Novel Compounds Targeting the RNA-Binding Protein HuR. Structure-Based Design, Synthesis, and Interaction Studies

Article abstract of DOI:10.1021/acsmedchemlett.8b00600

The key role of RNA-binding proteins (RBPs) in regulating post-transcriptional processes and their involvement in several pathologies (i.e., cancer and neurodegeneration) have highlighted their potential as therapeutic targets. In this scenario, Embryonic Lethal Abnormal Vision (ELAV) or Hu proteins and their complexes with target mRNAs have been gaining growing attention. Compounds able to modulate the complex stability could constitute an innovative pharmacological strategy for the treatment of numerous diseases. Nevertheless, medicinal-chemistry efforts aimed at developing such compounds are still at an early stage. As part of our ongoing research in this field, we hereby present the rational design and synthesis of structurally novel HuR ligands, potentially acting as HuR-RNA interferers. The following assessment of the structural features of their interaction with HuR, combining saturation-transfer difference NMR and in silico studies, provides a guide for further research on the development of new effective interfering compounds of the HuR-RNA complex.

Full text of DOI:10.1021/acsmedchemlett.8b00600

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Letter
Novel compounds targeting the RNA-binding protein HuR.
Struc-ture-based design, synthesis and interaction studies.
Serena Della Volpe, Rita Nasti, Michele Queirolo, Muhammet Yagiz Unver, Varsha
R. Jumde, Alexander Dömling, Francesca Vasile, Donatella Potenza, Francesca
Alessandra Ambrosio, Giosuè Costa, Stefano Alcaro, Chiara Zucal, Alessandro
Provenzani, Marcello Di Giacomo, Daniela Rossi, Anna K. H. Hirsch, and Simona Collina
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00600 • Publication Date (Web): 21 Jan 2019
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Novel compounds targeting the RNA-binding protein HuR.
Structure-based design, synthesis and interaction studies.
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†,#
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‡,§
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Serena Della Volpe, Rita Nasti, Michele Queirolo, M. Yagiz Unver, Varsha K. Jumde, ,
∥
¶,
¶
◆
Alexander Dömling, Francesca Vasile, * Donatella Potenza, Francesca A. Ambrosio, Giosué
◆
◆
⊥
⊥
†
Costa, Stefano Alcaro, Chiara Zucal, Alessandro Provenzani, Marcello Di Giacomo, Daniela
Rossi, Anna K. H. Hirsch, * Simona Collina. *
†
‡,§,
†,
†Department of Drug Sciences, Medicinal Chemistry and Technology Section, University of Pavia, Via Taramelli 12, 27100,
Pavia, Italy
‡Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI),
Department of Drug Design and Optimization and Department of Pharmacy, Saarland University, Campus building E8.1,
6
6123, Saarbrücken, Germany
§Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, NL-9747 AG Groningen
∥Department of Drug Design, University of Groningen, A. Deusinglaan 1, Groningen, 9713 AV, Netherlands
¶Department of Chemistry, University of Milan, Via Golgi 19, 20133, Milano, Italy
◆Department of Health Sciences, University “Magna Græcia” of Catanzaro, Viale Europa, 88100, Catanzaro, Italy
⊥Department of CIBIO, University of Trento, Via Sommarive 9, 38123, Povo, TN, Italy
KEYWORDS. RNA-binding protein; HuR–RNA complexes; STD-NMR; Virtual screening; multi-component reactions.
ABSTRACT: The key role of RNA-binding proteins (RBPs) in regulating post-transcriptional processes and their involvement in
several pathologies (i.e., cancer and neurodegeneration) have highlighted their potential as therapeutic targets. In this scenario,
Embryonic Lethal Abnormal Vision (ELAV) or Hu proteins and their complexes with target mRNAs have been gaining growing
attention. Compounds able to modulate the complex stability could constitute an innovative pharmacological strategy for the treatment
of numerous diseases. Nevertheless, medicinal-chemistry efforts aimed at developing such compounds are still at an early stage. As
part of our ongoing research in this field, we hereby present the rational design and synthesis of structurally novel HuR ligands,
potentially acting as HuR–RNA interferers. The following assessment of the structural features of their interaction with HuR,
combining saturation-transfer difference NMR and in silico studies, provides a guide for further research on the development of new
effective interfering compounds of the HuR–RNA complex.
