Substrate and inhibitor selectivity, and biological activity of an epoxide hydrolase from Trichoderma reesei

Article abstract of DOI:10.1007/s11033-018-4481-4

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

Full text of DOI:10.1007/s11033-018-4481-4

Molecular Biology Reports
https://doi.org/10.1007/s11033-018-4481-4
ORIGINAL ARTICLE
Substrate and inhibitor selectivity, and biological activity
of an epoxide hydrolase from Trichoderma reesei
Gabriel S. de Oliveira1 · Patricia P. Adriani1 · Hao Wu2 · Christophe Morisseau2 · Bruce D. Hammock2 ·
Felipe S. Chambergo1
Received: 19 August 2018 / Accepted: 7 November 2018
© Springer Nature B.V. 2018
Abstract
Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the correspond-
ing vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in
pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase
recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based
inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural sub-
strates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH
were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous
role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible
development of novel drugs for human diseases.
Keywords Trichoderma reesei · Epoxide hydrolase · EH inhibitors · Epoxy fatty acids · Growth inhibitory activity
Introduction
Epoxide hydrolases (EHs) are found in all organisms, includ-
ing mammals, invertebrates, plants, fungi and bacteria.
Electronic supplementary material The online version of this
They catalyze the hydrolysis of epoxides to the correspond-
ing vicinal diols by the addition of a molecule of water [1].
Biologically, EHs have three main functions: detoxification,
catabolism and regulation of signaling molecules [1, 2]. In
vertebrates, EH play an important role in disease develop-
ment because epoxy-fatty acids (EpFAs), which are EH sub-
strate, have vasodilator and anti-inflammatory function [1].
In humans, EHs are associated with the occurrence of can-
cer and other diseases, such as increased risk of developing
hepatocellular carcinoma [3]; ovarian cancer [4]; colorectal
adenoma, mainly among smokers [5, 6] and coronary artery
disease in caucasians [7]. Soluble epoxide hydrolase inhibitors
represent a therapeutic strategy for the treatment of cardio-
vascular diseases, reducing significantly the blood pressure
in rats [8], inhibit vascular smooth muscle cell proliferation
[9], present potent anti-inflammatory effects [10], show to be
effective against neuropathic diabetic pain in rodent models
[11] and against equine laminitis, which is a complex and often
fatal disease involving inflammation, hypertension, and severe
neuropathic pain [12]. Therefore, it is evident the importance
article (https://doi.org/10.1007/s11033-018-4481-4) contains
supplementary material, which is available to authorized users.
* Felipe S. Chambergo
fscha@usp.br
Gabriel S. de Oliveira
gabriel.stephani.oliveira@usp.br
Patricia P. Adriani
patricia.adriani@usp.br
Hao Wu
harwu@ucdavis.edu
Christophe Morisseau
chmorisseau@ucdavis.edu
Bruce D. Hammock
bdhammock@ucdavis.edu
1
Escola de Artes, Ciências e Humanidades, Universidade de
São Paulo, 1000 Av. Arlindo Bettio, Ermelino Matarazzo,
CEP: 03828-000 São Paulo, Brazil
2
Department of Entomology and Nematology, and UC Davis
Comprehensive Cancer Center, University of California, One
Shields Avenue, Davis, CA, USA
Vol.:(0123456789)
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Molecular Biology Reports
of the study of the EH metabolism of fatty acids and EH inhib-
itors for drug design.
Materials and methods
EH inhibitors may also be important in controlling patho-
gens. Recently, an EH from Mycobacterium tuberculosis (the
bacteria causing tuberculosis) was shown to be essential in
the biosynthesis of mycolic acid, an extra-long chain fatty
acid necessary for the pathogenicity of the bacteria [13]. The
study developed by Biswal et al. [14] reports that an EH from
M. tuberculosis are involved in a detoxification pathway and
could be among the potential drug targets for the development
of antituberculars, however more studies about in vivo activity
of these EH inhibitors are necessary. The study from Spillman
[15] shows that two EHs from Plasmodium falciparum (para-
sitic protozoan that causes malaria in humans) play an impor-
tant role in the infection process of humans, suggesting that
inhibitors for these enzymes may help fight against malaria.
However, studies about the role of EH inhibitors in the process
of malaria infection in humans have not yet been made. The
EH from Pseudomonas aeruginosa, a lung pathogenic bac-
teria, helps the microorganism established itself in the lungs
cavity [16], therefore further studies about the inhibition of
this EH are necessary.
