Interfering with HuR-RNA Interaction: Design, Synthesis and Biological Characterization of Tanshinone Mimics as Novel, Effective HuR Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01176

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone

Full text of DOI:10.1021/acs.jmedchem.7b01176

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Article
Interfering with HuR-RNA Interaction: Design, Synthesis and Biological
Characterization of Tanshinone Mimics as Novel, Effective HuR Inhibitors
Leonardo Manzoni, Chiara Zucal, Danilo Di Maio, Vito G. D'Agostino, Natthakan
Thongon, Isabelle Bonomo, Preet Lal, Marco Miceli, Vanessa Baj, Marta Brambilla,
Linda Cerofolini, Saioa Elezgarai, Emiliano Biasini, Claudio Luchinat, Ettore Novellino,
Marco Fragai, Luciana Marinelli, Alessandro Provenzani, and Pierfausto Seneci
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01176 • Publication Date (Web): 09 Jan 2018
Downloaded from http://pubs.acs.org on January 10, 2018
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Interfering with HuR-RNA Interaction: Design, Synthesis and
Biological Characterization of Tanshinone Mimics as Novel,
Effective HuR Inhibitors
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Leonardo Manzoni, Chiara Zucal, Danilo Di Maio, Vito G.
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D’Agostino, Natthakan Thongon, Isabelle Bonomo, Preet Lal, Marco
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Miceli, Vanessa Baj, Marta Brambilla, Linda Cerofolini, Saioa
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Elezgarai, Emiliano Biasini, Claudio Luchinat, Ettore Novellino, Marco
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8*
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Fragai, Luciana Marinelli, Alessandro Provenzani, and
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*
Pierfausto Seneci
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Institute of Molecular Science and Technology (ISTM), CNR, Via Golgi 19, 20133 Milan, Italy
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Povo (TN),
2
Italy
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Scuola Normale Superiore, Piazza dei Cavalieri 7, Iꢀ56126 Pisa, Italy.
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Chemistry Department, University of Milan, Via Golgi 19, 20133 Milan, Italy
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Consorzio Interuniversitario di Risonanze Magnetiche di Metallo Proteine (CIRMMP), Via L.
Sacconi 6, 50019 Sesto Fiorentino (FI), Italy
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CERM and Chemistry Department, University of Florence, Via della Lastruccia 3ꢀ13, 50019 Sesto
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Fiorentino (FI), Italy
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Istituto di Ricerche Farmacologiche Mario Negri, Milan, 20156, Italy
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Pharmacy Department, University of Naples, Via Montesano 49, 80131 Naples, Italy
*
To whom correspondence should be addressed:
Pierfausto Seneci Tel: +39 02ꢀ50314060; Fax: +39 02ꢀ50314074: pierfausto.seneci@unimi.it
Correspondence may also be addressed to:
Luciana Marinelli Tel: +39 081ꢀ679899; Fax: +39 081 676569; lmarinel@unina.it
Alessandro Provenzani Tel: +390461283176; Fax: +390461283239; alessandro.provenzani@unitn.it
+ The authors wish it to be known that, in their opinion, the first 3 authors should be regarded as joint
First Authors
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ABSTRACT:
The human antigen R (HuR) is an RNAꢀbinding protein known to modulate the expression of
target mRNA coding for proteins involved in inflammation, tumorigenesis and stress responses and is a
valuable drug target. We previously found that DihydrotanshinoneꢀI (DHTS, 1) prevents the
association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS
structure, we designed and synthesized an array of orthoꢀquinones (tanshinone mimics) using a
functionꢀoriented synthetic approach. Among others, compound 6a and 6n turned out to be more
effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined
approach of NMR titration and Molecular Dynamics (MD) simulations suggests that 6a stabilizes HuR
in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNAꢀ
Electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the
first time, the generation of structure activity relationships (SARs), thus providing a solid background
for the generation of highly effective HuR disruptors.
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INTRODUCTION
The Human antigen R (HuR), also known as HuA or ELAVL1, is an ubiquitously expressed
RNA binding protein that binds preferentially to adenineꢀ and uridineꢀrich elements (ARE) of target
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coding and nonꢀcoding RNAs. HuR is primarily localized in the nucleus, where it exerts postꢀ
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transcriptional functions such as splicing and alternative polyadenylation, although it shuttles to the
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cytoplasm carrying the targeted RNA to be spatioꢀtemporally regulated in translation and stability. As
a stressꢀresponse protein, HuR modulates the expression of target mRNA (containing AREs
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preferentially in their 3’UTR) coding for proteins involved in inflammation, cell division,
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tumorigenesis,
angiogenesis,
metastasis, senescence, apoptosis,
immune
or stress
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responses. The importance of HuR in inflammation and cancer has encouraged the research for
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inhibitors/modulators to interfere with its biological activity. Several compounds have been named as
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HuR disruptors, i.e. molecules that can inhibit the HuRꢀRNA complex formation.
