Screening and Phenotypical Characterization of Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) Inhibitors as Multistage Antischistosomal Agents

Article abstract of DOI:10.1021/acsinfecdis.9b00224

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus Schistosoma. The disease is endemic in tropical and subtropical areas of the world where water is infested by the intermediate parasite host, t

Full text of DOI:10.1021/acsinfecdis.9b00224

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Article
Screening and phenotypical characterization of
Schistosoma mansoni histone deacetylase 8 (SmHDAC8)
inhibitors as multi-stage antischistosomal agents
Fulvio Saccoccia, Margherita Brindisi, Roberto Gimmelli, Nicola Relitti, Alessandra Guidi, A. Prasanth
Saraswati, Caterina Cavella, Simone Brogi, Giulia Chemi, stefania butini, Giuliana Papoff, Johanna
Senger, Daniel Herp, Manfred Jung, Giuseppe Campiani, Sandra Gemma, and Giovina Ruberti
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00224 • Publication Date (Web): 29 Oct 2019
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Screening and phenotypical characterization of Schistosoma mansoni
histone deacetylase 8 (SmHDAC8) inhibitors as multi-stage anti-
schistosomal agents
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Fulvio Saccoccia,a,^ Margherita Brindisi,b,^ Roberto Gimmelli,a Nicola Relitti,c Alessandra
Guidi,a A Prasanth Saraswati,c Caterina Cavella,c Simone Brogi,c,d Giulia Chemi,c Stefania
Butini,c Giuliana Papoff,a Johanna Senger,e Daniel Herp,e Manfred Jung,e Giuseppe
Campiani,c,* Sandra Gemma,c,* Giovina Ruberti a,*
aInstitute of Biochemistry and Cell Biology (IBBC), National Research Council (CNR),
Campus A. Buzzati-Traverso, via E. Ramarini 32, 00015 Monterotondo (Rome), Italy
bDepartment of Excellence of Pharmacy, University of Napoli Federico II, via D. Montesano
49, 80131 Naples, Italy
cDepartment of Excellence of Biotechnology, Chemistry and Pharmacy, University of Siena,
via Aldo Moro 2, 53100, Siena, Italy
dDepartment of Pharmacy, University of Pisa, via Bonanno 6, 56126, Pisa, Italy
eInstitute
of
Pharmaceutical
Sciences, University
of
Freiburg, Albertstraße
25, 79104 Freiburg, Germany
*Corresponding authors:
campiani@unisi.it; gemma@unisi.it; giovina.ruberti@cnr.it
Keywords: Schistosoma mansoni, histone deacetylase inhibitors, SmHDAC8, hHDAC8,
screening, biological characterization
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Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by
platyhelminths of the genus Schistosoma. The disease is endemic in tropical and subtropical
areas of the world where water is infested by the intermediate parasite host, the snail. More
than 800 million people live in endemic areas and more than 200 million are infected and
require treatment. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment
and transmission control being safe and very effective against adult worms of all the clinically
relevant Schistosoma species. Unfortunately, it is ineffective on immature, juvenile worms,
therefore it does not prevent re-infection. Moreover, the risk of development and spread of
drug resistance due to the wide use of a single drug in such a large population represents a
serious threat. Therefore, research aimed to identifying novel drugs to be used alone or in
combination with PZQ are needed. Schistosoma mansoni histone deacetylase 8
(SmHDAC8) is a class I zinc-dependent HDAC which is abundantly expressed in all stages
of its life cycle, thus representing an interesting target for drug discovery. Through virtual
screening and phenotypical characterization of selected hits, we discovered two main
chemical classes of compounds characterized by the presence of a hydroxamate-based
metal binding group coupled to a spiroindoline or a tricyclic thieno[3,2-b]indole core as
capping groups. Some of the compounds of both classes were deeply investigated and
showed to impair viability of larval, juvenile, and adult schistosomes, to impact egg
production in vitro and/or to induce morphological alterations of the adult schistosome
reproductive systems. Noteworthy, all of them inhibit the recombinant form of SmHDAC8
enzyme in vitro. Overall, we identified very interesting scaffolds paving the way to the
development of effective anti-schistosomal agents.
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Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting more than
200 million people worldwide, mainly in tropical and sub-tropical areas, is caused by the
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Platyhelminthes of the genus Schistosoma.1 The pathology is due to eggs being trapped
into host tissues once released by female worms into the circulation. To date, praziquantel
(PZQ, 1, Figure 1) is the only drug approved for the treatment of schistosomiasis, being very
effective against adult worms of the most clinically relevant Schistosoma species (S.
mansoni, S. haematobium and S. japonicum).2 However, PZQ is poorly effective against the
juvenile parasites and reinfection is common.3-5 The increasing threat of drug-resistance
coupled to the need for drugs with wider efficacy towards different parasite life cycle stages
prompted the search for new druggable targets and compounds for the treatment of
schistosomiasis.
