Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs

Article abstract of DOI:10.1021/acsmedchemlett.5b00468

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC₅₀ < 500 nM) and good selectivity over the activity of human HDAC in cells (up to >50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.

Full text of DOI:10.1021/acsmedchemlett.5b00468

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Letter
Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs
Jesus Maria Ontoria, Giacomo Paonessa, Simona Ponzi, Federica Ferrigno, Emanuela Nizi, Ilaria
Biancofiore, Savina Malancona, Rita Graziani, David Roberts, Paul Willis, Alberto Bresciani, Nadia
Gennari, Ottavia Cecchetti, Edith Monteagudo, Maria Vittoria Orsale, Maria Veneziano, Annalise Di
Marco, Antonella Cellucci, Ralph Laufer, Sergio Altamura, Vincenzo Summa, and Steven Harper
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00468 • Publication Date (Web): 05 Mar 2016
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Discovery of a Selective Series of Inhibitors of Plasmodium
falciparum HDACs.
Jesus M. Ontoria,*,† Giacomo Paonessa,† Simona Ponzi,† Federica Ferrigno,† Emanuela Nizi,† Ilaria
Biancofiore,† Savina Malancona,† Rita Graziani,† David Roberts,‡ Paul Willis,‡ Alberto Bresciani,†
Nadia Gennari,† Ottavia Cecchetti,† Edith Monteagudo,† Maria V. Orsale,† Maria Veneziano,† Annalise
Di Marco,† Antonella Cellucci,† Ralph Laufer,† Sergio Altamura,† Vincenzo Summa,† and Steven
Harper†
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†Departments of Chemistry and Biology, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Rome, Italy.
‡Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland.
KEYWORDS: Malaria, Plasmodium falciparum, PfHDAC1, 4-arylimidazoles.
ABSTRACT: The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known
human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led
to the identification of compounds with sub-micromolar inhibition of P. falciparum growth (EC50 < 500 nM) and good selectivity
over the activity of human HDAC in cells (up to > 50-fold). Inhibition of parasital HDACs as the mechanism of action of this new
class of selective growth inhibitors is supported by hyperacetylation studies.
Infection with malaria parasites such as Plasmodium falci-
parum remains a devastating cause of death in tropical geog-
raphies with 40% of the world population at risk of acquiring
the disease. There are approximately 200 million clinical cases
of malaria every year leading to an estimated 600,000 deaths.1
The requirement for improved therapies to treat and to cure
malaria is an evident medical and humanitarian need that is
exacerbated by an alarming rise in parasite resistance to the
current standard of care.2,3 Drugs that operate via novel mech-
anisms of action for which no innately resistant parasites are
expected are therefore especially desirable.
DNA is tightly packed around histone proteins in the nucle-
us of eukaryotic cells with its transcription being regulated by
chemical modifications to the nucleosomal histone proteins
themselves. Histone deacetylases (HDACs) are zinc-
dependent enzymes that play crucial roles in modulating
mammalian cell chromatin structure, transcription and gene
expression.4-6 HDACs have also been identified as important
regulators of transcription in P. falciparum,7-10 and inhibition
of P. falciparum histone deacetylases (PfHDACs) has been
reported to both effectively kill the parasites (Vorinostat, Fig-
ure 1)11-16 and lead to efficacy in animal models of malaria
(compound 2).17 Such findings underscore the potential for
PfHDAC inhibitors to be used for malaria therapy.18-20
Figure 1. Structures of known human HDAC inhibitors.
Previous research in our labs led to a novel series of hetero-
cyclic inhibitors of human class-I HDAC’s represented by
compound 3 (Figure 1).23 This compound class features a cen-
tral heterocycle that displays the pharmacophore elements
common to many inhibitors of HDAC enzymes. Thus, a zinc
binding group (ZBG) believed to interact with the catalytic
metal ion of the HDAC enzyme is anchored by an alkyl linker.
The ZBG is complemented by two surface contact groups
(namely the aryl substituent and the amide group) that are
thought to interact close to the entrance of the substrate bind-
ing channel.24 With many compounds from this structural class
available, our efforts toward selective inhibitors of PfHDACs
logically began through screening a representative panel of
human HDAC1 inhibitors against a recombinant PfHDAC1
enzyme. The PfHDAC1 enzyme required for this campaign
was expressed in fusion with a flag protein tag, in either insect
or mammalian cell lines, and was purified with an Anti-Flag
affinity gel column.