For a detailed
description of the known inhibitors and theirs properties, an exhaustive perspective has been recently
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published. However, neither systematic Structure Activity Relationships (SARs) studies, nor
chemical synthesis of novel families of HuR inhibitors have been reported so far. From a structural
point of view, rational design of HuR inhibitors is rather challenging due to the protein conformational
32–34
plasticity.
Moreover, HuR switches between at least two conformations upon binding/unbinding of
its RNA substrate: an “open” (apo) conformation, which is characterized by almost no contacts
between its first two RNA recognition motif (RRM) domains, and a “closed” (holo) conformation,
which is instead characterized by a few interꢀresidue contacts between the RRM domains.
Recently, as a result of a high throughput screening on a set of antiꢀinflammatory agents, we
identified 1 (DihydrotanshinoneꢀI, DHTS, Figure 1), a low molecular weight natural product able to
27
interact with HuR, thus affecting its postꢀtrascriptional functions. Compound 1 is a major component
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of extracts from Danshen (Salvia miltiorrhiza) used in traditional Chinese medicine as a treatment for
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inflammation, cardiovascular and cerebrovascular diseases. Our detailed in vitro and in vivo
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characterization of 1 showed the HuR dependency of its mechanism of action and its potency on
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cancerꢀlinked HuRꢀmRNA interactions. Naturally occurring tanshinones 2ꢀ4 (Figure 1) were tested as
HuR inhibitors, observing a preference for an aryl condensed (compounds 1ꢀ2) vs. saturated D rings
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(compounds 3ꢀ4).
Figure 1. Naturally occurring tanshinones 1-4.
Structural complexity has long hindered the synthetic exploitation of natural products as drugꢀ
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oriented chemotypes. However, molecular editing through diverted total synthesis and functionꢀ
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oriented synthesis (FOS) are synthetic paths that help to transform a natural product to a simpler,
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equally active synthetic analogue.
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We applied a FOS approach to 1, starting from the bicyclic AꢀB scaffold 5 (Figure 2). It
contains the oꢀquinone group and a pyrrole A ring, to provide novel, 1ꢀinspired, synthetic tanshinone
mimics bearing R ꢀR substitutions. Here the synthesis of a small library of tanshinone mimics 6a-t,
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bearing substitutions in positions 1, 3, 6 and 7, is reported. Tanshinone mimics were tested for
inhibition of HuRꢀRNA interactions and SARs were established. The most potent HuR inhibitor 6a
(
Figure 2) was further characterized in a panel of in vitro and cellular assays and showed a direct K of
D
4.5 µM to HuR. The molecular interaction of 6a with HuR, and with the HuRꢀmRNA complex was
also elucidated via a combined approach of NMR and computational studies and grounded the path for
the next generation of HuR inhibitors.
RESULTS AND DISCUSSION
Synthesis
Retrosynthesis. A FOSꢀbased approach to natural products analogues entails the design of an
uncomplicated synthetic strategy towards equally active, significantly simpler compounds. We built
our strategy around a B ringꢀlike orthoquinone and we opted for a substituted, Nꢀsulfonylatedbicyclic
AꢀB scaffold 6 as a functionꢀoriented replacement for the tanshinone AꢀD ring system. The furanꢀ
pyrrole A ring switch was meant to provide HuR inhibitionꢀinspired novelty, as the Nꢀsubstituted
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indole MPT0B098 (7, Figure 2) is a negative modulator of HuR. This compound bears a substituted
sulfonamide in position 1, that was introduced on our A ring (scaffold 6) to increase potency and
further diversify our mimics from naturally occurring tanshinones.
We reasoned that a preliminary SAR around positions 1, 3, 6 and 7 on scaffold 6 could be
established by exploiting Nꢀsulfonylations (functionalization of Nꢀ1, ꢀSO R ), Suzuki couplings
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(
functionalization of Cꢀ3, ꢀR ), radical CH functionalizations and Michael additions (functionalization
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of Cꢀ7, ꢀR ) and DielsꢀAlder cycloadditions (functionalization of Cꢀ6 and Cꢀ7, ꢀcycloR ꢀR , Figure 2).
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Figure 2. Tanshinone mimics as HuR inhibitors: core scaffold, functionꢀoriented synthesis,
active analogues.
As to synthetic targets 6 (R , R , R ≠ H, or R ꢀ R ≠ H, Scheme 1), we envisaged that
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functionalization of Cꢀ6 and/or Cꢀ7 could be carried out on 1,3ꢀdisubstituted indoleꢀ4,5ꢀdiones 6 (R3,
R = H). Such compounds could be made by Oꢀdemethylation and oxidation of 1,3ꢀdisubstituted 5ꢀ
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methoxyindoles 9. Compounds 9 could be prepared by 3ꢀbromination of commercially available 5ꢀ
methoxyindole 8, followed by Nꢀsulfonylation and Suzuki coupling (Scheme 1).
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Scheme 1. Retrosynthetic analysis to tanshinone mimics 6.