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Screening of compound libraries in vitro and in vivo, drug repurposing and target-based drug
discovery have been used in order to identify novel compounds with anti-schistosomal
activity. Promising compounds with different scaffolds have been discovered and could
contribute to the development of new drugs (i.e. oxamniquine derivatives, biarylalkyl
carboxylic acid derivatives (BACADs), arylmethylamino steroids (AASs), N,N ́-diarylureas
and analogues, thioredoxin glutathione reductase (TGR) Inhibitors, kinase inhibitors,
cysteine protease inhibitors, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase
inhibitors and schistosomal histone deacetylase (HDAC) inhibitors).6,7
HDACs have emerged as promising drug targets in many research areas, including parasitic
diseases.8,9 HDACs are Zn2+- or NAD+-dependent lysine deacetylases that modulate cell
chromatin structure, transcription, and gene expression as well as several cytoplasmic
signaling pathways.10-13 S. mansoni HDAC8 (SmHDAC8) is a class I zinc-dependent HDAC,
which was demonstrated to be abundantly expressed in all S. mansoni life cycle stages;
therefore it has been recently proposed as a promising target for drug discovery.11,14
Noteworthy, the human homologue (hHDAC8) is poorly expressed in healthy cells, with a
potential as a therapeutic target in cancer cells.15 As a consequence, several efforts were
spent in order to identify promising antiparasitic hits that may target SmHDAC8. Recently
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reported SmHDAC8 inhibitors are depicted in Figure 1 (2, 3).16-21 In the present paper,
starting from structure-based virtual screening, we identified the effective compounds 4-12
(Figure 1). These were investigated in their ability to impair viability of S. mansoni larval
stage (schistosomula), juvenile schistosomes, and adult pairs in addition to their impact on
egg production and/or to induce alterations in the reproductive organs’ morphology. The
selected compounds demonstrated to inhibit recombinant form of SmHDAC8 in the low
micromolar range.
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Results and Discussion
Virtual screening
We performed a structure-based virtual screening campaign based on a High Throughput
Docking (HTD) approach as previously described22-24 with the aim of identifying novel
SmHDAC8 inhibitors. The crystal structure of SmHDAC8 (PDB ID: 4BZ7)16 was employed
to screen our in-house chemical library. This library is composed by ≈ 3000 compounds
containing a variety of heterocyclic and peptidomimetic templates rationally designed for a
broad range of biological targets, and is enriched with inhibitors of metallo-enzymes.23,25-27
Our proprietary database was screened using Glide software28 applying specific criteria
based on the evaluation of Glide extra precision (XP) score followed by a rescoring using
Glide standard precision (SP) scoring function. All the compounds showing a computational
score ≤ -9.00 kcal/mol, computed on the scoring and rescoring procedure, were kept as
potential SmHDAC8 inhibitors. The protocol provided twenty-nine compounds that were
selected to undergo biological screening. Remarkably, a number of compounds bearing
metal-binding groups (MBG) and arising from medicinal chemistry projects focused on the
development of anticancer agents, were retrieved.
Chemistry
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Compounds 4 and 5 (Figure 1) were synthesized as previously described.25 Compounds 6
and 7 were synthesized as described in Figure 2, panel A. The previous protocol was
adapted to the synthesis of 2-arylderivatives 6 and 7. Ketone intermediates 15a,b were
synthesized starting from the corresponding tosylhydrazone derivative 14 synthesized from
13 as reported.29 Ketones 15a,b underwent Fisher reaction with phenylhydrazine in the
presence of concentrated sulfuric acid. The double bond of the resulting indolenines was
reduced with sodium borohydride in methanol, and the secondary amines 16a,b thus
obtained were subjected to a reductive amination protocol with methyl 4-formylbenzoate.
The resulting ester intermediates were converted to racemic hydroxamic acids 6 and 7 upon
treatment with hydroxylamine hydrochloride.
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Compounds 8-10 were prepared starting from cinnamic acid 17 as described in Figure 2,
panel B. The 2-aryl-3,4-dihydro-2H-thieno[3,2-b]indole core was prepared according to a
reported procedure.30 Addition of triethylamine to a mixture of mercaptoacetic acid and
cinnamic acid 17 in 1,4-dioxane yielded the 3-[(carboxymethyl)thio]-3-phenylpropanoic acid,
which upon heating to 120 °C in the presence of acetic anhydride and sodium acetate,
afforded the cyclic keto-derivative 18. Intermediate 18 and the appropriate 4-
halophenylhydrazine were suspended in ethanol and subjected to microwave irradiation to
obtain tricyclic thienoindole derivatives 19a,b. Intermediates 19a,b were subjected to an
alkylation reaction with methyl 4-(bromomethyl)benzoate in presence of potassium tert-
butoxide giving the corresponding esters 20a,b. Starting from 19b, ester 21 was obtained
through an alkylation reaction using methyl 5-(bromomethyl)thiophene-2-carboxylate
(prepared as described in Scheme S1 of the Supporting Information). Esters 20a,b and 21
were finally subjected to a reaction with hydroxylamine and potassium hydroxide to afford
the final hydroxamic acids 8-10.
The synthesis of 11 (Figure 2, panel C) was accomplished reacting p-toluenesulfonyl
hydrazide 13 with acetone to obtain the tosylhydrazone intermediate 22. This compound
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was treated with 3-pyridinecarboxaldehyde in the presence of cesium carbonate to afford
the ketone 23, which was then used in the Fischer reaction with 4-methoxyphenylhydrazine
to provide the indolenine intermediate. The double bond of this latter compound was
reduced by catalytic hydrogenation and the corresponding amine 24 was subjected to a
reductive amination procedure with methyl 4-formylbenzoate. The ester derivative obtained,
was treated with hydroxylamine solution and potassium hydroxide to afford the final
compound 11.