RNA is an important regulatory element of many cellular processes
and, thus, so-called RNA-binding proteins (RBPs), play a
prominent role in affecting the fate of target messenger RNAs
compounds able to bind HuR and to inhibit the formation of the
HuR–RNA complex may have anticancer properties. So far,
various high-throughput screening campaigns afforded several
mRNAs) coding for proteins pivotal in key cellular functions.1-5
9-12
(
natural products.
Therefore, a dysregulation of RBPs may be related to the
2
,4,6
pathogenesis of several diseases.
In 2018, the first candidate
After studying the concept of druggability of ELAV proteins and
1
3-15
drug targeting RBPs, called H3B-8800, reached the clinical phase
for the treatment of acute myelogenous leukemia and chronic
myelomonocytic leukemia. Among RBPs, the family of ELAV
related complexes with mRNA,
we analyzed the literature
concerning the main findings on the ELAV–RNA complexes from
a medicinal-chemistry standpoint and defined the interaction
7
1
6,17
(Embryonic Lethal Abnormal Vision) proteins is involved in
features of HuR and a small series of natural products.
In this
controlling the functional activities of diverse RNA populations. In
particular, HuR regulates splicing, stability and translation of
thousands of coding and non-coding RNAs and is therefore
letter, we report on the structure-based design and the synthesis of
compounds with different “core structures” and the investigation of
their interactions with HuR using a combination of saturation-
transfer difference (STD)-NMR and in silico studies. While on the
RNA side the interaction of HuR and RNA is mediated by AU-rich
elements (AREs), on the protein side two consecutive RNA
recognition motif-type (RRM1 and RRM2) domains are involved;
8
considered a valid drug target for anticancer therapy. HuR is a
nuclear protein but, upon cell stress such as DNA damage, it
shuttles into the cytoplasm where it regulates the fate of cargo
mRNAs and determines the abundance of the encoded proteins.
Over-expression of HuR is associated with tumor progression and
these interact directly with target RNAs through highly conserved
ribonucleoprotein (RNP1 and RNP2) sequences.
8
18,19
poor prognosis in various cancer types. For this reason,
For the design
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of new compounds, we focused on a pocket-like region hosting
complex with the ARE sequence of RNAc-fos (PDB code 4ED5),
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small HuR ligands, formed by the two asymmetric units of the
protein, which belong to RNP1 and RNP2 of RRM1. Inspection of
the cocrystal structure of the HuR RRM1 and RRM2 domains in
confirms that this region corresponds to binding site of RNA
uridine residues 8 and 9 (U8-U9)(Figure 1a).
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Figure 1. a) HuR–RNA contacts (PDB code 4ED5) for U8-U9. Red lines show side-chain contacts, green lines show main-chain contacts.
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Distances are expressed in Å; b) Modification applied to the anchor to increase the predicted affinity. Dashed blue rectangles indicate
hydrogen-bond acceptors and dashed green rectangles hydrogen-bond donors on both the original and modified anchor, while the dashed
orange circle shows the anchor modification potentially allowing for additional hydrophobic interactions with the selected Tyr63 residue.
We based our search for new scaffolds for HuR ligands on the key
The computationally predicted structures can be easily prepared by
interactions shown in Figure 1a and employed the free web-based
a short synthesis using MCR or equally efficient processes.
TM 21-24
virtual screening platform AnchorQuery
.
This program is
specifically designed for targeting protein–protein interactions
(PPIs) with small molecules by combining the anchor concept with
one-pot multicomponent reaction (MCR) chemistry. Briefly, PPI
inhibitors are characterized by specific moieties able to mimic
amino acid side chains of the donor protein, called “anchor motifs”.
Since the contact surfaces involved in PPIs are typically large and
flat, similar to protein–RNA interactions, we used NucleoQuery, a
TM
nucleoside derivative of the web application AnchorQuery for
the rational structure‐based design of HuR–RNA targeting
compounds. We selected U8 and U9 as anchors, and investigated
whether one of the two nucleotides may be a pharmacophore in
terms of occupied position and interactions. To increase the
probability of discovering new ligands, the anchors were modified
so as to bear an additional phenyl group to establish a π–π-stacking
interaction with Tyr63 (Figure 1b). As a result of four runs in
NucleoQuery, a large library (800 molecules) featuring a wide
range of structurally diverse derivatives was obtained. To select the
candidates for synthesis, we relied on visual inspection and
molecular-recognition studies. This way, we selected 17
compounds featuring piperidinones, aromatic heterocycles, N,N-
disubstituted amides and sulfonamides as scaffolds (see SI, S1.2).