1-Alkyl/Arylsulfonyl-3-Aryl Indole-4,5-Diones 6a-6j. The retrosynthetic scheme leading to 1,3ꢀ
disubstituted indoleꢀ4,5ꢀdiones 6 (R , R ≠ H, R = R = H, Figure 1) was validated by synthesizing 1ꢀ
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phenylsulfonylꢀ3ꢀphenyl indoleꢀ4,5ꢀdione 6a (Scheme 2). 5ꢀMethoxyindole 8 was brominated in
position 3 (step a, compound 10) and treated with phenylsulfonyl chloride (step b). 3ꢀBromo
phenylsulfonamide 11 was reacted with phenyl boronic acid in a Suzuki coupling (step c) to provide,
after careful optimization of the experimental protocol, 1ꢀphenylsulfonylꢀ3ꢀphenylꢀ5ꢀmethoxyindole
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a. Demethylation (step d, compound 12a) and oxidation with IBX (step e) led to 1ꢀphenylsulfonylꢀ
3ꢀphenyl indoleꢀ4,5ꢀdione 6a (Scheme 2) with an overall ≈35% yield.
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ꢀBromo phenylsulfonamide 11 was reacted with o- (R = oꢀMePh, 9b) and pꢀsubstituted
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methylphenyl indoleꢀ4,5ꢀdione 6b and 1ꢀphenylsulfonylꢀ3ꢀpꢀdimethylaminophenyl indoleꢀ4,5ꢀdione 6c
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(
steps d,e, Scheme 2) as reported for 6a.
Scheme 2. Synthesis of 1ꢀphenylsulfonylꢀ3ꢀaryl indoleꢀ4,5ꢀdiones 6a-c.
+
ꢀ
Reagents and conditions: (a) Br , DMF, rt, 24 hrs, 74%; (b) PhSO Cl, nꢀBu N HSO , aqueous 50% KOH, CH Cl ,
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rt, 3 hrs, 90%; (c) arylboronic acid, Pd(PPh ) , dry DME/EtOH 4/1, N atmosphere, rt, reflux, 8 hrs, 83ꢀ92%; (d) 1M BBr
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in dry CH Cl , N atmosphere, ꢀ78°C to 5°C, 87ꢀ99%; (e) IBX, EtOAc (40°C) or DMF (rt), 2 to 24 hrs, 87ꢀ96%.
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The synthesis of pꢀsubstituted (R = pꢀOMe, 9d) and mꢀsubstituted (R = mꢀOMe, 9e) aryl
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ethers (Scheme 3) required demethylation of the 5ꢀmethoxy group on 3ꢀbromo phenylsulfonamide 11
step a) before Suzuki coupling (step b) and IBX oxidation (step c, Scheme 3) to avoid undesired
demethylation of the 3ꢀmꢀ or pꢀmethoxyphenyl group.
(
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Scheme 3. Synthesis of 1ꢀphenylsulfonylꢀ3ꢀmethoxyphenyl indoleꢀ4,5ꢀdiones 6d,e.
Reagents and conditions: (a) 1M BBr in dry CH Cl , N atmosphere, ꢀ78°C to 5°C, 86%; (b)
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methoxyphenylboronic acid, Pd(PPh ) , dry DME/EtOH 4/1, N atmosphere, rt, reflux, 8 hrs, 79ꢀ85%; (c) IBX, DMF, rt, 6
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to 48 hrs, 67ꢀ87%.
We attempted the synthesis of 1ꢀalkylsulfonyl or 1ꢀm/pꢀsubstituted arylsulfonylꢀ3ꢀphenyl
indoleꢀ4,5ꢀdiones 6f-l by replacing phenylsulfonyl chloride with alkylꢀ or m/pꢀarylsulfonyl chlorides
(
Scheme 2). Unfortunately, the Suzuki coupling protocol optimized for the synthesis of 9a was not
applicable as such to other sulfonamides. Thus, we synthesized compounds 6f-i,k,l according to the
longer, more efficient strategy depicted in Scheme 4.
1
ꢀPhenylsulfonylꢀ3ꢀphenylꢀ5ꢀmethoxyindole 9a was deꢀsulfonylated (step a, compound 14) and
treated with arylꢀ (step b) or alkylsulfonamides (step b’) to provide 1ꢀm/pꢀsubstituted arylsulfonylꢀ and
1ꢀmethylsulfonylꢀ3ꢀphenylꢀ5ꢀmethoxy indoles (respectively 9f-i and 9l) in good to excellent yields. 1ꢀ
pꢀNitrophenylsulfonylꢀ3ꢀphenylꢀ5ꢀmethoxyindole 9f was reduced (step c, amine 9j) and acetylated to
1
ꢀpꢀacetamidophenylsulfonylꢀ3ꢀphenylꢀ5ꢀmethoxyindole 9k (step d). Conversion of aryl ethers 9f-i,k,l
into 1ꢀm/pꢀsubstituted arylsulfonylꢀ or 1ꢀalkylsulfonylꢀ3ꢀphenyl indoleꢀ4,5ꢀdiones 6f-i,k,l (steps e and
f, Scheme 4) was carried out as previously described for 6a.