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Schistosomula assays for initial compounds screening
One of the major hurdles in structure-based drug design is the identification of enzyme
inhibitors that, despite a fair potency against the target enzyme, are also characterized by
high activity in whole cell or whole organisms, having suitable drug-like properties, such as
cell penetration26. To address this issue, compounds from our virtual screening campaign,
were screened using the ATP-based viability assay developed in our laboratory on S.
mansoni larval stage (schistosomula).31 A 60% viability reduction upon treatment at the
concentration of 50 μM was set up as cut-off value together with DMSO (100% viability) and
gambogic acid (0% viability) as negative and positive controls, respectively. Seven out of
twenty-nine compounds, namely 4-10 showed to be effective in vitro, decreasing the
schistosomula viability by 80-90% 72 h after treatment. Two more compounds, 11 and 12
showed to impact larvae viability to a lower extent, with a 35-40% reduction. Next, in order
to calculate the LC50, dose-response curves were designed by treating schistosomula with
each of the hits at concentrations ranging from 0.7 up to 100 µM. The LC50 of compounds
4-12 are reported in Figure 3, panel A, along with dose-response curves (Figure 3, panel B
for the most structurally diverse and representative compounds 4, 6, 8, 11 and Figure S1 for
compounds 5, 7, 9, 10, and 12).
This initial screening of compounds deriving from the virtually selected pool, was aimed at
identifying promising hit compounds able to kill the larvae with a good potency. It is well
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known that different stages of the schistosome life cycle have different susceptibility to drug
actions;32 therefore the activity of the most promising compounds was investigated against
adult and juvenile S. mansoni.
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Effects of the hit compounds on adult S. mansoni worm pair’s viability
In order to investigate the effects of compounds active on schistosomula toward other
parasite developmental stages, adult S. mansoni worm pairs were treated with the most
promising compounds 4-12 both at 20 μM and 50 μM; their impact on parasite viability was
monitored for seven days upon treatment. Viability of each schistosome was graded from 3
(normal) to 0 (severe effect) by assigning daily worm phenotype scores (plate-attachment,
movement, color, gut peristalsis, tegument damage).31 Six out of the nine compounds,
namely 6 to 11 showed to markedly decrease the viability of treated worms at 50 μM. In
particular, upon treatment with 9 viability dropped below 40% whereas with all others it was
below 10% within the seven days (Figure 4). Vehicle-treated worms were always included
in the assays and used for comparison. At 50 μM concentration, with the unique exception
of compound 11, the first 24 h showed to be critical, with the main drop on worm’s viability
exerted after the first day of treatment. Then, a slower decrease in viability was recorded.
Treatment with 6 and 7 at the highest concentration showed to give the aforementioned
immediate loss of viability, followed by a slower decrease in residual viability for the following
six days until parasites death. The same compounds used at 20 μM, behaved differently,
with 6 retaining a slightly higher potency than 7; parasites treated with both compounds
received an overall high score even though 6 appeared to interfere slightly more with the
viability status. The initial viability drop was also observed after treatment with compound
10 at both concentrations. Treatment with both 8 and 10 at 50 µM resulted in worm death
within five and seven days, respectively. On the other hand, compound 9 impacted viability
to a much lesser extent and did not induce complete parasites death. All the effective
compounds were also assayed on mammalian NIH-3T3 cells, by MTT assay, in a dose
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response curve. Compound 7 was the most toxic (IC50 2.37 μM ± 0.37 SEM), whereas
compounds 6, 9-11 showed an IC50 around 10 μM and compound 8 had an IC50 in the high
micromolar range (Figure 3, panel C).
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Egg production impairment
Compounds 6-11, showed to be the most effective in killing adult worms (viability less then
40% at day 7 upon treatment at 50 μM), were then evaluated for their impact on egg
production in vitro. First, the highest sub-lethal concentration which ensured 100% viability
and pairing was identified for each compound, hence, eggs of in vitro treated-worms were
counted at 72 h and normalized to the number of couples. In particular, worm pairs were
treated using the following concentrations for each selected compound: 6, 7 and 11 were
used at 10 μM, 8 at 20 μM, 9 at 30 μM, and 10 at 5 μM. The above conditions resulted in
fully viable couples despite compounds 8, 9, and 10 clearly showed reduced egg laying in
vitro (Figure 5, panel A); compounds 8 and 9 treated samples also showed evident signs of
egg damage (Figure 5, panel B). Worms treated with sub-lethal concentration of compounds
6 and 7 resulted in the presence of vitelline and germ cells with egg fragments in the culture
medium. These were very abundant in compound 7-treated samples, where damaged eggs
were also detected (Figure 5, panel B). This turned our focus into assessing if any damage
could be detected in the reproductive systems of the parasites.
Alterations of the reproductive systems of S. mansoni worm pairs treated with sub-
lethal doses of selected compounds
Adult worm couples were analyzed by means of carmine red staining followed by confocal
laser scanning microscopy (CLSM). Sub-lethal doses of compounds were chosen to
preserve the overall parasite viability and pairing. For each compound, three to five couples
were analyzed and images of ootype, ovary, vitellarium, testis and parenchyma recorded.
All observations were compared with the morphology as well as organs and tissues
organization of control (DMSO) worms (Figure 6 and Figure S2, S3).
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Worm pairs treated with compound 6 (10 μM) showed either an empty ootype or vitelline
cells and oocytes accumulation (Figure 6). In three out of four samples no properly,
developed eggs were detected. The overall morphology of ovary and vitellarium was
preserved in compound 6-treated females however cell degeneration was observed in the
ovary, especially in the immature compartment, and in the vitellarium in all samples (Figure
6). Treatment with compound 6 resulted also in an increase in the number of sperm cells in
testis and relative seminal vesicle (Figure 6). No alterations in the sub-muscle layers were
detected. Pairs treated with compound 7 (10 μM) showed phenotypic alterations similar to
those of compound 6-treated schistosomes (Figure S2). Interestingly, treatment with
compound 6 and 7 did not impact the number of eggs layed in vitro, nevertheless egg-shell
fragments, vitellocytes, oocytes and sperms were detected in the medium and some eggs
were damaged in compound 7-treated samples (Figure 5B). These observations, along with
the CSLM analysis, suggest the possibility that the eggs were properly released within the
first 48 h; after that time a possible drop in egg production might occur due to reproductive
organs damage.