For all compounds, the anchor shows good overlap with the
corresponding uracil ring, preserving the same hydrogen bonds
with Arg97 and Lys92, while the additional aromatic rings should
afford the desired interaction with Tyr63. Moreover, selected
compounds are engaged in additional interactions with the protein,
including hydrophobic interactions with residues Ile23, Asn25,
Phe65, Ile103, Ile133 (docking poses of exemplary compounds for
each scaffold can be found in SI, S1.3). Taking into account the
synthetic feasibility and commercial availability of the
corresponding starting materials, we selected the most promising
scaffolds and replaced the uracil moiety withan easy-to-handle
starting material. We evaluated several hydrophobic/aromatic
portions as anchors, potentially able to interact with HuR in the
position occupied by U8 and preserving its main contacts. Thus, we
designed and synthesized compounds 1–4 (Figure 2), characterized
by different scaffolds.
Figure 2. Structures of the four compounds selected for the
experimental study. The modified anchors are highlighted in light
blue. The different scaffolds (piperidinones, heterocycles, N,N-
disubstituted amides) are highlighted in bold.
Thus, compound 1 was synthesized by a Castagnoli-Cushman
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reaction (CC-3CR) followed by amidation; 2 by a Groebke-
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Blackburn-Bienaymé (GBB-2CR); 3 by a Van Leusen reaction
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(vL-3CR), and 4 by a reductive amination followed by
acylation. Herein, we will briefly describe the synthetic strategies
adopted to obtain compounds 1–4. Further details on protocols
employed and compound characterization are reported in the
supporting information (SI, S2).
Amidation of the acid intermediate trans (±)-9 with amine 10
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afforded compound (±)-1 as racemate. The key intermediate trans
-oxopiperidine-3-carboxylic acid (9) was obtained in goodyield
and diastereoselectivity, applying a microwave-aided one-pot CC-
CR on benzaldehyde (6), isobutylamine (7), and glutaric
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anhydride (8) (Scheme 1a). A microwave-assisted GBB-2CR,
involving isocyanide 12, aldehyde 6 and amino-thiazole 13
produced compound 2. Isocyanide 12 was obtained starting from
amine 11, which was converted into the corresponding formamide
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and subsequently dehydrated (Scheme 1b). A vL-3CR27 on
The interaction of compounds 1, 3 and 4 with HuR was studied by
STD-NMR spectroscopy. This technique, based on the nuclear
Overhauser effect, is well-established epitope-mapping
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commercially available aldehyde 18, isobutylamine (7) and
substituted TosMIC derivative 17 led to compound 3. This MCR
allows to access functionally rich imidazoles in a single pot via
cycloaddition of TosMIC reagents on imines, generated in situ
from an aldehyde and an amine under mildly basic conditions. We
synthesized 17 starting from p-toluenesulfinic acid (14), which
afforded the intermediate formamide derivative upon reaction with
commercially available 3-phenoxybenzaldehyde (15), formamide
(16) and chlorotrimethylsilane; subsequent dehydration furnished
a
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methodology for studying the target–ligand interactions. Briefly,
the method relies on the selective irradiation of the protein, which
allows magnetization to be transferred to the bound ligand; the
observation of the ligand signals in the NMR spectrum provides an
indication of the interaction. Those ligand protons that are nearest
to the protein are more likely to become highly saturated, and
therefore show the strongest signal in the mono-dimensional STD
spectrum. Therefore, the intensity of the STD signal (expressed as
absolute STD percentage) reflects the proximity of the ligand to the
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isocyanide 17 (Scheme 1c). Compound 4 was obtained by
reductive amination of aldehyde 20 followed by an amide coupling
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with acid 23 followed by removal of the THP protecting groups
protein surface.
The group epitope mapping illustrates which
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(
Scheme 1d). Prior to the interaction study, compounds 1–3 were
chemical moieties of the ligand are key for molecular recognition
in the binding site. The analysis of STD data shows that compounds
1 (Figure S3.2.1 and Table S3.2.1), 3 (Figure S3.2.2 and Table
S3.2.2), and 4 (Figure 3 and Table S3.2.3) interact with the protein.
converted into the corresponding hydrochloride salts, and their
solubility in both the buffer and time-ranges required for STD-
NMR experiments was evaluated. Under these conditions,
compound 2 proved to be poorly soluble and had to be excluded
from the interaction study.
