Discovery of a Biologically Active Bromodomain Inhibitor by Target-Directed Dynamic Combinatorial Chemistry

Article abstract of DOI:10.1021/acsmedchemlett.8b00247

Target-directed dynamic combinatorial chemistry (DCC) has emerged as a strategy for the identification of inhibitors of relevant therapeutic targets. In this contribution, we use this strategy for the identification of a high-affinity binder of a parasite

Full text of DOI:10.1021/acsmedchemlett.8b00247

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Letter
Discovery of a biologically active bromodomain inhibitor
by target-directed dynamic combinatorial chemistry
Paula García, Victoria L. Alonso, Esteban Serra, Andrea Marta Escalante, and Ricardo L. E. Furlan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00247 • Publication Date (Web): 11 Sep 2018
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Discovery of a biologically active bromodomain inhibitor by target-
directed dynamic combinatorial chemistry.
Paula García,# ‡ Victoria L. Alonso,#,¥ ‡ Esteban Serra,#,¥ Andrea M. Escalante,# and Ricardo L. E. Furꢀ
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lan# *
# Facultad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario, CONICET. Suipacha 531,
S2002LRK Rosario (Argentina).
¥ Instituto de Biología Molecular y Celular de Rosario (IBR). Universidad Nacional de Rosario, CONICET. Ocampo y Esꢀ
meralda, 2000 Rosario (Argentina).
Keywords: dynamic combinatorial libraries • target-directed chemistry • Trypanosoma cruzi • bromodomain inhibitor
Abstract: Targetꢀdirected dynamic combinatorial chemistry (DCC) has emerged as a strategy for the identification of inhibitors of
relevant therapeutic targets. In this contribution, we use this strategy for the identification of a highꢀaffinity binder of a parasite
target, the Trypanosoma cruzi bromodomainꢀcontaining protein TcBDF3. This protein is essential for viability of T. cruzi, the proꢀ
tozoan parasite that causes Chagas disease. A small dynamic library of acylhydrazones was prepared from aldehydes and acylhyꢀ
drazides at neutral pH in presence of aniline. The most amplified library member shows: (a) high affinity for the template, (b) interꢀ
esting antiparasitic activity against different parasite forms and (c) low toxicity against Vero cells. In addition, parasites are rescued
from the compound toxicity by TcBDF3 overexpression, suggesting that the toxicity of this compound is due to the TcBDF3 inhibiꢀ
tion i.e. the binding event that initially drives the molecular amplification, is reproduced in the parasite leading to selective toxicity.
The identification of smallꢀmolecule modulators of protein
function is a key activity in modern drug discovery and chemꢀ
ical biology. For targets with limited biostructural information
available, random hitꢀidentification strategies are frequently
used, wherein target affinity is measured to select the best
binder out of large compound libraries.[1] Target binding can
also be exploited for the molecular recognition of reactive
small molecule building blocks and for both the chemical
ligand assembly and the identification of highꢀaffinity building
block combinations integrating in this way chemical synthesis
and library screening.[2] When this ligand assembly is achieved
through dynamic covalent chemistry, reversibility allows
proofreading to increase the yield of good binders.[3,4]
One limitation of covalent DCC as a tool for the identificaꢀ
tion of drug discovery relevant ligands is the limited number
of reversible reactions that can be carried out under biomꢀ
acromoleculeꢀcompatible conditions and form bonds that are
stable under physiological conditions.[14] The exploitation of
hydrazones in biomacromolecule compatible DCC was preꢀ
cluded for more than a decade because of the required acid
conditions for their exchange.[12,13,15] The introduction of aniꢀ
line[16,17] and related amines as catalysts has paved the way
towards the few examples of biomacromolecule induced adapꢀ
tation of hydrazone based DCLs.[6,9,11,16,18] Another potential
limitation for the exploitation of DCC in drug discovery is the
unproductive ligand amplification, i.e. a concentration inꢀ
crease driven by nonꢀcovalent interactions with the template
that do not affect the biomolecule functionality. This type of
interaction cannot be detected by DCL composition analysis in
presence and in absence of the template. It requires a parallel
analysis of the effect of the DCL on the template.
Dynamic combinatorial chemistry (DCC) allows the combiꢀ
nation of building blocks through reversible reactions. Since
the product distribution of these dynamic combinatorial librarꢀ
ies (DCLs) is dictated by the stability of the library members,
it can adapt to environmental changes that affect such stability.