Reagents
All reagents and solvents were purchased from Sigma
Aldrich Chemical (USA) or Fisher Scientific (USA).
The radioactive substrates were synthesized as described
by Borhan et al. [36], except the juvenile hormone (JH-III),
which was purchased from PerkinElmer (USA). The fluo-
rescent substrates were synthesized as described by Jones
et al. [37] and Morisseau et al. [38]. The Inhibitors were
synthesized as described by Shen and Hammock [39]. All
their structures are given in tables, and boldface numbers
throughout the text refer to these compounds.
The mix of EpFAs was prepared as described by Moris-
seau et al. [40].
Cloning, expression and purification of TrEH
Recombinant TrEH was prepared as described by de Oliveira
[33]. The concentration of the purified protein was estimated
using the method described by Whitakerand e Einargranum
[41].
The filamentous fungus genus Trichoderma belongs to the
Ascomycota division, and has more than 100 species iden-
tified. They are usually found in soils worldwide, occurring
on root surfaces of plants and other organic materials [17].
Trichoderma species are economically used for the commer-
cial production of enzymes (cellulases, glucanases, pectinases,
xylanases and others), as a biological control agent of phy-
topathogenic fungi, and in the food industry [18]. However,
Trichoderma genus also have negative effects, like infection
of Trichoderma species causing the destruction of cultivated
mushrooms [19, 20], and they can be opportunistic pathogens
of immunocompromised mammals, including humans, and
may even cause death of the host. There are reports of fatal
and treatable infections by the species Trichoderma longibra-
chiatum [21–24], Trichoderma viride [25, 26], Trichoderma
harzianum [27, 28], Trichoderma pseudokoningii [29, 30],
Trichoderma citrinoviride [21] and Trichoderma koningii [31,
32]. Thus, the importance to identify and characterize new
target for antifungal agents in this genus of fungus. Toward this
end, our group identified and determined the 3D structure of
a soluble EH from Trichoderma reesei QM9414 (TrEH) [33,
34], that is a model organism of the Trichoderma genus and
a widely used industrial host organism for protein production
[35]. In this study, we determine the substrate and inhibitor
selectivity of TrEH, and test the role of this enzyme in the
fungus growth.
Assay based on radioactive substrates
The specific activity of TrEH in reactions with radioactive
substrates were determined using tritium-labeled substrates:
trans-diphenylpropene oxide (1, t-DPPO), cis-diphenylpro-
pene oxide (2, c-DPPO), trans-stilbene oxide (3, t-SO), cis-
stilbene oxide (4, c-SO), and juvenile hormone (5, JH-III).
The assays were performed as described by Morisseau et al.
[42] in glass test tubes containing 99 µL of diluted TrEH
enzyme ([Enzyme]final = 1.3 µg/mL) in sodium phosphate
buffer with BSA (Na2HPO4/NaH2PO4 100 mM, 0.1 mg/
mL bovine serum albumin (BSA), pH 7,4), and 1 µL stock
solution of tritium-labeled substrates 1–5 (5 mM, diluted in
Dimethylformamide (DMF)) ([Substrate]final = 50 µM) was
added. After 10 min of incubation at 37 °C, the reaction was
stopped with the addition of 250 µL of isooctane, which
extracts the remaining epoxide from the aqueous phase. The
samples were vortexed and then centrifuged at 3000 rpm
for 5 min so that 30 µL of the aqueous phase were collected
for analysis. The activity was measured by the amount of
radioactive diol released in the aqueous phase using a liquid
scintillation counter (Wallac model 1409, USA).
Assay based on fluorescent substrates
The screening of fluorescent substrates was performed
with (3-phenyloxiranyl)-acetic acid cyano-(6-methoxy-
naphthalen-2-yl)-methyl ester (6, PHOME); cyano(6-meth-
oxy-naphthalen-2-yl)methyl oxiran-2-ylmethyl carbonate
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Molecular Biology Reports
(7, CMNGC), cyano(6-methoxy-naphthalen-2-yl)methyl
trans-[(3-phenyloxiran-2-yl)methyl] carbonate (8, MNPC),
cyano(6-methoxy-naphthalen-2-yl)methyl 2-(3-ethyloxiran-
2-yl)acetate (9, MNEEpp), cyano(6-methoxy-naphthalen-
2-yl)methyl (2-(oxiran-2-yl)ethyl) carbonate (10, MNCEpB),
cyano-(6-methoxy-naphthalen-2-yl)-methyl-trans-((3-
ethyl-oxiran-2-yl)methyl) carbonate (11, MNPEC), cyano-
(6-methoxy-naphthalen-2-yl)-methyl 3,3-dimethyl-oxiranyl-
methyl carbonate (12, MniPC), as substrates.