Figure 3. STD-NMR spectrum (bottom) and 1H-NMR (top) of
compound 4. The strong interaction observed for aromatic rings A,
B and C is evidenced.
For ligand 1, the isopropyl group shows the strongest interaction
(0.40 STD %), while the STD signal related to ring B is the least
intense (0.03 STD %); ring A contributes to the interaction (0.20
STD %), while the protons of the amino chain (H14, H15 and H16)
do not give STD signals. The binding of compound 3 to HuR is
mediated by the pyridone anchor (1.20 STD %) and by the
isopropyl moiety (0.30 STD %). For compound 4, we observed
STD signals of similar intensity for the three aromatic rings (0.40
% for ring A, 0.36 % for ring B and 0.25% for ring C, respectively),
suggesting that they are positioned within a protein pocket.
In parallel, we performed docking studies on the crystal structure
of HuR RRM1-2 domains according to the approach we
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published. We used STD-NMR in combination with in silico
studies (applied to the “closed” HuR conformations, see SI) to
enable a more detailed description of the ligand–protein behavior
in solution. Molecular docking simulations were performed to
elucidate the binding mode and the interactions between
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compounds 1, 3, and 4 with HuR protein, using Maestro tools .
We observed that the anchor moiety of each compound is predicted
to be superimposed with U8, establishing pivotal interactions with
the HuR protein. The interactions of compounds 1 and 3 with HuR
is described in the SI (Section S4.3, Figures S4.3.1 and S4.3.2). As
for compound 4, we report strong hydrophobic interactions for all
aromatic rings as evidenced by STD data. The amide carbonyl
group establishes double hydrogen bonds with Arg97 and
hydrophobic interactions with the same residue and Ile103 (Figure
Scheme 1. Synthetic pathways of compounds 1–4. Reagents and
conditions: a) Compound (±)-1: i) 6, 7, HCOOH, ACN, mw
1
20°C, 50 W, 30 min; ii) 8, p-xylene, reflux, 10 h, yield 62%; iii)
TBTU, DIPEA, THF, rt, 12 h, yield 71%. b) Compound 2: i) Ethyl
formiate, 10h; ii) TEA, POCl , DCM, rt, 4 h, yield 62%; iii) 6, 13,
HCOOH, ACN, mw 120°C, 30 min; iv) 12, ZrCl , 80 °C, 10 h,
yield 21%. c) Compound 3: i) H O, 30 min; ii) HCl, MTBE, 30
min; iii) 15, 16 Me SiCl, ACN, Toluene, N , 50 °C, 5 h; iv) 14, 16
h, yield 62%; v) POCl , triethylamine, THF, 10°C, 45 min, yield
5%; vi) 18, 7 dry DMF, rt, 4 h; vii) 17, K CO , rt, 72 h, yield 15%.
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). Regarding ring B, it establishes a π–π-stacking interaction with
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Phe65 and hydrophobic interactions with Arg97, Pro98 and Phe65.
Ring C is involved in a double hydrogen bond with Arg153 and
Ile103 and different hydrophobic interactions. The zoomed-in view
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in Figure 4 highlights the full overlap of U8 and the anchor motif
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(ring A) of compound 4.
*
*
S.C. e-mail: simona.collina@unipv.it
F.V. e-mail: francesca.vasile@unimi.it
*A.K.H.H. email: Anna.Hirsch@helmholtz-hzi.de
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
#
manuscript. S.D.V and R.N. contributed equally to the work.
Present Addresses
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R. N.: Department of Environmental Science and Policy (ESP),
University of Milan, 20133 Milano-Italy M.Q.: Agro business
Park 10, 6708 PW Wageningen, The Netherlands
Funding Sources
A.K.H.H. gratefully acknowledges funding from the Helmholtz-
Association’s Initiative and Networking Fund. A.P. acknowledges
Associazione Italiana per la Ricerca sul Cancro (AIRC) [21548]
Figure 4: 3D representation of HuR–4 interactions. Hydrogen
bonds and hydrophobic interactions are displayed as yellow and
dark dashed lines, respectively; a π–π-stacking interaction is shown
as a blue dashed line. RNA and HuR are represented as a brown
and yellow cartoon, respectively. Compound 4 is shown in green
sticks.