Therefore, appropriate analysis of a DCL response to a given
stimulus can give useful information about some properties of
library members.[5] Addition of a template molecule is a popuꢀ
lar strategy to induce composition changes in DCLs. It has
been used to discover synthetic receptors for guest templates
as well as ligands for biomacromolecular templates,[3] includꢀ
ing proteins such as enzymes,[6ꢀ10] lectins[11] and transportꢀ
ers.[12ꢀ13] Although some of these proteins are potential theraꢀ
peutic targets, examples of biologically active molecules reꢀ
sultant of template induced molecular amplification from
dynamic combinatorial libraries are very rare.[6]
Bromodomains are proteinꢀinteraction modules that bind to
acetylꢀlysine residues of histone and nonꢀhistone proteins. In
recent years, human bromodomains have become attractive
drug targets for the treatment of a variety of diseases.[19] The
inhibition of eukaryotic pathogens bromodomains is
considered a promising strategy to treat infections, even
though it continues to be almost unexplored.[20,21] Several
bromodomainꢀcontaining proteins have been identified in
different protozoan parasites. Since some of them are essential
for viability, they represent new opportunities for the discovꢀ
ery of antiparasitic drugs.[21ꢀ25] We have characterized three
bromodomainꢀcontaining coding sequences in Trypanosoma
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cruzi, the protozoan parasite that causes Chagas disease.[22ꢀ25]
TcBDF3 the effect of the DCL on some of the properties of the
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TcBDF3 is a rare cytoplasmic bromodomain containing proꢀ
tein that interacts with acetylated αꢀtubulin and is involved in
flagella morphogenesis and differentiation. These distinct
features makes it an interesting drug target against T.
cruzi.[23,24]
template was analyzed.
S
H
O
N
N
O
HO
A1B4
5
Here we report the preparation and TcBDF3ꢀinduced
adaptation of a DCL of hydrazones. The most amplified
library member binds to the acetylꢀlysine recognition pocket
of the template and shows high cytotoxicity against different
T. cruzi parasite forms. On the contrary, it shows low toxicity
against Vero cells and TcBDF3 overexpressing parasites.
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The library was prepared from a set of six aldehydes (A1ꢀ
A6) and four acylhydrazides (B1ꢀB4) (Figure 1). Their combiꢀ
nation can produce hydrazones that include, aromatic, aliphatꢀ
ic, polyhydroxylated and heterocyclic (including N, O or S)
moieties, with phenol, methoxyl, carboxyl and alcohol substitꢀ
uents as potential recognition groups. One or more of these
recognition groups are present in most of the reported comꢀ
pounds that possess affinity for different human bromoꢀ
domains.[26]
Figure 2. Amplification factors (AFs) observed for library
members in the presence of the template TcBDF3. AFs were
calculated by dividing the LCꢀMS peak area of each comꢀ
pound in the templated and untemplated libraries. Black: AF
higher than 2. Dark grey: AF between 1.5ꢀ2. Light grey: AF
lower than one.
TcBDF3 possesses a tryptophan residue (W117) placed in a
hydrophobic pocket that is the acetylꢀlysine binding site. The
intrinsic fluorescence of this aromatic residue can be affected
by compounds that enter the TcBDF3 hydrophobic pockꢀ
et.[20,24] In the presence of the DCL a significant decrease in
the TcBDF3 maximum intrinsic fluorescence was observed
(Figure 3A), suggesting that one or more library members
could enter the hydrophobic pocket. In order to support this
observation, the effect of the library was also measured with a
mutated version of TcBDF3 (TcBDF3m) that includes two
point mutations (Y123A and L130A). TcBDF3m was designed
to retain its secondary structure while losing its ability to bind
to its acetylated ligand in vitro.[24] Neither the maximum inꢀ
trinsic fluorescence of TcBDF3m was affected by the library
(Figure 3A) nor the A1B4 concentration was affected by the
presence of TcBDF3m (Figure 3B), suggesting a specific bindꢀ
ing of A1B4 to the binding pocket of the wild type protein.[29]
Figure 1. Building blocks used for DCL generation.