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Regarding compound 8 (20 µM), the ootype was found empty in four out of five females
(one contained only vitelline cell aggregates) (Figure 6); the ovary morphology was
unaffected, even though cellular degeneration, in particular in the immature compartment,
was observed. Moreover, black spots (alterations previously observed upon treatment with
other synthetic compounds33,34) were detected in a large number of mature oocytes (Figure
6). The vitelline cells appeared to be irregular and disordered and dark areas resembling
hole-like structures within the vitellarium were also detected in all samples, likely due to lipid
aggregation in vitelline cells (Figure 6).35 Within the seminal duct and seminal vesicle of
three male parasites an accumulation of sperm cells was found (Figure 6), whereas one
specimen showed a collapsed seminal vesicle and in one male no clear accumulation of
spermatozoa, with respect to control worms, was observed. Moreover, a generalized
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damage for the male parenchymal tissue with numerous unstained dark areas was present
(Figure 6).
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Couples treatment with compound 9 (20 μM) was not associated to alterations within the
vitellarium and ovary; yet the ootype was empty in two out of three samples (Figure S3a);
as for worms treated with compound 8 (Figure 6). It is worth noting that control worms
(DMSO-treated) displayed eggs within the ootype in almost all the specimens investigated.
On the contrary, alterations similar to the ones described for compound 8, were detected in
some of the worms treated with compound 9 at slightly higher concentrations (30 μM),
namely: cell degeneration in the ovary in half of the samples; sperms accumulation in one
out of the three samples in testis; numerous unstained, dark, hole-like areas in the vitellarium
(all samples) and in the parenchyma in two out of three (Figure S3b). These observations,
along with the diminished egg counts (Figure 5), lead us to hypothesize that the reproductive
systems are the main site of action of compounds 8 and 9.
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The small but significant decrease in egg-laying detected in worm pairs treated with
compound 10 at 5 μM (Figure 5A) was not associated to CSLM morphological alterations
within the reproductive organs in 3 samples (Figure S2).
Effect of selected compounds on male juvenile worms
All compounds, effective on adult parasites, were also tested against juvenile schistosomes
(4-week-old parasites) poorly sensitive to PZQ.32 Male parasites were subjected to the same
treatment conditions as for adult pairs, namely 20 and 50 µM of the selected compounds for
up to 7 days. At these conditions, only compound 11 did not show any striking effect,
whereas the remaining compounds, especially at 50 µM, showed a strong lethal phenotype
already three days after treatment, with parasites death occurring 5 days post treatment
(Figure 7). In addition, 6 and 7 exerted the same lethal effect also using low compound
concentrations, resulting more active on juvenile parasites than on the adult ones (Figure
4). Noteworthy treatment with both concentrations showed an initial drop in parasite’s
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viability as observed for adult worms (50 µM). This implies that almost all the effects in terms
of viability and phenotypic changes occurred within the first 24 h, after which viability slowly
decreases throughout. In general, the overall sensitivity to the compounds is shared
between adult and juvenile stages with the latter one showing a higher susceptibility, with
the exception represented by compounds 9 and 11 having major activity on adults (Figure
4). Taken together, these data suggest that compounds 8, 9 and 10, sharing a thienoindolyl
core, not only have similar effects in killing all the developmental parasite stages of the
definitive host (schistosomula, juvenile, and adult schistosomes) but were also able to
impact egg-laying at sub-lethal doses. Compound 6 showed to be very effective in killing
adult and juvenile worms at 20 μM; its isomer 7 was less potent on adult parasites.
Compounds 8 and 9 were not able to kill the parasite at lower concentrations; rather, their
main action was exerted by impairing the egg-laying.
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Activity of hit compounds on recombinant SmHDAC8 and docking studies of selected
inhibitors with SmHDAC8
In order to investigate the mechanism of action of molecules 4-12, SmHDAC8 enzymatic
assays were performed in vitro. A recombinant form of the SmHDAC8 was produced in E.
coli and assayed for its residual activity upon pre-incubation with each of the selected
compounds. The activity assays were performed by using commercially available
fluorescence-based kit, and residual activity of the enzyme was evaluated with respect to
the untreated enzyme. The residual activity was also measured for the human HDAC8
(hHDAC8) treated with the same compounds. An initial screening, performed at 50 μM for
the whole set of selected compounds, demonstrated that only compounds 6-11 were
inhibitors of both homologue forms. Hence, a dose-response curve was designed for the
same compounds and the alleged IC50 values were calculated; the results are shown in
Figure 8. The data indicate that compounds 6-11 are inhibitors of both SmHDAC8 and
hHDAC8 enzymes, with the whole set of IC50 values in the low micromolar range. Since
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hHDAC8 belongs to class I histone deacetylases, to further characterize the new HDAC
inhibitors, we tested compounds 6-11 against HDAC1, a nuclear enzyme isoform that
belongs to the same class. Two- to three-fold lower IC50 values were recorded on hHDAC1
for compounds 6, 8, and 11, while similar values were observed for inhibitors 7 and 9.
Gratifyingly, compound 10 showed only 44% hHDAC1 inhibition even at 50 µM (Figure 8).