Of the five HDAC encoding genes known in P. falciparum
one has homology to mammalian class
I
isoforms
(PfHDAC1), two are similar class II (PfHDAC2 and 3) mam-
malian HDACs, while the remaining two are class III HDACs,
or silent information regulator 2 (SIR2) proteins.19 In light of
the close sequence homology between PfHDAC1 and human
class I HDACs21 an undesired effect of the use HDAC inhibi-
tors is potential toxicity resulting from induction of epigenetic
changes in host mammalian cells. Approved cytotoxic anti-
cancer agents such as Vorinostat operate via inhibition of hu-
man HDAC enzymes (hHDACs), and can cause mechanism
based toxic side effects.22 Thus, the development of selective
inhibitors for PfHDAC’s is viewed as necessary to circumvent
potential toxicity issues in the host, and we report here our ef-
forts in this direction. This work led to the development of the
first selective series of inhibitors of PfHDAC’s reported to
date.
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In addition, PfHDAC1 enzyme mutated in a Tyr residue measurement of nuclear histone hyperacetylation in both hu-
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(Y301H) located in the putative catalytic site was expressed.
Mutation of the corresponding Tyr-to-His in human class-I
HDACs has been demonstrated to severely reduce their enzy-
matic activities without affecting the overall protein structure,
demonstrating that Y301 is essential for catalytic turnover.25
Y301H PfHDAC1 in fusion with a flag tag was expressed and
purified under analogous conditions to the wild-type enzyme.
Both purified proteins showed a low, though detectable,
deacetylase activity in in-vitro assays with fluorogenic sub-
strates.
man and parasite treated cells. The inclusion of histone hyper-
acetylation experiments, as well as screening against the
hHDAC1 enzyme amounts to an approach that adds weight to
data obtained from simple comparison of cell-based assay da-
ta.
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Compound 3 was tested head to head on mutant and wild
type PfHDAC1. No loss of enzyme activity nor inhibition was
observed with the mutant enzyme, despite its (presumed) lack
of catalytic efficiency. While it cannot be excluded that the
role of Y301 in the catalytic mechanism of PfHDAC1 differs
to that observed for the corresponding residue in human class-
I HDACs, these results (together with the low in-vitro activi-
ties observed across a variety of expression vectors in both
mammalian and insect cell lines) added to our concern that the
biological activities being measured were not in fact attributa-
ble to the PfHDAC1 enzyme. This conclusion was ultimately
supported by the finding that both hHDAC1 and hHDAC2
could be clearly detected by Western Blot using specific anti-
human HDAC antibodies following the expression and purifi-
cation of either mutant or wild-type PfHDAC1 from human
HeLa cells (Figure 2). The weight of evidence indicates that
PfHDAC1 is expressed as an inactive enzyme that likely needs
endogenous co-factors for its biological activation, and conse-
quently that biological results reported to date on inhibition of
PfHDAC1 are potentially false positives stemming from the
presence of co-purified host HDAC’s from the cellular expres-
sion vectors, likely associated in complex with PfHDAC, at
least in the expression systems used.
The apparent lack of catalytic activity following expression
of PfHDAC1 posed a significant challenge for the develop-
ment of selective inhibitors of this enzyme. Although cell
based assays were available to measure either inhibition of P.
falciparum growth in erythrocytes (Pfgrowth assay) or block-
ade of human class-I HDAC’s in HeLa cells, exclusive use of
the comparison of these two assays to determine reliable en-
zyme selectivities was viewed as precarious. Differences in
cell type and assay protocol are likely to influence properties
such as protein binding and cell penetration, and together with
the inherently larger variability in cell-based assay data as
compared to that from biochemical assays may significantly
impair interpretations based on direct comparisons. Although
in-vitro assays measuring cell-penetration and protein binding
might aid interpretation of results, issues with compound
throughput under such a screening paradigm were viewed as
problematic.
Figure 2. Western Blot analysis of purified wt and Y301H
mutant PfHDAC1s using anti-Flag, anti-hHDAC2 or anti-
hHDAC1. aArrows indicate the position of the probed protein.
M, molecular weight marker.