Aldehydes A1-A6 (75 ꢀM each) and acylhydrazides B1ꢀB4
(75 ꢀM each) were dissolved in ammonium acetate buffer
(100 mM, pH 6.5) in the presence of aniline (3.75 mM). After
10 hours of reaction, the system reached a constant composiꢀ
tion. LCꢀMS analysis showed the presence of thirty hydraꢀ
zones with unique molecular weights (Table S1). When the
dynamic library was exposed to recombinant TcBDF3 (100
ꢀM), a shift in the composition was observed. The templateꢀ
induced response of the DCL favored mainly the formation of
hydrazone A1B4 that showed a 2.4 fold increase in concentraꢀ
tion (Figure 2). Another five library members increased their
concentrations to a lesser extent, between 1.5 and 1.8 times,
whereas the rest of library members either increased slightly
or decreased their concentrations.
Figure 3. (A) Normalized intrinsic fluorescence of recomꢀ
binant TcBDF3 or TcBDF3m (10 µM) in ammonium acetate
buffer (100 mM, pH 6.5) in absence and in presence of the
DCL. (B) LCꢀMS peak area for A1B4 in the untemplated DCL
and in the DCLs in presence of TcBDF3 or TcBDF3m (100
µM).
A template induced molecular amplification in DCC is idealꢀ
ly driven by specific nonꢀcovalent interactions between the
amplified ligand and the template. However, different factors
can disrupt the correlation between amplification and affinity
of the amplified compound to the template.[27,28] In particular,
when a biomacromolecule is used as a template to drive ampliꢀ
fication of relatively small ligands, interactions between liꢀ
brary members with different regions of the template are posꢀ
sible, and some of those interactions may not affect the biologꢀ
ical function of the template biomolecule. To gain insight into
the type of interaction between the library members and
Hydrazone A1B4 was then prepared individually and its
interaction, as well as the interaction of each of its component
building blocks A1 and B4, with the template TcBDF3 were
evaluated by fluorescence quenching and by Differential
Scanning Fluorimetry (DSF) or Thermal Shift. Individually,
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To gain insight into the specificity of A1B4 for TcBDF3 in
building blocks A1 and B4 (100 ꢀM) did not produce any
detectable change in fluorescence. However, the maximum
intrinsic fluorescence of TcBDF3 decreased regularly in the
presence of increasing concentrations of A1B4 (Figure 4). The
fluorescence data were analyzed by SternꢀVolmer, modified
SternꢀVolmer and double logarithmic plots. The SternꢀVolmer
plot showed a negative deviation (towards the xꢀaxis) as preꢀ
viously reported for TcBDF3 with bromodomain inhibiꢀ
tors.[20,24] From the double logarithmic plot a dissociation
constant value of 1.7 ꢀM was obtained. In addition, binding to
A1B4 significantly increased the thermal stability of TcBDF3
with ꢁTm observed of 3.43°C whereas for TcBDF3m ꢁTm
was 0.4ºC, which further supports the binding of A1B4 to
TcBDF3 (Figure S8).
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vivo, the sensitivity of bromodomainꢀoverexpressing lines was
evaluated. T. cruzi epimastigotes overexpressing TcBDF3
through a tetracyclineꢀinduced plasmid were treated with
A1B4, at a concentration around its IC50 value, in the absence
and in the presence of tetracycline (Figure 5). Overexpression
of TcBDF3 completely rescued epimastigotes from the growth
inhibition produced by A1B4, suggesting that the toxicity of
this compound is due to the TcBDF3 inhibition.
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Figure 5. Relative growth of T. cruzi Dm28c epimastigotes
transfected with pTcINDEXGWꢀBDF3HA, uninduced (ꢀtet)
and induced (+tet) with Tetracycline (tet), untreated (control)
and treated with A1B4 (25 µM). The experiment was perꢀ
formed in triplicate and cell growth was determined after 72 h
of culture by counting viable forms. The values obtained were
normalized to the condition without compound (control). The
bar graph represents the mean SD; **P < 0.05 (unpaired, twoꢀ
tailed Student’s t test). In the experiment with tetracycline, the
observed difference in relative growth in absence (+tet conꢀ
trol) and in presence of inhibitor (+tet +A1B4) is not significaꢀ
tive.
Figure 4. Fluorescence spectra of TcBDF3 (10 ꢀM) alone and
with increasing amounts of A1B4. λex = 295 nm. FI, fluoresꢀ
cence intensity; A.U., arbitrary units.
To support these results, the modelled 3D structure of
TcBDF3 was used to perform docking predictions using the
Swissdock server.[30] The best prediction located the hydraꢀ
zone A1B4 inside the hydrophobic pocket of TcBDF3 (Figure
S9).