In order to gain insights into the binding mode of compounds 6-11 we performed SmHDAC8
docking experiments. All the presented compounds reported a docking score below -9.00
kcal/mol that represents the minimal requirement for selecting molecules in our virtual
screening procedure (see Experimental section for further details). In particular, in Figure 9
the poses of the compounds most effective on schistosomes are reported. They belonging
to two structural templates, namely indolines such as compound 6 (Figure 9 A, B for R- and
S-enantiomers, respectively) and thieno[3,2-b]indoles as for inhibitor 10 (Figure 9 C,D for
R- and S-enantiomers, respectively). Binding poses of compounds 7-9 are reported in the
Supporting Information (Figure S4). For each compound, the potential coordination bond is
established by the carbonyl moiety of the hydroxamic portion of the molecule with Zn2+
through a monodentate coordination. The R-enantiomer of 6 (panel A) was differently
accommodated as compared to the corresponding S-enantiomer. In particular, the phenyl
ring of the pendant benzyl moiety of the R-6 can form two different π-π stackings with F151
and H188 of the enzyme. Two additional stackings, with H292 and with F216 are also
established through the chlorophenyl moiety and the indoline ring. In terms of polar contacts,
the R-enantiomer of compound 6 can form an H-bond with G150, as depicted in Figure 6
and a salt bridge through its protonated methylpiperidine with D100. The main difference in
the binding mode of the S-enantiomer of compound 6 is represented by the different
accommodation of the cap group in the binding site, which now establishes a cation-π
stacking with H292 through its protonated piperidine N-atom, while the central portion of the
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molecule maintains the same contacts.
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On the other hand, the R- and S- enantiomers of 10 were similarly accommodated inside
the binding site. The MBG of the two enantiomers can form an H-bond with G150 through
the NH moiety of the hydroxamic acid and another H-bond with Y341 through the carbonyl
moiety of the MBG. The thiophene ring establishes two π-π stackings with F151 and H188,
while the two phenyl rings of the cap group can establish two π-π stacking with F215 and
F216 for R-enantiomer, while the S-enantiomer lack the stacking with F215, due to its
different conformation with respect to R-enantiomer (Figure 9 panel D).
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In addition, the aforementioned compounds were evaluated for their potential capability to
behave as pan-assay interference compounds (PAINS), namely compounds that frequently
give positive results in screening campaigns due to nonspecific reactions with several
biological targets.36 This calculation was performed by means of FAFDrugs4.0
(http://www.fafdrugs4.mti.univ-paris-diderot.fr/ access date December 2018).37 Remarkably,
the compounds filtered for PAINS indicated that none of them was labeled as potential
“frequent hitter”. For compound 11 the software highlighted the presence of the
methoxydialkylaniline as a low-risk structural alert with a number of occurrences below the
threshold. Due to this low risk, compound 11 was retained for evaluation of its biological
activity.
HDAC inhibitors treatment causes histones hyperacetylation
In order to investigate the effects of the selected compounds on HDACs activity, acetylation
of lysine residues within histone proteins was evaluated. Worm pairs were treated with sub-
lethal doses (20 μM) of selected compounds; after 72 h, histone-enriched fractions of
parasite lysates have been subjected to SDS-PAGE and western blot analysis (Figure 10).
Treatment with the pan inhibitor TSA (1 μM) was used as positive control of histones
hyperacetylation. A strong signal was detected for samples treated with compounds 8-10
whereas no changes in acetylation signal was associated to compounds 6, 7 and 11
parasites-treatment (Figure 10).
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It is worth noting that schistosomes treated with compounds 6 and 7 did not show any
detectable increase in histones acetylation, even though they behave as inhibitors of
recombinant SmHDAC8 activity in vitro. However, it has been recently reported that HDAC8
can also deacetylate non-histone substrates12,38,39 and several nuclear and cytoplasmic
acetylated proteins have been also identified in Schistosoma.40
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Conclusions
We deepen the investigation of the biological effects associated to treatment of Schistosoma
with two main chemical classes of HDAC inhibitors characterized by the presence of a
hydroxamate-based MBG coupled to a (spiro)indoline or a tricyclic thieno[3,2-b]indole core
as capping groups (compounds 4-7 and 8-10, respectively). Compounds of both classes
were shown to impair the viability of multiple life cycle stages of S. mansoni (schistosomula,
juvenile and adult parasites); compounds 8-10 also decrease egg-laying in vitro and
treatment with compounds 6-9 has been associated to morphological alteration in the
reproductive organs of adult pairs. Compounds 6-11 demonstrated to inhibit the enzymatic
activity of recombinant form of SmHDAC8 in the low micromolar range. Based on these
results, following studies should be aimed at optimizing potency and selectivity of the new
(hetero)arylhydroxamate-based molecules in order to proceed to their evaluation in murine
models of infection. Moreover, based on the innovative mode of action of SmHDAC8
inhibitors, with respect to currently available drugs, combination therapy should be
considered early in compounds optimization phase as a strategy to improve efficacy on
multiple developmental stages including juvenile schistosomes, and to avoid selection of
drug-resistant parasites.
Experimental Section
Chemistry
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General procedures. Starting materials and solvents were purchased from commercial
suppliers and used without further purification. Reaction progress was monitored by TLC
using silica gel 60 F254 (0.040-0.063 mm) with detection by UV. Silica gel 60 (0.040-0.063
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mm) was used for column chromatography H NMR and C NMR spectra were recorded
on a Varian 300 MHz, or a Bruker 400 MHz spectrometer using the residual signal of the
deuterated solvent as internal standard. Splitting patterns are described as singlet (s),
doublet (d), triplet (t), quartet (q), and broad (br); the value of chemical shifts () are given
in ppm and coupling constants (J) in Hertz (Hz). Mass spectra were recorded utilizing
electron spray ionization (ESI) Agilent 1100 Series LC/MSD spectrometer. Melting points
were determined using a Büchi® Melting point B-540. Yields refer to purified products and
are not optimized. All moisture-sensitive reactions were performed under argon atmosphere
using oven-dried glassware and anhydrous solvents. All compounds that were tested in the
biological assays were analyzed by combustion analysis (CHN) to confirm the purity > 95%.