Screening results for a subset of our available compound
collection in both the P. falciparum growth and human class-I
HDAC cellular assays highlighted an apparently low degree of
selectivity, with most compounds inhibiting parasite growth at
concentrations comparable with their human class-I HDAC
inhibition EC50. However, a group of outliers that featured
significantly improved selectivity for parasite growth inhibi-
tion and which shared a common structural feature were clear-
ly evident. The bulk of compounds tested (e.g. compound 4,
Table 1) featured either ketone or hydroxamic acid zinc bind-
ing moieties but the outliers in contrast were based upon a
secondary amide as ZBG (e.g. compound 5). This finding was
somewhat surprising based on the conserved nature of the ac-
tive site across different HDACs. An often asserted view is
that selectivity between P. falciparum and human HDACs is
likely to be realizable by modification of the surface contact
groups in the pharmacophore, while our observation was that a
change to the zinc binding group was impacting apparent se-
lectivity.21,26,27 In line with our goal of optimizing away from
potent human HDAC enzyme inhibitors an attractive feature
was the much lower hHDAC1 enzyme inhibition shown by
compound 5 that contains a methylamide as ZBG. Compound
5 is around an order of magnitude weaker than 4 in terms of
parasite growth inhibition. However, in line with the lower
hHDAC enzyme inhibition for the amide, a much stronger
drop in cellular inhibition of human class-I HDACs was noted,
resulting in an overall improvement in the apparent selectivity.
That the binding orientations of 4 and 5 are similar, and that
the methylamide (rather than the imidazole ring) in 5 is func-
tioning as the ZBG is supported by several pieces of un-
published data. Notably, the imidazole ring in compound 5 can
be replaced with alternative ring systems that are weak Zn2+
binders without loss of parasite growth inhibition (data not
shown).
To alleviate the risks associated with our need to use a cell-
based assay as a front-line screen for the development of se-
lective inhibitors of PfHDACs we have undertaken an ap-
proach outlined as follows: i) comparison of parasite growth
inhibition in erythrocytes with human class-I HDAC inhibition
in HeLa cells was used as a preliminary readout of selectivity
with multiple repetitions of the cell based assays in order to
minimize interpretation errors resulting from assay variability;
ii) structure activity relationships were focused on improving
parasite growth inhibition while reducing activity against the
human HDAC1 enzyme; iii) apparent selectivities measured in
cell-based assays were validated at the biochemical level by
In view of the expected high metabolic turnover for com-
pounds containing a naphthyl ring as the aryl substituent,23 the
2-methoxyquinoline analog 6 was prepared.28 This structural
change (naphthyl to 2-methoxyquinoline) has previously been
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employed to favor reduced rodent clearance for human halogenation protocol that furnished the desired mono-
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HDACi’s and pharmacokinetic studies of 5 and 6 in mouse
indeed demonstrated that plasma clearance was ameliorated
for the latter compound (60 mL/min/kg vs. 10 mL/min/kg).
However, neither of these analogs achieved detectable plasma
exposure following oral administration, with portal vein sam-
pling studies confirming that poor intestinal absorption was an
issue for the methylamide ZBG analogs. Detectable oral expo-
sure was however achievable by installing neutral functionali-
ty in place of the basic amine group present in 6. Thus, both
compounds 7 and 8 showed a degree of oral bioavailability (F
= 10% and 20%, respectively) in mouse. The neutral amide
analogs (7 and 8) showed weaker P. falciparum growth inhibi-
tion and/or failed to show any apparent selectivity, but taken
as a whole the overall characteristics of the methylamide ZBG
analogs in Table 1 showed potential as a route toward selec-
tive and potentially orally available inhibitors of P. falciparum
growth.
iodinated compound 11. The methyl amide ZBG was installed
by ester deprotection and subsequent coupling with methyla-
mine to give the synthetically versatile intermediate 12. Re-
moval of the Cbz protecting group in this compound and in-
stallation of the thiazole amide furnished the substrate for final
Suzuki reactions. Cross couplings between 13 and boronic ac-
ids (or esters) proceeded smoothly when PdCl2 was employed
as a pre-catalyst,30 furnishing (after HPLC purification) com-
pounds 16-26, 28 and 29 (Scheme 1). Compounds 14,28 15, 27
and 30 where synthesized using alternative procedures that are
described in the Supporting Information.