In view of the promising biophysical data and considering
the novelty of TcBDF3 as a therapeutic target for Chagas
disease, the effect of A1B4 on the different life cycle stages of
T. cruzi was evaluated in vitro. Amastigotes are present in the
mammalian host cells, whereas epimastigotes and the infective
metacyclic trypomastigotes are present in the gut of the insect
vector. The cytotoxicity was also studied in the Vero cell line
to evaluate selectivity. A1B4 shows a trypanocidal effect on
the three life cycle stages of T. cruzi with IC50 values between
13 and 23 µM (Table 1). Interestingly, its toxicity against
Vero Cells was low, with an IC50 value higher than 200 µM,
giving a selectivity index ≥ 9 depending on the parasite form.
In order to evaluate if amplification of A1B4 is somehow
related to the properties shown by this compound, a poorly
amplified compound was prepared and analyzed (A4B4).
Compared to A1B4, A4B4 was amplified in a lesser extent
(AF=1.21, Figure 2), produced a smaller change in the intrinꢀ
sic fluorescence and thermal shift of TcBDF3 in vitro, and
showed lower cytotoxicity against the parasite in vivo (Figure
S12).
In summary, a hydrazone based DCL was prepared under
reaction conditions that are compatible with the biologically
relevant template TcBDF3. When mixed, DCL and template
affected each other: the template induced changes in the relaꢀ
tive concentration of the library members, and the library
produced changes in the intrinsic fluorescence of a tryptophan
residue placed in the acetylꢀlysine recognition pocket of the
bromodomain. Blockage of the entrance to this pocket by
specific mutations deleted both effects. Binding of the most
amplified library member, A1B4, to the template TcBDF3 was
studied by fluorescence quenching and thermal shift observing
a Kd of 1.7 µM, slightly lower than the value for the best
TcBDF3 inhibitor reported.[20] The binding process observed
in vitro also seems to be possible in cell since the compound
A1B4 shows interesting toxicity activity against different
parasite forms but it is not toxic for other eukaryotic cells,
Table 1. Cytotoxicity of A1B4 on the different life cycle stages of
T. cruzi and Vero cells.
IC50 (ꢀM)[a,b] (SI[c]
)
compound
Epimastigotes
23 ± 3.8 (>9)
18.16±5.13 (2)
8 ± 3 (14)
Trypomastigotes
17.8±2.29 (>11)
27.07 ± 4.23 (1)
7 ± 2 (16)
Amastigotes
13.1±1.28(>15)
3.92±1.24 (8)
3 ± 1.5 (38)
Vero cells
A1B4
BZN[d]
NFX[e]
>200 ± 6
30.17±12
115 ± 12
[a] The results are averages of three separate determinations. [b] IC50 is the concenꢀ
tration required to give 50% inhibition, calculated by nonꢀlinear regression analysis from
betaꢀgalactosidase activity at the used concentrations (0ꢀ200 ꢁM). [c] Selectivity index
(SI) is the ratio between IC50 on Vero cells toxicity and IC50 activity of extracellular or
intracellular forms of the parasite. [d] MTT assay on Vero cells incubated 72 h with
BZN (0ꢀ250 ꢁM). [e] Values obtained from literature.[31] BZN, benznidazol; NFX,
nifurtimox (reference drugs for the treatment of Chagas disease).
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[8]
Soubhye, J.; Gelbcke, M.; Van Antwerpen, P.; Dufrasne,
such as Vero cells, or for parasites wherein TcBDF3 has been
overexpressed. Hydrazone A1B4 showed very interesting
antiparasitic activity and selectivity index.
F.; Boufadi, M. Y.; Nève, J.; Furtmüller, P. G.; Obinger, C.; Zouaoui
Boudjeltia, K.; Meyer, F. From Dynamic Combinatorial Chemistry to
in Vivo Evaluation of Reversible and Irreversible Myeloperoxidase
Inhibitors. ACS Med. Chem. Lett. 2017, 8, 206–210.
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These results illustrate how biologically active smallꢀ
molecules can be identified through a DCC casting approach
by using mild reaction conditions, biologically relevant temꢀ
plates and appropriate analysis of DCLꢀadaptation and DCLꢀ
effect on the template. In addition, the biophysical and biologꢀ
ical properties observed for compound A1B4 give relevance to
bromodomainꢀcontaining proteins as attractive targets for the
discovery of selective antiparasitic molecules.