column (2.1 × 150 mm, 1.8 µM particle size). Water with
0.1% glacial acetic acid was used as mobile phase (A)
Acetonitrile:methanol (84:16) with 0.1% glacial acetic acid
was used as mobile phase (B) The detection was carried
out by monitoring the selected-reaction transitions using a
4000 QTrap tandem mass spectrometer (Applied Biosystems
Instrument Corporation, CA) equipped with an electrospray
source (Turbo V). The quantification was performed using
external calibration followed by normalization of diols and
epoxides to recoveries of corresponding internal standards
CUDA. Results are expressed as means standard devia-
tions from three separate assays.
The determination of the specific activity of TrEH with
fluorescent substrates was performed according to Moris-
seau et al. [38]. The assays were performed in black 96-well
plates, to which 170 µL of TrEH enzyme solution (1.17 µg/
mL in sodium phosphate buffer with BSA) was added. Sub-
sequently, 30 µL of work solution of substrate [prepared
with the 270 µL mixture of substrate stock solution (5 mM
diluted in DMSO) and 3.730 µL of sodium phosphate buffer
with BSA was added to each well ([Enzyme]final = 1 µg/mL;
[Substrate]final = 50 µM)]. Fluorescence, emitted by the
TrEH reaction with the substrates 6–12, was measured using
Gemini EM fluorescent plate reader (Molecular Devices,
USA), with the excitation wavelength of 330 nm and emis-
sion wavelength of 465 nm for 10 min at 30 °C. Assays were
run under conditions where product formation was linearly
dependent both on the concentration of enzyme and on the
time for the course of the assay.
Screening of EH inhibitors by fluorescent assay
High‑throughput screening assay
The screening to find inhibitors for TrEH was performed
as described by Morisseau et al. [38], with some modifica-
tions: The 96-well fluorescence plates were prepared prior
to the assay, with the addition of 20 µL DMSO solution (1%
DMSO/sodium phosphate buffer with BSA) on the column
1 of the plate. In each well of the remainder of the plate (col-
umns 2–12), a solution was added with one of the inhibitors
of the library (10 µM inhibitor/1% DMSO/sodium phosphate
buffer with BSA). 30 plates were prepared, following the
above description to test all inhibitors. The plates, already
prepared, were stored in a refrigerator.
LC‑MS/MS analysis of TrEH activity on epoxy fatty
acids
In these pre-prepared plates, 150 µL of sodium phos-
phate buffer with BSA in the A1–D1 wells (these four wells
were used as background control) and 150 µL enzyme solu-
tion (150 ng/mL) diluted in sodium phosphate buffer with
BSA were added to the rest of the wells of the plate (wells
E1–H1 were used as full activity control). Subsequently,
30 µL of the solution of compound 6 (PHOME, 150 µM)
in sodium phosphate buffer with BSA / DMSO (97: 3)
([Substrate]final = 22.5 µM; [Enzyme]final = 112.5 ng/mL;
[inhibitor]final = 1 µM). The plate was incubated at room
temperature for 20 min in the dark. The amount of 6-meth-
oxy-2-naphthaldehyde formed was measured by fluorescence
detection using SpectraMax GEMINI EM fluorescent plate
reader (Molecular Devices, USA) with excitation wave-
length of 330 nm and wavelength of emission of 465 nm.
With these last assays, we choose 90 compounds with
the highest inhibitiory activity (> 90%) and prepared plates
with 20 µL of five concentrations of these inhibitors (10 µM,
1 µM, 100 nM, 10 nM and 1 nM). The assays were per-
formed as described above and we find the six best inhibitors
(compounds 13–18).