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Scheme 4. Synthesis of 1ꢀalkyl/arylsulfonylꢀ3ꢀphenyl indoleꢀ4,5ꢀdiones 6f-i,k,l.
+
ꢀ
Reagents and conditions: (a) Aqueous 3M NaOH, 2/1 MeOH/THF, 80°C, 2 hrs, 98%; (b) R SO Cl, nꢀBu N HSO ,
1
2
4
4
50% KOH, CH Cl , rt, 3 hrs, 87ꢀ92%; (b’) NaH, mesyl chloride, dry DMF, N atmosphere, 0°C to rt, 3 hrs, 59%; (c);
2
2
2
SnCl .2H O, 1/1 THF/MeOH, 80°C, 2hrs, 95%; (d); Ac O, pyridine, CH Cl , rt, 22 hrs, 82%; (e) 1M BBr in dry CH Cl ,
2
2
2
2
2
3
2
2
N atmosphere, ꢀ78°C to 5°C, 73ꢀ99%; (f) IBX, EtOAc (40°C) or DMF (rt), 2 to 24 hrs, 87ꢀ96%.
2
1
-Phenylsulfonyl-3-Phenyl-7-Thioaryl Indole-4,5-Diones 6m-6o. The retrosynthetic scheme
leading to 1,3,7ꢀtrisubstituted indoleꢀ4,5ꢀdiones 6 (R , R ≠ H, R = SꢀAr, R = H, Figure 2) was
1
2
3
4
validated by synthesizing 1ꢀphenylsulfonylꢀ3ꢀphenylꢀ7ꢀthiophenylindoleꢀ4,5ꢀdione 6m (Scheme 5) via
4
1
Michael addition of substituted benzenethiols on oꢀquinones. Namely, 1ꢀphenylsulfonylꢀ3ꢀphenyl
indoleꢀ4,5ꢀdione 6a was treated with thiophenol (step a), providing 1ꢀphenylsulfonylꢀ3ꢀphenylꢀ7ꢀ
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thiophenylindoleꢀ4,5ꢀdione 6m after oxidation of the reduced form (step b, Scheme 5) in moderate
yield after extensive optimization. The optimized experimental protocol was used with pꢀ
methoxybenzenethiol (6n) and pꢀcarboxymethylbenzenethiol (6o), observing moderate two step yields
for both quinones.
0
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Scheme 5. Synthesis of 1ꢀphenylsulfonylꢀ3ꢀphenylꢀ7ꢀthioaryl indoleꢀ4,5ꢀdiones 6m-6o.
Reagents and conditions: (a) aryl thiol, DMF, 2ꢀ3 hrs, rt, 62ꢀ88%; (b) IBX, DMF, 2 hrs, rt, 52ꢀ56%.
1-Phenylsulfonyl-3-Phenyl-7-Aryl Indole-4,5-Diones 6p-6t. The retrosynthetic scheme leading
to 1,3,7ꢀtrisubstituted indoleꢀ4,5ꢀdiones 6 (R , R , R ≠ H, R = H, Figure 1) was validated by
1
2
3
4
synthesizing 1ꢀphenylsulfonylꢀ3,7ꢀdiphenylindoleꢀ4,5ꢀdione 6p (Scheme 6) via Mn(III)ꢀmediated
4
0,42
radical addition of boronic acids.
1ꢀPhenylsulfonylꢀ3ꢀphenyl indoleꢀ4,5ꢀdione 6a was treated with
phenylboronic acid and Mn(OAc) (step a), providing 1ꢀphenylsulfonylꢀ3,7ꢀdiphenylindoleꢀ4,5ꢀdione
3
6
p (Scheme 6). The experimental protocol required extensive optimization, and a moderate yield was
finally obtained. The optimized experimental protocol was then used with arylꢀ (6q-s) and alkylboronic
acids (6t), adapting the reaction time to each substrate (Scheme 5) and observing poor to moderate
reaction yields.
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Scheme 6. Synthesis of 1ꢀphenylsulfonylꢀ3ꢀphenylꢀ7ꢀaryl indoleꢀ4,5ꢀdiones 6p-6t.
Reagents and conditions: (a) boronic acid, Mn(OAc) , 1,2ꢀdichloroethane, 80°C, 30 to 150 minutes, 14ꢀ36%.
3
Diels-Alder Cycloadducts 6u-6w. Validation of the retrosynthetic scheme to 1,3,6,7ꢀ
tetrasubstituted indoleꢀ4,5ꢀdiones 6 (R , R ≠ H, cyclo R R , Figure 1) targeted 1ꢀphenylꢀ3ꢀ
1
2
3
4
phenylsulfonylꢀ6ꢀmethylphenantro[1,2ꢀb]pyrroleꢀ10,11ꢀdione 6v. We envisaged a DielsꢀAlder
cycloaddition between 1ꢀphenylsulfonylꢀ3ꢀphenyl indoleꢀ4,5ꢀdione 6a and 6ꢀmethylꢀ1ꢀ
vinylcyclohexene 15a, followed by DDQ dehydrogenation/aromatization of tetrahydrocycloadduct 6u
4
3
to aromatic 6v (Scheme 7). Unfortunately, we could not obtain pure diene 15a in reasonable amounts
43
following the published synthetic procedure.