Computational Details
Database and protein preparation and High Throughput Docking protocol. Our
proprietary database was built in Maestro suite41. All the structures were minimized by
means of MacroModel42 with OPLSAA_2005.43 The GB/SA model was used for simulating
the solvent effect. PRCG method (10000 maximum iterations and 0.001 gradient
convergence threshold) was employed. Compounds were then submitted to LigPrep
program44 producing a possible ionization state taking also into account all enantiomers and
tautomers at pH 7.4 ± 0.2.
The three dimensional structure of SmHDAC8 was taken from the PDB database (PDB ID:
4BZ7, Crystal structure of SmHDAC8 complexed with M344, DOI: 10.2210/pdb4BZ7/pdb)
and submitted to Protein Preparation Wizard protocol implemented in Maestro suite 2015
for obtaining a suitable protein structure for the screening protocol.22,23 Water molecules and
compounds used in the crystallization process were removed maintaining the Zn2+ ion and
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the ligand M344. After the preparation protocol, the refined protein was used in HTD as
reported in the next paragraph.
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The HTD protocol was carried out employing Glide software45 implemented in Maestro46
suite using the database and the protein prepared as above-mentioned. Energy grid was
prepared using default value of protein atom scaling factor (1.0 Å) within a cubic box
centered on the crystallized inhibitor M344. After grid generation with the introduction of
metal constrains25, the ligands were docked into the enzyme. The number of poses entered
to post-docking minimization was set to 50. In order to select the most promising compounds
from our library, we used extra XP scoring function, coupled with a rescore procedure
employing SP scoring function, applying a score cutoff of -9.00 kcal/mol.
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Considering that our in-house database contains compounds with one or more stereogenic
center, an additional criterion for molecules selection was that the enantiomers of a molecule
must have a docking score below -9.00 kcal/mol. The assessment for potential Pan Assay
Interference Compounds (PAINS) in the set of selected molecules was conducted by means
of FAFDrugs4 (http://www. fafdrugs4.mti.univ-paris-diderot.fr/ access date December
2018).36,37
Schistosoma mansoni studies
Reagents. Dimethyl sulfoxide (DMSO), percoll, Nonidet P-40 (NP40), foetal bovine serum
(FBS), thimerosal, gambogic acid, trichostatin A (TSA), bovine serum albumin (BSA),
carmine-red and Canada balsam, thrombin from bovine plasma, protease inhibitor cocktail,
DNase, were purchased from Sigma-Aldrich (Saint Louis, USA) and talon superflow
histidine-tagged protein purification resin from GE Healthcare Life Sciences (Marlborough,
USA); CellTiter-Glo (CTG) reagent from Promega (Madison, USA); Dulbecco-Modified
Eagle’s Medium (DMEM) with or without phenol red, HEPES, L-glutamine from Lonza
(Basel, Switzerland); Trizol reagent, glycogen, RNA grade, T4 DNA Ligase, SuperScript™
III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase, antibiotic-
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antimycotic reagent (100×) from Thermo Fisher Scientific (Waltham, USA); the primary
monoclonal anti-α-tubulin antibody (DM1A) from Sigma-Aldrich; the anti-acetylated-lysine
(Ac-K2-100) from Cell Signaling Technology (Danvers, USA); the goat anti-mouse and anti-
rabbit IgG (H+L)-horseradish peroxidase secondary antibodies from Bio-Rad Laboratories
(Hercules, USA). Fluor De Lys HDAC8 Fluorimetric Drug Discovery Kit (BML-AK518) was
from Enzo Lifescience (Farmingdale, USA), in the form of the BML-AK518 fluorimetric drug
discovery kit.
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SmHDAC8 cloning. Total RNA was extracted from 8-10 adult S. mansoni worms washed
3 times in 1.0 mL PBS resuspended in Trizol reagent (100 μL), flash frozen in liquid nitrogen
and stored at −80°C for 1 h. During thawing, samples were homogenized with disposable
pellet pestles served by cordless motor. Next, Trizol reagent was added up to 500 µL and
samples extracted with 200 μL of chloroform-isoamyl alcohol mix (24:1). The aqueous phase
was then transferred to a new 1.5 mL microfuge tube and RNA was precipitated with 250
μL of 2-propanol. RNA was further purified by two washes in 75% ethanol and pellet dried
at room temperature was resuspended in fresh milliQ water. The full-length cDNA for
SmHDAC814 was PCR amplified with the oligonucleotides GR581 5’-
CCCCCCATGGGATCTGTTGGGATCGTTTATGG-3’
and
GR583
5’-
CCCCGAGCTCCATACCAGTTAAATTATATAATTG-3’ and the SuperScript™ III one-step
RT-PCR system. Next, the PCR product was purified and cloned between the NcoI and SacI
sites of the pET-52b(+) plasmid encoding a C-terminal thrombin cleavage site followed by a
His-tag. The recombinant plasmid was checked by nucleotide sequencing (Eurofins
Genomics service). Overexpression of the His-tagged SmHDAC8 was carried out in E. coli
BL21(DE3) cells.