The determination of TrEH activity on EpFAs was per-
formed by LC–MS/MS analysis. The enzymatic reactions
were performed as described by Morisseau et al. [40]. In
glass tubes, 99 µL of TrEH enzyme solution at 2 µg/mL
(diluted in sodium phosphate buffer with BSA) and 1 µL
of the mix of epoxy fatty acids (14 regioisomers of arachi-
donic acid, (ARA), linolenic acid (LA), eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA), diluted in etha-
nol, all with a final concentration of 1 µM). The sample was
incubated at 30 °C in a water bath with stirring, and the
reaction was quenched by the addition of 400 µL of metha-
nol. Concentration of enzymes and incubation time were
optimized to yield < 5% conversion of the substrates in the
mixture. Then, 1 µL of 12-(3-cyclohexylureido) dodecanoic
acid (CUDA, 100 µM in methanol) was added to the solution
as an internal standard (200 nM of CUDA in a total volume
of 500 µL solution). The tubes were vortexed for 5 s and
centrifuged at 3000 rpm for 5 min. The supernatant was
collected for LC–MS/MS analysis.
The reactions were analyzed using Agilent 1200 SL
liquid chromatography series (Agilent Corporation,
USA) with an Agilent Eclipse Plus C-18 reversed-phase
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IC50 determination
dextrose agar (PDA) containing the TrEH inhibitors 13, 15,
17 and 18 diluted in DMSO, however we did not test the
compounds 14 e 16 because they show low solubility in the
fungual medium. 10 µL of the spore solution (0.9% NaCl)
with 1×106 spores/mL concentration was deposited in the
center of the plate in three culture conditions: PDA (control);
PDA + 2% DMSO (control); PDA + 2% DMSO + 1 mM
TrEH inhibitor (assay).
The IC50 was determined as described by Morisseau et al.
[38] using the six best inhibitors (compounds 13–18). In
96-well plates for fluorescence assays, 170 µL of puri-
fied TrEH (132.3 ng/mL) sodium phosphate buffer with
BSA, 2 µL of the inhibitors (100 nM ≤ [Inhibitor]≤5 mM
diluted in DMSO). The mixture was incubated for 5 min
at 30 °C. Afterwards, 30 µL of substrate 6 (150 µM)
in sodium phosphate buffer with BSA/DMSO (97:3)
([Substrate]final = 22.5 µM; final [Enzyme]final = 112.5 ng/
mL; 1 nM ≤ [Inhibitor]final ≤ 50 µM). The activity was moni-
tored for 10 min at 30 °C by measuring the formation of
6-methoxy-2-naphthaldehyde as described above. IC50 val-
ues were determined by regression of at least six reference
points in the linear region of the curve, with a minimum of
two data points on both sides of the IC50 values. Results are
expressed as means standard deviations from three sepa-
rate assays.
Radial growth was monitored for 5 days at 30 °C and
hyphae development was measured in millimeters for deter-
mination of fungal growth in the presence of each TrEH
inhibitor. The assays were performed in triplicate and the
results are expressed as means standard deviations.
Results and discussion
Assay based on radioactive substrates
The data in Table 1 shows that the purified TrEH enzyme
was active on all radioactive substrates tested, with the high-
est specific activity on substrate 1. When compared to the
purified sEHs from mammalian EHs (Table 1), it is possible
to observe that TrEH catalysis is better than human sEH
[43], for compounds 1, 2 and 3. While among the enzyme
TrEH inhibitors effects in the growth of T. reesei
After the identification of the best TrEH inhibitors, T. reesei
QM9414 (ATCC26921) growth analysis was performed on
petri dishes (60 mm diameter) with fungal medium potato
Table 1 Selectivity of TrEH for radioactive surrogate substrate, comparison to mammalians sEH
Substrate structure
No
Specific activity (nmol/min/mg)a
TrEH
Mouse sEHb
Human sEHb
4,500 200
Rat sEHb
1
14,400 2,100
17,000 300
10,200 700
2
3
950 70
190 20
51 17
814.5 31.5
54 4.5
214.2 10.2
102 10.2
476 17
4
5
5.8 1.6
130 35
68 17
9.4 2.7
8.1 1
1.071 153
283.5 40.5
357 40.8
aResults are means standard deviations of three separate measurements
bData from [42]
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Molecular Biology Reports
tested, TrEH turns over substrate 2 the fastest, the mouse
sEH hydrolyzes substrate 3 two fold greater than TrEH,
finally, for substrates 4 and 5, TrEH has the lowest specific
activity compared to the other organisms studied.
substrates, which could be advantageous for bioorganic syn-
thesis application.
TrEH has a high specific activity for substrate 6, which
was designed for the screening of compounds library [44],
and it was selected for the identification of TrEH inhibitors.