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Scheme 1. Chemistry for SAR at aryl substituenta
Table 1. SAR of compounds 4-8a,18
HeLa
Pf
class I
HDA
C
hHDAC1
growth
b
b
b
cpd
4
Structure
EC50
IC50
IC50
SIc
0.038
0.094
0.0014
2.5
± 0.008 ± 0.03
± 0.0004
0.31
3.77
0.34
12
4.7
1.6
3.8
5
6
7
8
± 0.08
± 1.04
± 0.14
0.52
2.45
0.25
a
± 0.03
± 0.64
± 0.08
Reagents and conditions: (a) (i) MeNHOMe.HCl, HBTU,
DIPEA; DMF, RT; (ii) LiAlH4, THF, -20 °C; (iii) Glyoxal, NH3,
MeOH, RT; (b) (i) NIS, CH3CN, -10 °C; (ii) Na2SO3, Bu4NHSO4,
dioxane-water, 160 °C, microwave, 30 min; (c) (i) TFA, DCM,
RT; (ii) MeNH2, HBTU, DMF, RT; (d) (i) HBr in AcOH, DCM,
0 °C to RT; (ii) thiazole-2-carboxylic acid, EDC.HCl, HOBt,
DIPEA, DMF, RT; (e) ArB(OH)2, PdCl2(dppf), K2CO3, DME-
water, 110 °C.
0.57
0.89
0.20
± 0.19
± 0.30
± 0.07
1.76
6.65
1.76
± 0.36
± 2.18
± 0.38
The results in Table 2 highlight that both Plasmodium
growth potency as well as selectivity with respect to inhibition
of human class-I HDACs in HeLa cells were strongly influ-
enced by the nature of the aryl substituent attached to the im-
idazole core. Although clear SAR trends were difficult to iden-
tify, there were indications that the aryl group attached to the
imidazole ring is involved in a specific interaction with
PfHDAC. Similar to the trend observed between compounds 5
and 6, the 2-naphthyl compound 14 was a marginally more
potent and selective inhibitor of Plasmodium growth than 7.
However, introduction of a methyl group at position 1 of the
naphthyl ring (compound 15) completely abolished P. falcipa-
rum growth inhibition. Such a strong influence on activity re-
sulting from introduction of a single methyl group likely
points to a change in enzyme inhibition rather than a physico-
chemical effect that impacts cell-based activity. Similarly,
while many 3,4-fused bicyclic heterocycles generated sub-
aIC50 and EC50 values in µM. Compound 3 was used as stand-
ard reference compound (see data in Supplementary Table 1).
bIC50 and EC50 values are the average of at least three individual
measurements ± SD. SI: Selectivity Index (HeLa class I HDAC
(µM)/Pfgrowth (µM)).
c
Given of the detrimental effect of basic functionality on oral
absorption the electro-neutral thiazole amide fragment was
retained during efforts to align potency, selectivity and oral
absorption. In order to allow efficient exploration of SAR at
the aryl substituent on the imidazole core a new synthetic
strategy was developed based the introduction of the aryl moi-
ety by Suzuki-Miyaura cross-coupling chemistry as the last
step of the synthesis. Starting from the known amino acid 9
(Scheme 1),28 the free carboxylic acid was transformed into an
imidazole ring in three synthetic steps.29 Iodination of the im-
idazole was accomplished by employing a halogenation/de-
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micromolar P. falciparum growth inhibition (compounds 5-7,
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8.28
1
2
3
4
5
6
7
8
14) no active compounds based on a 2,3-fused bicyclic ring
system were found throughout the course of the program. The
weak micromolar growth inhibition shown by compound 16
was illustrative of results typically obtained for 2,3-fused het-
erocycles. A wide variety of 3,4-fused bicyclic heterocycles
were evaluated, and compounds 17-20 highlight the somewhat
empirical nature of this exploration. The indole and indazole
analogs (17 and 18, respectively) retained sub-micromolar
growth inhibition and showed 10-fold improved selectivity
with respect to the 2-methoxyquinoline analog 7 that was our
starting point. Furthermore these compounds provided a first
indication that P. falciparum growth inhibition in erythrocytes
that is stronger than activity measured in the human HDAC1
biochemical assay could be achieved. In contrast, the benzim-
idazole analog 19 and the benzotriazole analog 20 were weak
and poorly selective inhibitors of Plasmodium growth.
Throughout the program we were unable to correlate trends
between activities and measured or calculated physicochemi-
cal properties (e.g. logD) that may account for such differ-
ences in profile. Very different results were again obtained
within a set of isomeric quinoline/isoquinoline analogs. Of the
5 isomers that were prepared, quinoline analog 21 proved to
be a very weak inhibitor while the 4-isoquinoline 22 emerged
as the most promising. 22 showed sub-micromolar Plasmodi-
um growth inhibition and 18-fold selectivity with respect to
the human HDAC cellular assay. Substituted analogs of 22
were explored, with 23 and 24 both showing improved growth
inhibition in the 100 nM range and comparable selectivity. In
line with a specific enzyme interaction for the aryl group as
previously described, the positioning of the quinoline substitu-
ent was crucial, with the 3-substituted analog 25 showing
weak P. falciparum growth inhibitory activity. Compounds 23
and 24 represent the most potent inhibitors that were prepared,
but further improvements in selectivity with respect to human
cellular HDAC inhibition were achieved.