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ime libraries addressing the neuronal GABA transporter GAT1.
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Supporting Information
TcBDF3 and TcBDF3m purification, Library preparation, Library
analysis, Synthesis and characterisation of A1B4 and A4B4.
Binding Experiments, Biological Activities and Docking predicꢀ
tion is available in Supporting Information.pdf file.
AUTHOR INFORMATION
Corresponding Author
[14] Miller. B. J. Dynamic covalent chemistry: Catalysing dyꢀ
namic libraries. Nat. Chem. 2010, 2, 433–434.
* Eꢀmail: rfurlan@fbioyf.unr.edu.ar.
[15] Hydrazone based libraries were preꢀequilibrated and neuꢀ
tralized, stopping the exchange, before addition of the biomolecular
template.
[16] Bhat, T.; Caniard, A. M.; Luksch, T.; Brenk, R.; Campopiꢀ
ano, D. J.; Greaney, M. F. Nucleophilic catalysis of acylhydrazone
equilibration for proteinꢀdirected dynamic covalent chemistry. Nat.
Chem. 2010, 2, 490–497.
[17] Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E.
Nucleophilic Catalysis of Hydrazone Formation and Transimination:ꢂ
Implications for Dynamic Covalent Chemistry. J. Am. Chem. Soc.
2006, 128, 15602–15603.
[18] Clipson, A. J.; Bhat, V. T.; McNae, I.; Caniard, A. M.;
Campopiano, D. J.; Greaney, M. F. Bivalent Enzyme Inhibitors Disꢀ
covered Using Dynamic Covalent Chemistry' Chemistry ꢀ A Europeꢀ
an Journal. Chem. Eur. J. 2012, 18, 10562–10570.
[19] Hewings, D. S.; Rooney, T. P. C.; Jennings, L. E.; Hay, D.
A.; Schofield, C. J.; Brennan, P. E.; Knapp, S.; Conway, S. J. Proꢀ
gress in the Development and Application of Small Molecule Inhibiꢀ
tors of Bromodomain–Acetylꢀlysine Interactions. J. Med. Chem.
2012, 55, 9393−9413.
[20] Ramallo, I. A.; Alonso, V. L.; Rua, F.; Serra, E.; Furlan, R.
L. E. A Bioactive Trypanosoma cruzi Bromodomain Inhibitor from
Chemically Engineered Extracts. ACS Comb. Sci. 2018, 20, 220−228.
[21] Jeffers, V.; Yang, C.; Huang, S.; Sullivan, W. J. Jr. Bromoꢀ
domains in Protozoan Parasites: Evolution, Function, and Opportuniꢀ
ties for Drug Development. Microbiol Mol Biol. 2017, 81, e00047ꢀ16,
DOI: 10.1128/MMBR.00047ꢀ16.
[22] Villanova, G. V.; Nardelli, S. C.; Cribb, P.; Magdaleno, A.;
Silber, A. M.; Motta, M. C. M.; Schenkman, S.; Serra, E. Trypanosoꢀ
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[23] Alonso, V. L.; Villanova, G. V.; Ritagliati, C.; Machado
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domain Factor 3 Binds Acetylated αꢀTubulin and Concentrates in the
Flagellum during Metacyclogenesis. Eukaryotic Cell 2014, 6, 822–
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Author Contributions
‡These authors contributed equally to this work.
Funding Sources
This work was supported by FONCYT (PICT2015ꢀ3574),
CONICET (PIP 695 and 797) and Universidad Nacional de Roꢀ
sario.
ACKNOWLEDGMENT
P.G and V.A. acknowledge to CONICET for her postdoctoral
fellowships. The authors thank CIBION for HRMS results.
ABBREVIATIONS
DCC, Dynamic combinatorial chemistry; DCL, Dynamic combiꢀ
natorial library; TcBDF3, Trypanosoma cruzi bromodomain.
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[29] The observed selectivity in amplification and binding for
A1B4 with TcBDF3 or TcBDF3m, and the following biological
results are particularly important taking in consideration that the
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Discovery of a biologically active bromodomain inhibitor by target-directed dynamic combinatorial chemistry.
Paula García, Victoria L. Alonso, Esteban Serra, Andrea M. Escalante, Ricardo L. E. Furlan
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