Assay based on fluorescent substrates
LC–MS/MS analysis of TrEH activity on epoxy fatty
acids
TrEH activity was measured for several fluorescent sub-
strates previously developed for mammalian EH [37]. The
results (Table 2) show that TrEH has greater specific activity
with substrates 6, 8, 11 and 12 than the human and mouse
sEH [37]; however, the specific activity of TrEH was deter-
mined with a higher concentration of substrate (Table 2).
compounds 7 and 9 are also good substrate for TrEH while
10 yields a much lower activity. The activity of TrEH seems
to be enhanced by the presence of an aromatic group near
the epoxide ring, and concurred with the results obtained
with the radioactive substrates. Put together the results on
all the surrogate substrates (Tables 1, 2) indicate that TrEH
active site can accommodate a wild variety of exogenous
Finally, we test the ability of TrEH to hydrolyze natural
substrates, the epoxy fatty acids. To quickly determine the
selectivity of TrEH for this family of epoxides, we deter-
mine the amount of dihydroxy-fatty acids (DiHFAs, which
are the product of the EH reaction) formed from the mix of
EpFAs. In the Fig. 1, the data of human sEH enzyme activity
[40] were also plotted. The results indicate that TrEH is first
active on this kind of substrate, with a strong preference for
epoxide derived from longer fatty acid chain, especially n-3
such as DHA. The fact that TrEH hydrolyzes natural epox-
ides suggest a biological role for this enzyme in fatty acids
metabolism in the fungus.
Table 2 Selectivity of TrEH for radioactive surrogate substrate, comparison to mammalians sEH
Substrate structure
No
TrEH
Human sEHb
Mouse sEHb
[Substrate] Specific activitya
[Substrate] Specific activitya
[Substrate] Specific activitya
(µM)
(µM)
(µM)
6
7
50
50
17,316.2 453.7 10
714 23
10
10
214 63
ND
11,051.7 141.4 10
ND
8
9
50
50
9501.7 733.0 10
6818.4 200.3 10
2689 44
ND
10
10
781 129
ND
10
11
12
50
50
50
1937.7 583.8 10
882.3 380.8 10
ND
10
10
10
ND
408 14
11.7 0.2
125 32
8.4 0.1
427.1 61.6
10
aResults are means standard deviations of three separate measurements and are shown in nmol/min/mg
bData from [36]
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Table 3 Screening of inhibitors for TrEH
Inhibitor structure
No
13
IC50 (nM)a
33.6 10.7
14
15
99.0 8.5
104.9 17.6
16
17
138.8 14.4
259.3 71.3
Fig. 1 Selectivity of TrEH and human sEH on a mixture of EpFAs.
Data of the formation of diol (product of the EH enzyme reaction)
carried out by TrEH and human sEH [40] with the mix of EpFAs (14
regioisomers of arachidonic acid (ARA), linolenic acid (LA), eicosa-
pentaenoic acid (EPA), docosahexaenoic acid (DHA) at 1 µM each).
Results are expressed as means standard deviations from three sepa-
rate assays
18
357.9 57.6
aResults are averages of triplicate experiments
Epoxy-fatty acids are ubiquitous chemicals found in ver-
tebrates, invertebrates, plants and microorganisms [1]. The
role of epoxy-fatty acids in humans is well studied because
of their role in health [45]. The pharmacological inhibition
of EH is investigated to reduce inflammation and pain. In
plants, recent studies have shown the role of epoxy-fatty
acids in cuticule biosynthesis and structure as well as in
host-defenses [46]. The fact that TrEH hydrolyzes natural
epoxy-fatty acid suggest a biological role for this enzyme
in fatty acids metabolism in the fungus, but also how the
fungus interacts with the targeted host plant.
function, which are probably mimicking epoxy fatty acids.
The best compounds were used to investigate the biology of
TrEH in the fungus.
TrEH inhibitors effects in the growth of T. reesei
T. reesei QM9414 was grown in medium containing TrEH
inhibitors. The culture of the plate containing PDA (con-
trol condition) reached 100% of growth (it means the total
diameter of the plate) after 3 days of incubation (Fig. 2a).
On the other hand, the fungus cultured on plates contain-
ing inhibitors 13, 15, 17 and 18 showed an inhibition of
growth during the whole incubation time, when compared
the control conditions (Fig. 2). The Plates with inhibitors 13
demonstrated the lowest percent of fungal growth (38.2%,
Fig. 2c, d). Globally, the growth inhibition potency of the
compounds tested relates to their potency against TrEH, sug-
gesting that the biological effect observed is related to TrEH
inhibition.