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Scheme 7. Attempted synthesis of 6,7,8,9ꢀtetrahydroꢀ1ꢀphenylꢀ3ꢀphenylsulfonylꢀ
phenanthro[1,2ꢀb]indoleꢀ10,11ꢀdione 6v.
Due to the inhibitory activity observed with bicyclic indoleꢀ4,5ꢀdione 6a and some of its
congeners, a tetracyclic, tanshinoneꢀlike core should not be necessarily needed to prevent HuRꢀmRNA
interactions. Thus, cycloadditions on dienophile 6a were targeted to introduce potencyꢀoriented
(additional interactions with the binding site on HuR) and/or “druggability”ꢀoriented substitutions on
Cꢀ6 and Cꢀ7 (modulation of selectivity, solubility and lipophilicity, etc.).
DielsꢀAlder cycloaddition between 1,3ꢀcyclohexadiene 15b and dienophile 6a provided a
mixture of desired orthoꢀquinone 6w and diphenol 16b (step a, Scheme 8). Oxidation (step b)
converted the mixture to pure 6w.
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Scheme 8. Synthesis of cycloadduct 6w.
Reagents and conditions: (a) Cat. dry ZnCl , dry CH Cl , Ar atmosphere, 0°C, 5 min, 88%; (b) CAN, 2/1
2
2
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MeCN/H O, 0°C, 10 min, quantitative.
2
A more systematic effort towards tanshinoneꢀlike 1,3,6,7ꢀtetrasubstituted indoleꢀ4,5ꢀdiones 6
(
R , R ≠ H, cyclo R R , Figure 1) will be carried out, and reported in future.
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2
3
4
Biochemical characterization
Compounds 6a and 6n are more effective than 1 in inhibiting the HuR-RNA complex
formation. Tanshinone mimics 6a-i, 6k-t and 6w were evaluated using a previously developed
biochemical tool based on Amplified Luminescent Proximity Homogenous Assay AlphaꢀScreen
2
7,28
technology.
Recombinant Hisꢀtagged HuR (rHuR) bound to nickel chelate acceptor beads was
incubated with a biotinylated single strand AUꢀrich RNA probe (BiꢀAU), recognized by streptavidinꢀ
coated donor beads. When rHuR binds to the BiꢀAU, the beads are brought into proximity and a
fluorescence signal can be detected. We evaluated the ability of tanshinone mimics to inhibit the rHuRꢀ
BiꢀAU complex formation in saturation binding conditions. Knowing that the K value for the rHuRꢀ
D
27
BiꢀAU interaction is 2.5 nM , we fitted on AlphaScreen saturation curves the Ki values, quantifying
the inhibitory efficiency of tested compounds from high to low nanomolar range (Table 1). Among
tanshinone mimics showing Ki with a percentage of inhibition >50%, compound 6a (Ki = 12.8 nM)
and 6n (Ki = 15 nM) were more effective than 1, while compounds 6h, 6k, 6l, 6r and 6s showed
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similar activity (Figure 3 and Table 1). Consistently with previous data , Ki value of our compounds
change according to the host in which the recombinant protein is produced (Supporting figure 1).
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Figure 3. Ki calculation by Alpha screen assessing specific binding of Hisꢀtagged HuR and the
AUꢀrich biotinylated RNA. Ki were calculated with respect to a K of 2.5 nM for the rHuRꢀBiꢀAU
D
interaction, and normalized to control (DMSO). Fitting curves show nonlinear regression fits of the
data according to a 1ꢀsite binding model in GraphPad Prism. Plotted bars are mean ±SD of two
independent experiments.
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Table 1. Abilities of tanshinone mimics to inhibit the rHuR-Bi-AU complex formation.
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1
Concentration (nM) leading to halfꢀmaximal inhibition of rHuRꢀBiꢀAU complex.
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Cpd
1
Structure
Ki, nM
Cpd
6a
Structure
Ki, nM
50
12.8
6
b
Interfering* 6c
>100
6
d
>200
6e
>200
6f
Interfering* 6g
>100
>100
6h
48
6i
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k
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6l
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6m
Interfering* 6n
15
6o
Interfering* 6p
>100
6
q
>100
6r
6t
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6
s
55
>200
6w
>300
*
(
interfering with the fluorescence spectra of excitationꢀemission of Donor and Acceptor beads
histidine (nickel) chelate detection kit)
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Tanshinone mimics 6b, 6f, 6m and 6o resulted as interferers with the emitted fluorescence in
44,45
AlphaScreen,
thus we proceeded with a second independent, orthogonal assay protocol for these
and a few other tanshinone mimics (Figure 4 and Supporting Figures 2 and 3). We evaluated their
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inhibitory ability via a nonꢀdenaturing and nonꢀcrosslinked REMSA.