SmHDAC8 expression in E. coli and purification. The recombinant enzyme was
produced accordingly with Marek et al.47 with minor changes in the purification procedure.
Briefly, freshly transformed E. coli BL21 (DE3) cells were spread on a LB agar plate
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containing 100 μg/mL ampicillin and the bacteria film grown on the plate was resuspended
and grown up to 20h in sterile Terrific Broth medium containing ampicillin, 0.17 M KH2PO4
and 0.72 M K2HPO4. Then, 500 μM IPTG and 100 μM ZnCl2 were added to the culture in
order to induce expression of the recombinant His-tagged protein. After 1 h, the E coli cells
were harvested and the bacterial pellet resuspended in ice-cold purification buffer A (50 mM
Tris, pH 8.0, 150 mM KCl, protease inhibitor cocktail, and 10 μg/mL DNase). After the lysis,
the suspension was centrifuged at 28000 x g. From this point ahead, all of the purification
procedure was conducted at 4°C. Major modifications in the already published procedure
were as follows. For the protein purification, the supernatant was incubated 1 h in a glass
flask containing 0.4 mL/L of Talon superflow affinity resin, previously equilibrated in buffer
A. The resin was then loaded into a disposable plastic column and twashed with 150 mL of
buffer A. Then, 60 U of thrombin in 6 mL of buffer A were added to the resin and left to
incubate up to 16 h under shaking in order to release the protein. The resin was loaded into
disposable plastic column and the eluate was collected followed by one or more washing
step. The fractions before and after the thrombin cleavage were analyzed by 12% SDS-
PAGE and those containing only the cleaved protein were pooled together and loaded into
a pre-equilibrated (with buffer A) 1 mL HiTrap Q FF column. Elution was achieved by a step-
wise gradient with increasing concentration of KCl (from 50 mM up to 600 mM) in 50 mM
Tris, pH 8.0. Purity was were assessed by means of 12% SDS-PAGE. Usually SmHDAC8
eluted between 250 mM and 450 mM KCl. The fractions containing the purified protein were
pooled and diafiltrated 3 to 4 times against buffer B, 10 mM Tris, pH 8.0, 50 mM KCl, 2 mM
DTT, using AMICON ULTRA 30 KDa MWCO and concentrated up to 2.5 mg/mL. The
purified protein was then used within few days from the purification, in order to minimize
unfolding events and/or metal leaking, otherwise a decrease in its activity was observed.
Activity and inhibition assay of recombinant form of human and S. mansoni HDAC8.
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Recombinant human HDAC8 was an item of the Fluor de Lys HDAC8 fluorimetric drug
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discovery kit (Enzo Life Sciences, BML-AK518). Inhibition assays were performed as
already described in previous publications16,47,48 with minor modifications. Increasing
concentrations of selected compounds were used in order to draw scattered curves by which
IC50 value for each compound could be inferred with respect to human and S. mansoni
enzymes. DMSO concentration was kept constant to 0.5% and for each compound, a control
curve (enzymes incubated with DMSO only) was added. To keep safe from the possibility
of a slow-binding interaction between the inhibitor and the target, each enzyme was pre-
incubated with the selected compounds 15 minutes before substrate addition to the mixture.
Fluor de Lys Substrate was used at 50 μM. Then, compounds, substrate and enzymes (1.7
μM SmHDAC8 or 1 μM for hHDAC8) were incubated and the reaction left to proceed for 1
h at 30°C; subsequently, 2 μM TSA within 50 μl of 1x Developer II was added and the mixture
was further incubated for 1 h at 30°C to quench the reaction. Fluorescence was measured
in a plate reader (Varioskan Lux, Thermofisher Scientifica) with excitation at λ = 370 nm and
emission at λ = 450 nm. IC50 was obtained after non-linear regression curve fit performed
by means of GNU/Octave, according to a generalized form of a dose-response curve
equation, as reported by Copeland (2000; eq 8.25, p. 287 in the original text):
(1)
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where y is the residual activity of the enzyme in the presence of inhibitor at concentration
[I]; ymax is the maximum observed value for the enzyme at zero inhibitor concentration; ymin
is the minimum observed value for the enzyme at the highest inhibitor concentration. The
model did fit three parameters, IC50, ymax, ymin, while assuming that the dose response curves
has a Hill slope of -1.0.
HDAC1 assay. OptiPlate-96 black microplates (Perkin–Elmer) were employed with an
assay volume of 60 μL. Human recombinant HDAC1 (BPS Bioscience, Catalog #: 50051)
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was diluted in incubation buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2 and 1 mg/mL BSA). A total of 52 μL of this dilution were incubated with 3 μL of
different concentrations of inhibitors in DMSO and 5 μL of the fluorogenic substrate ZMAL
(Z-(Ac)Lys-AMC)48,49 (126 μM) at 37°C. After 90 min incubation time, 60 μL of the stop
solution (33 μM Trichostatin A and 6 mg/mL trypsin in trypsin buffer [Tris-HCl 50 mM, pH
8.0, NaCl 100ꢀmM]), were added. After a following incubation at 37°C for 30 min, the
fluorescence was measured on a BMG LABTECH POLARstar OPTIMA plate reader (BMG
Labtechnologies, Germany) with an excitation wavelength of 390 nm and an emission
wavelength of 460 nm [54,76].49,50,51
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Western blot analysis. Ten adult pairs were treated with selected HDAC inhibitors for 72
h. Parasites were processed by using a modified version of Dubois et al.52 protocol as
previously described.34 Samples were analyzed by 15% SDS-PAGE and immunoblotting
using α-tubulin DM1A (1:5000) or α-acetylated-lysine (1:4000) antibodies according to
manufacturer’s instructions. A Chemidoc XRS Bio-Rad with a chemi-luminescent camera
and ImageLab 4.0 software were used for acquisition of images. Each western blot analysis
was calibrated by Ponceau-staining.