> 25
> 2.5
3
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
± 1.59
0.33
6.33
1.28
19
12
± 0.10
± 1.42
± 0.22
0.63
7.63
2.12
9
± 0.09
± 0.50
± 0.35
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12
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16
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21
22
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29
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12.04
> 25
> 25
> 25
> 2.5
> 5
2.1
2.5
6.6
18
± 1.70
10.05
± 5.39
3.81
> 5
± 0.50
0.27
4.75
0.60
± 0.12
± 1.44
± 0.15
0.10
2.04
0.29
20
± 0.02
± 0.71
± 0.04
0.10
1.37
0.39
14
± 0.05
± 0.38
± 0.02
4.86
20.14
2.62
4.1
3.3
14
Table 2. SAR of compounds 7, 14-30.a
± 3.09
± 2.91
± 1.57
7.57
> 25
> 5
± 0.95
0.57
8.13
2.16
HeLa
class I
HDAC
± 0.07
± 0.01
± 0.30
Pf
hHDAC1
growth
0.90
20.49
2.49
b
b
b
cpd
7
R
EC50
IC50
IC50
SIc
23
± 0.23
± 0.43
± 1.02
0.57
0.89
0.20
1.6
± 0.19
± 0.30
± 0.07
0.45
16.63
1.85
37
± 0.08
± 3.26
± 0.31
0.30
1.70
0.32
5.7
1
14
15
± 0.12
± 0.57
± 0.08
0.5
> 25
> 2.5
54
± 0.17
23.73
23.94
aIC50 and EC50 values in µM. Compound 3 was used as stand-
ard reference compound (see data in Supplementary Table 1).
bIC50 and EC50 values are the average of at least three individual
> 25
± 1.50
± 0.89
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In summary, we have reported findings that cast doubt on
measurements ± SD. cSI: Selectivity Index (HeLa class I
HDAC(µM)/Pfgrowth(µM)).
1
2
PfHDAC1 enzyme inhibition data reported to date in the liter-
ature,31 and have described a screening approach that guided
the discovery of what we believe to be selective inhibitors of
Plasmodium falciparum HDACs. The leading compounds
from this work show sub-micromolar inhibition of P. falcipa-
rum growth (EC50 < 500 nM), have good selectivity (> 50-
fold) over human HDACs in cells and are weak inhibitors of
human HDAC enzymes. Support for the mechanism of action
of this compound class was provided by histone hyperacetyla-
tion studies.
Very weak inhibition of Plasmodium growth was measured
for numerous phenyl-substituted imidazoles, including the bi-
phenyl analog 26, which is a 7 µM inhibitor. However, 6,5-
biaryl systems turned out to show improved activity with the
4-(2,4)-thiazole analog 27 and 4-(1,2)-pyrazole analog 28 both
showing sub-micromolar P. falciparum growth inhibition and
selectivities around 15-20 fold. A further improvement in se-
lectivity was achieved with the isomeric pyrazole 29 which
was almost 40-fold selective for parasite growth. The most
selective compound overall was the 2-hydroxy-3-quinoline
analog 30 which in multiple tests consistently failed to show
activity in our human HDAC assays. The weak activity of 30
against human cellular class-I HDACs meant that no IC50 val-
ue was determined; the apparent selectivity of the compound
is estimated as being greater than 50-fold (based on the upper
concentration at which it was tested in the assay). Notably,
compounds 29 and 30 have hHDAC1 enzyme inhibition 4-5
fold weaker than activity in cell-based Plasmodium growth
assay, supporting the notion that these are selective inhibitors
of PfHDACs and in line with our goal of optimizing away
from strong hHDAC1 enzyme inhibition.