After mixing at least 10 fold
excess of rHuR with 75 fmol of 5′ꢀDY681ꢀlabeled AUꢀrich RNA probe (DY681ꢀAU) or with 25 nM of
FAMꢀRNA probe, we observed the formation of the higher, oligomeric molecular weight complex
between protein and RNA. The concomitant addition of active tanshinone mimics (5µM concentration)
caused a reduction of the shifted RNA probe, allowing qualitative estimation of their inhibitory ability
towards the BiꢀAU ligand at equilibrium. We noticed a concordance between the two biochemical
assays for compounds 6a, 6c, 6k, 6n, 6p-6t and 6w. Tanshinone mimics 6b, 6f, 6m and 6o were
therefore classified as inhibitors endowed with intermediate potency (Figure 4). Compound 6a was
tested for binding to RNA probe via REMSA and circular dichroism (CD) (Supporting Figure 4A and
4
B), showing no interference with the fluorescent probe and no change of the RNA conformation, thus
suggesting the absence of an interaction with RNA.
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Figure 4. REMSA characterization of selected tanshinone mimics. REMSA assay performed
with at least 10ꢀfold excess of recombinant HuR incubated for 30 min with 75 fmol of 5′ꢀDY681ꢀ
labeled RNA probe. Incubation with RNA probe only (ꢀ), with rHuR, RNA probe and DMSO (+) used
as positive control of the binding, and incubation with tanshinone mimics 6 (5µM).
Tanshinone mimic 6a directly binds to HuR protein and modulates its binding with
intracellular target mRNAs. Compound 6a was selected among the most potent tanshinone mimics for
further evaluation. It showed a similar mechanism to 1 in interacting with the truncated form of HuR
comprising the first two RRM domains (RRM1ꢀRRM2) but not with the third domain (RRM3) and not
with the RNA probe (Supporting Figure 4 and 5).
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Dynamic mass redistribution (DMR) analysis revealed, in a labelꢀfree format, a direct
protein:6a interaction at the equilibrium (Figure 5). Fullꢀlength rHuR was immobilized onto the surface
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of labelꢀfree microplates by amineꢀcoupling chemistry. Different amounts of 6a (0.03ꢀ100 ꢁM) were
added to the wells and the mass of molecular complexes was detected after a 30 min incubation. Doseꢀ
dependent binding of 6a to rHuR was observed in the 0.3ꢀ10 ꢁM range, sufficient to obtain saturation.
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The estimated affinity constant (K ) was ≈ 4.5 ꢁM. The same experiment was performed with 1, but it
D
27
was impossible to evaluate the K due to its poor solubility.
D
Figure 5. 6a binds to recombinant HuR. Different concentrations of 6a were added to labelꢀfree
microplate wells on which aliquots of fullꢀlength protein had previously been immobilized.
Measurements were performed before (baseline, proteinꢀcoated wells) and after (final) adding the
compound. The response (pm) was obtained subtracting the baseline output from the final output
signals. The output signal for each well was obtained by subtracting the signal of the proteinꢀcoated
reference area from the signal of uncoated area. The data (red dots) were fitted (black line) to a
2
sigmoidal function using a 4 parameter logistic (4PL) nonlinear regression model: R = 0.944; p =
0
.009.
We then determined if 6a was interfering on HuRꢀRNA binding in MCF7 cells. We performed
4
7
an RNA immunoprecipitation (RIP) assay on MCF7 extracts testing three validated HuR transcripts.
We clearly observed either a subsequent decrease of the relative number of mRNA copies and a
decreased expression level of such mRNAs (ERBB2, CTNNB1, VEGF) but not of nonꢀtarget genes
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(
RPLP0, HPRT1) (Figure 6A, B). Therefore, compound 6a directly binds to HuR both in vitro and in
cellular context, in a region contained between the first two RRM domains.
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Figure 6. RIP and quantitative realꢀtime PCR (qRTꢀPCR). A) RIP was performed in MCF7
cells, lysed after 6 h treatment with DMSO (CTRL), 1 (1 µM) or 6a (5µM). HuR antibody (IP) and an
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IgG isotype (IgG) were used for RNA precipitation. Changes in the mRNAs bound to HuR in the
control or treatment condition were assessed by qRT PCR, and compared with the ones obtained with
IgG precipitation, used as negative control. The relative values (Fold enrichment) were normalized to
IgG, considering the value of the housekeeping gene RPLP0. B) MCF7 were treated with 6a (5µM) for
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h to evaluate changes in total RNA levels. Expression level of ERBB2, CTNNB1, VEGF, RPLP0 and
HPRT1 were measured by qRTꢀPCR and normalized to RNA18s. Data are presented as mean ±SD of a
biological triplicate (*p<0.05 and **p<0.01 versus CTRL).
NMR and Molecular Modeling.