Ethics statement. Animal work was approved by the National Research Council, Institute
of Cell Biology and Neurobiology animal welfare committee (OPBA) and by the competent
authorities of the Italian Ministry of Health, DGSAF, Rome (authorization no. 25/2014-PR
and no. 336/2018-PR). All experiments were conducted in respect to the 3R rules according
to the ethical and safety rules and guidelines for the use of animals in biomedical research
provided by the relevant Italian law and European Union Directive (Italian Legislative Decree
26/2014 and 2010/63/EU) and the international guiding principles for biomedical research
involving animals (Council for the International Organizations of Medical Sciences, Geneva,
Switzerland).
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Maintenance of the S. mansoni life-cycle. A Puerto Rican strain of S. mansoni was
maintained by cycling within albino Biomphalaria glabrata, as the intermediate host, and ICR
(CD-1) outbred female mice as the definitive host as previously described.31 Briefly, 2 to 3
months old-female mice (purchased from Envigo, Udine, Italy) were housed with the
following conditions: 22 °C, 65% relative humidity, 12/12 h light/dark photocycle, standard
food and water ad libitum. Then, they were infected with 200-250 single sex or double sex
S. mansoni cercariae by the tail immersion technique. Juvenile (single sex) and adult
parasites (double sex) were recovered from mice 4 or 8 weeks after infection by reversed
perfusion of the hepatic portal system and mesenteric veins.
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Schistosomula viability assay. Schistosomula were obtained by mechanical
transformation of cecariae and percoll gradient. This stem from an optimized version of the
protocol of Brink et al.53 previously described by Protasio et al.54 and adapted in our
laboratory.31 The viability assay was performed as already described31 with minor
modification. In brief, the schistosomula (150-200/wells) were incubated in DMEM medium
(w/o phenol red) with compounds dissolved in DMSO for 72 hours, 37°C in 5% CO2, in 96-
well black plates. Then 50 μL of CellTiterGLO reagent (CTG) was added to each well and
relative luminescence units (RLU) signal was recorded after 30 minutes with Varioskan Lux.
Data analysis was achieved using GraphPad Prism v 7.0 software and IC50 was obtained
after non-linear regression curve fitting performed according to log (inhibitor) vs. normalized
response with variable slope curve model.
Viability assays on adult and juvenile worms and egg counts. For juvenile and adult
worm assays, 10 juvenile worms or 5 adult pairs were incubated with selected compounds
in 3 ml DMEM complete medium. For each compound, three experiments were carried out
and compounds were given to parasites only once without medium addition and/or
replacement. DMSO (vehicle) treated worms were used as control frame. Viability was
monitored daily under a Leica MZ12 stereomicroscope for 7 days and scores assigned, as
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previously reported.31 In particular the following phenotype scoring criteria were adopted: 3,
plate-attached, good movements, clear; 2, slower or diminished movements, darkening,
minor tegumental damage; 1, movements heavily lowered, darkening, tegument heavily
damaged; 0, dead, lack of any movement. For each sample the total score was determined
by the ratio of the sum of worm scores for the total number of worms. Vehicle-treated
samples were always included in the assays. Three days upon compound treatment, the
number of eggs laid in vitro by worm couples was counted using an inverted Leica DM IL
microscope (Leica Microsystems, Wetzlar, Germany) with a gridded-plate as supporting
tool.
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Confocal microscopy analysis. Carmine red staining was performed as previously
described.35 Images were acquired on an Olympus FV1200 confocal laser scanning
microscope using a UplanFLN 40x immersion oil lens (NA = 1.30) with optical pinhole at 1
AU and a multiline argon laser focused. Excitation was at 488 nm and fluorescence was
recovered in the range 500 nm to 700 nm. Images were collected as single stack.
Ancillary Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Scheme S1, Figure S1, Figure S2, Figure S3, Molecular Docking for compound 7 and 8,
Figure S4, Chemistry.
Author Information
Corresponding Authors
G. Campiani, Email: campiani@unisi.it; S. Gemma, Email: gemma@unisi.it; G. Ruberti,
Email: giovina.ruberti@cnr.it
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Author contribution
^F.S. and M.B. contributed equally to this work
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Acknowledgments
This work was supported by Ministero dell’Istruzione, dell’ Università e della Ricerca (MIUR)
(PRIN Project 20154JRJPP to G.C. and G.R.), by the CNR (National Research Council)-
CNCCS (Collezione Nazionale di Composti Chimici e Centro di screening) “Rare, Neglected
and Poverty Related Diseases - Schistodiscovery Project” (DSB.AD011.001.003 to G.R.),
and the Deutsche Forschungsgemeinschaft (DFG, Ju295/13.1). A special thanks to Stefania
Colantoni for mouse husbandry and Pierluigi Palozzo for dishwashing lab technical support.
Abbreviations used
PZQ, Praziquantel; HDAC, Histone deacetylase; BACAD, biarylalkyl carboxylic acid
derivative; AAS, arylmethylamino steroid; TGR, thioredoxin glutathione reductase; HMG-
CoA, 3-hydroxy-3-methyl-glutaryl-CoA; HTD, High Throughput Docking; MBG, metal-
binding group; CSLM, laser scanning microscopy; PAINS, pan-assay interference
compound; ESI, electron spray ionization; DMSO, Dimethyl sulfoxide; RLU, relative
luminescence units.
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