Screening of EH inhibitors by fluorescent assay
Using substrate 6, a library of around 3000 compounds,
developed previously as mammalian sEH inhibitors was
screened. The first screening with all the inhibitors allowed
us to find 90 compounds with the highest inhibitiory activity,
so they were tested at five concentrations (supplementary
material), with the aim of selecting the six molecules with
the highest inhibitory activity on TrEH. The IC50 of these
six inhibitors was determined (Table 3). All selected mol-
ecules have urea or amide in their structure, and It is widely
recognized that these groups mimic the epoxide group and
bind to the catalytic site of the EHs, preventing the catalysis
[39]. In these six best inhibitors: five have urea (compounds
13, 14, 15, 17, 18) and one has amide (compound 16) in
their structures. Interestingly, several compounds (13, 15 and
16) have long aliphatic chain with a terminal acid or amide
For the M. tuberculosis bacteria, treatment with an EH
inhibitors also reduced the growth of the microorganism
[14], suggesting potential pharmaceutical usage. Out of
the six best TrEH inhibitors found, only compound 18 was
reported before, and used for the treatment of ischemic heart
disease [47], and in experiments with a cellular model of
adipocytes, compound 18 maintained the cellular cholesterol
homeostasis, which brings benefits, because adipocyte dys-
function and its cholesterol imbalance are associated with
obesity [48].
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Molecular Biology Reports
Fig. 2 TrEH inhibitors effect in
the growth of T. reesei QM9414
was grown in medium contain-
ing TrEH inhibitors after 36 h of
cultivation. a Photograph of the
plate containing PDA with T.
reesei culture; b Photograph of
the plate containing PDA+2%
DMSO with T. reesei culture; c
Photograph of plate containing
PDA+2% DMSO+1 mM of
the compound 13 with T. reesei
culture; d Graph of the fungal
growth rate after 36 h of culti-
vation in the control conditions
(PDA and PDA+2% DMSO)
and conditions with the inhibi-
tors (PDA+2% DMSO+1 mM
of the compounds 13, 15, 17
or 18). Results are expressed
as means standard deviations
from three separate assays
The T. reesei is a model organism of the genus Tricho-
derma, which presents some pathogenic species with high
resistance to antifungal compounds [22] and are related to
systemic diseases that represent important causes of mor-
bidity and mortality among immunologically compromised
patients. In this sense, it is important to study TrEH inhibitory
molecules, since they cause the inhibition of T. reesei growth,
and may lead to the development of new antifungal agents.
It is important to note that, although the tested molecules are
good inhibitors of TrEH activity in vitro, it is possible that
their in vivo anti-growth activity is through a mechanism par-
tially or totally independent of TrEH. It should be noted that
there are several EH genes in the T. reesei genome. Therefore,
inhibition of growth can be caused by the set of inhibitory
actions on various enzymes. Inactivation studies of the TrEH
genes can be carried out in the future for the determination of
their relation to the inhibition of fungal growth. However, the
identification and characterization of novel chemicals that alter
the growth pattern of the fungus has the potential to lead to the
development of new antifungals.
Conclusions
The data presented here show that TrEH can hydrolyze
a wide variety of substrate, which can make it useful for
bioorganic preparation of epoxides and/or diols. Also,
this enzyme is able to hydrolase endogenous epoxy fatty
acids, which could be related to some biological activity
in the fungus. Toward testing this hypothesis, we identi-
fied potent inhibitors of this enzyme. Interestingly, these
compounds alters the growth pattern of the fungus, again
suggesting a role of TrEH in the microorganism biology.
The results and tools discovered here will be essential to
decorticate the biological role of fungal EH, and perhaps
for the discovery of new antifungal target and molecules.
Acknowledgements This work was supported by National Institute of
Environmental Health Sciences (Grant No. NIEHS-R01 ES002710),
the Superfund Program NIEHS (Grant No. P42 ES 04699), and São
Paulo Research Foundation (Grant Nos. FAPESP-2014/24107-1,
2017/25705-8). G.S.O. and P.P.A. is supported by a scholarship from
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Molecular Biology Reports
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants performed by any of the authors.
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hydrolases modulate erythrocyte vasoactive lipids during Plas-
modium falciparum infection. MBio. https://doi.org/10.1128/
mBio.01538-16
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