Due to the lack of a reliable biochemical assay in which to
measure enzymatic PfHDAC inhibition directly, the mecha-
nism of action by which the optimized inhibitors elicited their
anti-parasitic activity was studied by histone hyperacetylation
experiments. Assuming the inhibitors block the histone
deacetylation process in parasites, then a phenotype that shows
hyperacetylation of parasite histone proteins would be ex-
pected. Treatment of P. falciparum parasites with increasing
concentrations of 29, one our most potent and selective inhibi-
tors, and analysis of histone H4 lysine 8 clearly confirmed hy-
peracetylation at a concentration (EC50 = 350 nM) close to the
compounds EC50 measured in the parasite growth assay (EC50
= 450 nM, Supplementary Figure 2 in the Supporting Infor-
mation section). Furthermore, little histone hyperacetylation of
histone H4 was observed at concentrations up to 25 µM when
human HeLa cells were treated with 29, in agreement with the
HDAC activity measured in this cell line (EC50 = 16.7 µM).
These results – which were reproduced with several additional
compounds (data not shown) – support P. falciparum HDAC
inhibition as the mechanism of action and also appear to cor-
roborate the idea that comparison of P. falciparum growth in-
hibition and human cellular class-I HDAC inhibition data can
provide meaningful insight with regard to biochemical events
within the cells. Further profiling of compound 29 against a
panel of human HDAC enzymes (hHDAC 1-7, 9) showed that
for most isoenzymes no inhibition was observed up to 5 µM,
though hHDAC3 was inhibited with IC50 = 330 nM. No signif-
icant toxicity was observed for 29 in HeLa or HUVEC cells
(CC50 > 25,000 nM) nor did the compound bind to hERG ion
channels (IC50 > 30,000 nM). While 29 showed moderate in-
trinsic clearances in rat, human and mouse liver microsomes
(Clint = 27, 19 and 12 µL/min/mgP, respectively) it had clear-
ance above liver blood flow following i.v. administration in
mouse (Clp = 247 mL/min/kg). Efforts to understand the ap-
parent extra-hepatic clearance, which does not appear to stem
from instability in mouse plasma, and combine potency and
selectivity with oral exposure remain under investigation in
our laboratories.
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ASSOCIATED CONTENT
Supporting information
The supporting Information is available free of charge on the ACS
Publication website at DOI: XXX.
Synthetic experimental details and characterization data, de-
scription of primary biological assay protocols, and PK pro-
tocols (PDF)
AUTHOR INFORMATION
Corresponding Author
*For J. M. O.: E-mail: j.ontoria@irbm.it. Phone: +39 06
91093279.
Current Address
*For R. L.: Teva Pharmaceutical Industries Ltd, Discovery and
Product Development, P.O. Box 8077, Netanya 4250843, Israel.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
Funding Sources
IRBM Science Park acknowledges financial support from Medi-
cine for Malaria Venture (MMV) and National Collection of
Compounds and Chemical Screening Center (CNCCS Consorti-
um).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank Dr. Pietro Alano and Dr. Francesco Silvestrini
for the malaria parasite, Dr. Sergio Wittlin and Christian Scheurer
for helping to set up the Pf growth assay and Merck & Co. for
allowing us to test a panel of HDAC inhibitors from its compound
collection.
ABBREVIATIONS
DNA, Deoxyribonucleic acid; HDAC, histone deacetylase; ZBG,
zinz-binding group; SAR, structure-activity relationship; hERG,
human Ether-a-go-go-Related Gene; HUVEC, Human Umbilical
Vein Endothelial Cell; Cbz, carboxybenzyl; HBTU, O-
(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
phosphate; DIPEA, N,N-diisopropylethylamine; TFA, trifluoroa-
cetic acid; EDC, N-ethyl-N’-(3-
dimethylaminopropyl)carbodiimide; HPLC, high performance
liquid chromatography. PdCl2(dppf), [1,1’-
Bis(diphenylphosphino)ferrocene]dichloropalladium (II).
hexafluoro-
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Discovery of a Selective Series of Inhibitors of
Plasmodium falciparum HDACs
.
Jesus M. Ontoria,*,† Giacomo Paonessa,† Simona Ponzi,† Federica Ferrigno,† Emanuela Nizi,† Ilaria
Biancofiore,† Savina Malancona,† Rita Graziani,† David Roberts,‡ Paul Willis,‡ Alberto Bresciani,†
Nadia Gennari,† Ottavia Cecchetti,† Edith Monteagudo,† Maria V. Orsale,† Maria Veneziano,† Annalise
Di Marco,† Antonella Cellucci,† Ralph Laufer,† Sergio Altamura,† Vincenzo Summa,† and Steven
Harper†
†Departments of Chemistry and Biology, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Rome, Italy.
‡Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland.
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