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Tanshinone mimic 6a blocks HuR in a “closed” conformation. The 2D Hꢀ N HSQC
spectrum of RRM1ꢀRRM2 domains showed wellꢀdispersed signals in accordance with a folded protein
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structure, whose residues, including those of the linker region, have been previously assigned. The
48
resonances of residues forming the RRM1 domain are almost the same in the isolated domain and in
the RRM1ꢀRRM2 construct. The large superimposition of the signals in the isolated RRM1 and in the
tandem domains is in agreement with the relaxation data that show as the two domains move
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independently in solution in the absence of RNA. In line with the previously reported crystal
structures of HuR, each domain in the RRM1ꢀRRM2 construct is constituted by two αꢀhelices and four
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βꢀstrands.
The molecular interaction of 6a with RRM1ꢀRRM2 tandem domains of HuR was evaluated
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through solution NMR. Compound 6a shows improved solubility with respect to 1. Its effects on the
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protein is appreciable in the 2D Hꢀ N HSQC in the presence of 0.6 equivalents of the ligand, while
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with 1 comparable effects were observed after the addition of 4 equivalents. As also reported for 1, a
generalized decrease in signal intensity was observed for the protein resonances, with few residues
(Thr20, Leu22, Val66, Ser94, Tyr95, Ile103, Asn107, Leu108, Tyr109, Ile133, Val137, Leu138,
Val139, Ser146) experiencing a stronger effect (Figure 7). Tanshinone mimic 6a and 1 interact with the
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protein in the same region, i.e. the βꢀplatform of both domains. In particular, eight amino acids (Thr20,
Ser94, Tyr95, Asn107, Leu108, Ile133, Val137, Leu138) experience a decrease in signal intensity with
both ligands.
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Figure 7. Compound 6a stabilized recombinant HuR in a “closed” conformation. A) Graphical
representation of the intensity changes of RRM1ꢀRRM2 HuR protein per residues in the presence of
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.6 equivalents of 6a. The residues exhibiting the highest decreases in signal intensities are colored in
red. The secondary structures of the domains are reported on the graph. B) Surface representation of the
closed (PDB ID: 4ED5) conformation of HuR. The residues exhibiting the largest decrease in signal
intensities in the presence of 0.6 equivalents of 6a are shown in red.
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The generalized decrease of signal intensity, together with the distribution of affected residues
over the large surfaces of the βꢀplatform in each domain suggests an alteration of the equilibrium
between “closed” and “open” conformations upon ligand binding. Specifically, the decrease of signal
intensity was consistent with a mechanism where compound 6a stabilizes a “closed” conformation of
HuR. Collectively, NMR analysis indicates that 1 and 6a bind the HuR protein approximately in the
same region, producing similar effects on protein dynamics. However, it is interesting to note that one
residue (Ile103) of the interꢀdomain linker (hereafter referred to as “hinge” loop) is sensitive to 6a and
not to 1. This experimental evidence would suggest for 6a a binding site in a more close proximity of
the hinge loop, with respect to 1. To better explore this possibility a molecular modeling study was
performed.
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To this purpose, a combined approach of docking calculations and extended molecular
dynamics (MD) simulations was applied. Specifically, we first attempted a “blind” docking to the
entire HuR surface, using two different docking software to better sample the binding space
(AutoDock4.2 and Glide 6.5). Most of the highestꢀscore poses of 6a suggested by AutoDock were
located within the RNA binding cleft (residues 18 to 95 of RRM1 and 107 to 185 of RRM2) and in
proximity of the “hinge” loop. On the other hand, docking results with Glide converged towards one
solution, which was different from those predicted by Autodock, though it was placed in proximity of
the “hinge” loop as well. Therefore, albeit these results seem to indicate the region surrounding the
“
hinge” loop as the most likely binding region for 6a, docking failed to unequivocally pinpoint one
privileged binding mode, likely owing to omission of full receptor flexibility from the stateꢀofꢀtheꢀart
docking software. To account for the missing receptor flexibility, we carried out multiple extended MD
simulations on a reasonable number of 6a binding modes, for a total simulation time of 6 ꢁs, and
assessed their relative stability. Specifically, we opted for the binding pose predicted by Glide (Figure
8A) and the three best ranked and most diverse poses (in terms of root mean square deviation (RMSD)
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predicted by AutoDock) (Figure 8B,C,D). In all four cases, 6a drifted away from its starting docking
position and explored a significant portion of the HuR surface, as can be observed by following the
movement of the center of mass of 6a (Figure 8AꢀD), its RMSD vs time (Supporting Figure 6A) or its
distance from the center of mass of the two RRM domains (Supporting Figure 6B). After about 1 ꢁs,
each starting docking pose got stabilized and evolved to different final binding modes (Figure 8EꢀH)
which remain individually stable for almost 500 ns. Specifically, out of the four final binding poses,
one was located outside the RNA binding cleft (the Glide predicted binding pose, Figure 8E) while the
other three were located within the latter pocket, in correspondence or in close proximity of the “hinge”
loop. In these final poses 6a stabilizes HuR in a conformational state that is structurally incompatible
with RNA binding. In fact, in each case the two RRM domains were found to be more in contact with
each other than in the HuRꢀRNA complex crystal structure (Figure 8EꢀH). Accordingly, we observed
an increase in both the number of nonꢀnative interꢀdomain contacts and the amount of surface area
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“buried” between the two RRM domains (see respectively Supporting Figure 7A and 7B). These results
indicate that binding of 6a to HuR is correlated with a closure of the RNA binding cleft and,
consequently, with an overall decrease in the amount of interꢀdomain space accessible for RNA
binding.
