draft, Supervision, , Writing - review & editing
Renata C. Pascon: Resources, Funding acquisition, Investigation, Writing - original
draft, Supervision, Writing - review & editing
DECLARATIONS OF INTEREST
The authors declare no competing interest.
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