ACKNOWLEDGMENT
F.V. and S.C. acknowledge Federico Sala for his collaboration in
the NMR laboratory. S.D.V. and S.C. thankfully recognize Scuola
di Alta Formazione Dottorale (Pavia University) for the mobility
research scholarship. R.N. and M.Q. acknowledge the Erasmus
Traineeship program for the experience opportunity at the Stratingh
Institute for Chemistry, University of Groningen.
In this letter, we report for the first time on the structure-based
design and synthesis of potential HuR ligands. The structure-based
design strategy used in the present study enabled the identification
of three novel compounds that interact with HuR, establishing
contacts with the RNP regions of RRM1 and RRM2 domains of the
protein. Targeting this region is pivotal for the inhibition of the
protein activity and so the identification of compounds able to bind
to this site plays a critical role in the development of new potential
HuR inhibitors. Biophysical experiments (STD-NMR) are in
agreement with computational results, confirming that the designed
compounds 1, 3, and 4 are indeed binders of HuR. Furthermore,
theoretical experiments carried out by molecular dynamics and
docking studies confirm that compound 4 is the best binder of HuR
protein, in terms of theoretical binding affinity (SI, S4.2). The
identified hit compounds will be be further optimized in
forthcoming studies, building an in silico focused library. Our
findings represent a step forward in discovering novel compounds
potentially useful to counteract several pathologies such as cancer,
inflammation, and neurodegeneration, in which ELAV–RNA
complexes play a pivotal role.
ABBREVIATIONS
ELAV, Embryonic lethal abnormal vision; RBP, RNA-binding
protein; STD-NMR, saturation transfer difference-NMR; RRM,
RNA recognition motif-type; RNP, ribonucleoprotein; MCR,
multi-component reaction; CC-3CR, Castagnoli-Cushman
reaction; GBB-2CR, Groebke-Blackburn-Bienaymé reaction, vL-
3CR, Van Leusen reaction.
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1) http://anchorquery.csb.pitt.edu/
2) Koes, D.; Khoury, K.; Huang, Y.; Wang, W.; Bista, M.;
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Figure 1. a) HuR–RNA contacts (PDB code 4ED5) for U8-U9. Red lines show side-chain contacts, green lines
show main-chain contacts. Distances are expressed in Å;20 b) Modification applied to the anchor to increase
the predicted affinity. Dashed blue rectangles indicate hydrogen-bond acceptors and dashed green
rectangles hydrogen-bond donors on both the original and modified anchor, while the dashed orange circle
shows the anchor modification potentially allowing for additional hydrophobic interactions with the selected
Tyr63 residue.
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Figure 2. Structures of the four compounds selected for the experimental study. The modified anchors are
highlighted in light blue. The different scaffolds (piperidinones, heterocycles, N,N-disubstituted amides) are
highlighted in bold.
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Figure 3. STD-NMR spectrum (bottom) and 1H-NMR (top) of compound 4. The strong interaction observed
for aromatic rings A, B and C is evidenced.
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Figure 4: 3D representation of HuR–4 interactions. Hydrogen bonds and hydrophobic interactions are
displayed as yellow and dark dashed lines, respectively; a π–π-stacking interaction is shown as a blue
dashed line. RNA and HuR are represented as a brown and yellow cartoon, respectively. Compound 4 is
shown in green sticks.
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Scheme 1. Synthetic pathways of compounds 1–4. Reagents and conditions: a) Compound (±)-1: i) 6, 7,
HCOOH, ACN, mw 120°C, 50 W, 30 min; ii) 8, p-xylene, reflux, 10 h, yield 62%; iii) TBTU, DIPEA, THF, rt,
1
2 h, yield 71%. b) Compound 2: i) Ethyl formiate, 10h; ii) TEA, POCl3, DCM, rt, 4 h, yield 62%; iii) 6, 13,
HCOOH, ACN, mw 120°C, 30 min; iv) 12, ZrCl4, 80 °C, 10 h, yield 21%. c) Compound 3: i) H2O, 30 min; ii)
HCl, MTBE, 30 min; iii) 15, 16 Me3SiCl, ACN, Toluene, N2, 50 °C, 5 h; iv) 14, 16 h, yield 62%; v) POCl3,
triethylamine, THF, 10°C, 45 min, yield 85%; vi) 18, 7 dry DMF, rt, 4 h; vii) 17, K2CO3, rt, 72 h, yield 15%.
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table of content
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