Nevertheless, among the four poses issuing from our modeling approach, the one depicted in Figure 8F
and more in detail in Figure 9 seems to be more in agreement with both the NMR data and the SARs
reported here. Specifically, 6a was found between the RRM1 beta sheets (β1, β2, β3), the Nꢀterminal
part of the RRM2 α2 helix and the “hinge” loop. In this binding arrangement (Figure 9), the phenyl
ring in R is accommodated in a narrow, laterally open, hydrophobic pocket, shaped by Ile103, Ser99,
1
Lys104 and Lys156 residues, with which it establishes several CHꢀπ interactions. Notably, one
sulfonyloxygens establishes a waterꢀbridged Hꢀbond with the backbone C=O of Ala96, while the
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phenyl ring in R , forms a cationꢀπ interaction with Lys156 and several CHꢀπ interactions with the CH2
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groups of Ser48, Lys50, Asn67 and Lys156. The indoleꢀ4,5ꢀdione moiety is inserted in a solvent
exposed pocket, where it establishes CHꢀπ interactions with Ala96, Lys156, Ser158 and, a πꢀstacking
interaction with Phe65. In this regard, the quinoneꢀoxygens, which point to the solvent exposed part of
the pocket, likely play a crucial role in strengthening the πꢀstacking interaction with Phe65.
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As compared to the other poses, in the aboveꢀdescribed binding mode, 6a is in close proximity with a
larger number of HuR residues exhibiting the highest decreases in NMR signal intensity (Figure 7A).
Precisely, these residues are Leu22, Val66, and Ile103. Noteworthy, NMR pinpointed I103 in the
“hinge” loop as a residue sensitive to binding of 6a but not of 1, which is known to stabilize HuR in a
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closed form without stably interacting with the “hinge” loop. As compared to the other binding poses,
which are located either outside the RNA binding cleft or in more solvent exposed regions, this binding
mode (Figure 9) would be in line also with SARs studies. It would explain why substitutions on the
phenyl ring in R (6f, 6g, 6h, 6i, 6k, 6l), though still causing a drop in the activity, are generally better
1
tolerated than those on the phenyl ring in R (6b, 6c, 6d, 6e). In fact, thanks to the additional lateral
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space in the pocket hosting the phenyl ring in R , this ring could slightly rotate around the SꢀN bond so
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as to allow the attachment of various substituents, even large ones as in the case of 6k. This would not
be possible at position R , owing to potential steric clashes with residues shaping the pocket where it is
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hosted. This binding mode would also explain why the addition of electronꢀdrawing substituents on the
phenyl group in R (6f, 6g, 6h, 6i), particularly at the meta position (6g, 6i), also causes a drop in the
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activity. In fact, these substitutions would likely weaken the aforementioned waterꢀbridged Hꢀbond
with Arg97. Finally, SARs indicate that the addition of rigid and bulky substituents at position 6ꢀ7 (see
6w) or 7 (6p, 6q, 6r, 6s, 6t) of the bicyclic scaffold (B ring) is also generally detrimental to binding.
Steric clashes with the adjacent sulfonamidic group are very likely to arise as a result of their
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introduction, which would force a rotation around the SꢀN bond. That, according to our model, would
in turn lead to a rupture of the waterꢀbridged Hꢀbond with Ala96 and of the hydrophobic interactions of
the phenyl ring in R . In the case of 6q, but especially of 6r and 6s, the presence of a Hꢀbond donor at
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position 7 may partially compensate for these detrimental effects through the potential formation of a
Hꢀbond with the near Arg97 side chain. The only exception to this trend is represented by 6n, where
the presence of a sulfur atom directly linked to the scaffold likely increases the rotational flexibility and
makes the addition of a bulky group well tolerated.
In conclusion, our NMR and molecular modeling data provide useful insights into the binding mode
and mechanism of action of this family of compounds, suggesting that they most likely bind HuR at the
“hinge” region between the two RRM domains and stabilize HuR in a peculiar closed conformation,
which is incompatible with RNA binding.
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Figure 8. A-D) 6a exploration of the HuR RNAꢀbinding pocket for each simulated pose. HuR
is shown as purple cartoons, while the 6a center of mass is shown as spheres colored according to the
simulation time. E-F) Global view of the HuRꢀ6a complexes in each final MD simulation pose. Note
how the binding of 6a (green sticks) to HuR and the further closure of the mRNA binding cleft, as
compared to the mRNAꢀbound conformation (yellow), prevent the accommodation of the mRNA
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