Identification of Quinolinols as Activators of TEAD-Dependent Transcription

Article abstract of DOI:10.1021/acschembio.9b00786

The transcriptional co-regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are the vertebrate downstream effectors of the Hippo signaling pathway that controls various physiological and pathological processes. YAP and TAZ pair with the TEAD (TEA domain) family of transcription factors to initiate transcription. We previously identified a tractable pocket in TEADs, which has been physiologically shown to bind palmitate. Herein, a TEAD-palmitate interaction screen was developed to select small molecules occupying the palmitate-binding pocket (PBP) of TEADs. We show that quinolinols were TEAD-binding compounds that augment YAP/TAZ-TEAD activity, which was verified using TEAD reporter assay, RT-qPCR, and RNA-Seq analyses. Structure-activity relationship investigations uncovered the quinolinol substituents that are necessary for TEAD activation. We reveal a novel mechanism where quinolinols stabilize YAP/TAZ protein levels by occupying the PBP. The enhancement of YAP activity by quinolinols accelerates the in vivo wound closure in a mouse wound-healing model. Although small molecules that occupy the PBP have been shown to inhibit YAP/TAZ-TEAD activity, leveraging PBP to activate TEADs is a novel approach.

Full text of DOI:10.1021/acschembio.9b00786

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Article
Identification of Quinolinols as Activators of TEAD-dependent Transcription
Ajaybabu V. Pobbati, Tom Mejuch, Sayan Chakraborty, Hacer Karatas, Sakshibeedu
R. Bharath, Stéphanie M. Guéret, Pierre-Alexis Goy, Gernot Hahne, Axel Pahl, Sonja
Sievers, Ernesto Guccione, Haiwei Song, Herbert Waldmann, and Wanjin Hong
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00786 • Publication Date (Web): 19 Nov 2019
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Identification of Quinolinols as Activators of TEAD-dependent Transcription
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Ajaybabu V. Pobbati1,*, Tom Mejuch2, Sayan Chakraborty1, Hacer Karatas2,
Sakshibeedu R. Bharath1, Stéphanie M. Guéret3,4, Pierre-Alexis Goy1,5, Gernot
Hahne2, Axel Pahl2, Sonja Sievers2, Ernesto Guccione1, Haiwei Song1, Herbert
Waldmann2,6*, Wanjin Hong1,*
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1Department of Multi-Modal Molecular (M3) Biology, Institute of Molecular and Cell
Biology, 61 Biopolis Drive, 138673, Singapore
2Department of Chemical Biology, Max Planck Institute for Molecular Physiology,
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
3AstraZeneca−Max Planck Institute Satellite Unit, Department of Chemical Biology,
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
4Medicinal Chemistry, Research and Early Development Cardiovascular, Renal and
Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
5Department of Biochemistry, Yong Loo Lin School of Medicine, National University
of Singapore, Singapore.
6Technische Universität Dortmund, Faculty of Chemistry and Chemical Biology,
Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
*Correspondence:
ajaybabuvp@imcb.a-star.edu.sg, herbert.waldmann@mpi-dortmund.mpg.de
mcbhwj@imcb.a-star.edu.sg
Keywords
TEAD, YAP, TAZ, Hippo, quinolinol, regenerative medicine
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ABSTRACT:
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The transcriptional co-regulators YAP (Yes-associated protein) and TAZ
(Transcriptional co-activator with PDZ-binding motif) are the vertebrate downstream
effectors of the Hippo signaling pathway that control various physiological and
pathological processes. YAP and TAZ pair with the TEAD (TEA domain) family of
transcription factors to initiate transcription. We previously identified a tractable
pocket in TEADs, which has been physiologically shown to bind palmitate. Herein, a
TEAD-Palmitate interaction screen was developed to select small molecules
occupying the palmitate-binding pocket (PBP) of TEADs. We show that quinolinols
were TEAD-binding compounds that augment YAP/TAZ-TEAD activity, which was
verified using TEAD reporter assay, RT-qPCR and RNA-Seq analyses. Structure
activity relationship investigations uncovered the quinolinol substituents that are
necessary for TEAD activation. We reveal a novel mechanism where quinolinols
stabilize YAP/TAZ protein levels by occupying the PBP. The enhancement of YAP
activity by quinolinols accelerates the in vivo wound closure in a mouse wound-
healing model. Although small molecules that occupy the PBP have been shown to
inhibit YAP/TAZ-TEAD activity, leveraging PBP to activate TEADs is a novel
approach.
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The evolutionarily conserved Hippo signaling pathway plays key roles in organ size
maintenance, cell fate determination, myogenesis, cancer progression and tissue
homeostasis.1,2 Diverse upstream regulators that sense the cellular internal and
external cues control the activities of the Hippo pathway components. The prime role
of the Hippo pathway is to inhibit the function of its two component proteins - the
transcriptional co-regulator YAP (Yes-associated protein) and its homolog TAZ
(transcriptional co-activator with PDZ-binding motif).
YAP/TAZ possess a C-terminal transcriptional activation domain; they partner
with various transcription factors to control the expression of multitude of genes in a
context-specific manner and promote several cellular processes. Among the
transcription factors that functionally interact with YAP/TAZ, the TEAD (TEA
domain) family members have emerged as the most prominent players.3-6 The TEAD
family in mammals comprises four highly similar proteins (TEAD 1−4) with the same
domain architecture.7 TEADs possess a N-terminal DNA-binding domain called the
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TEA domain - named after the three proteins; TEF-1, Tec1 and abaA were the domain
was initially defined. At their C-terminus, TEADs possess a co-regulator binding
domain (CBD) that binds co-regulators like YAP, TAZ and Vgll proteins.8-11 The
DNA-binding domain of TEADs and the transcriptional activation domain of
YAP/TAZ are required for the transcriptional outcome, therefore, both YAP/TAZ and
TEAD need to pair to initiate transcription.
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YAP/TAZ-TEAD activity plays crucial roles in both physiological and
pathological processes as in tissue development or in cancers, respectively.
Dysregulated Hippo pathway,12 YAP/TAZ copy number increase13 or YAP/TAZ gene
fusions14,15 enhance YAP/TAZ activities in cells. In almost all solid human tumors,
increased YAP/TAZ activity is implicated in cancer progression, metastasis or drug
resistance.3,16 Therefore, inhibition of YAP/TAZ-TEAD activity was suggested as a
viable option for cancer therapy. On the other hand, stimulation of YAP/TAZ-TEAD
activity helps in the intrinsic repair of functionally damaged tissues or organs.
YAP and TAZ play a critical role in liver regeneration, and their depletion
hampers regeneration after partial hepatectomy.17 Although YAP/TAZ is non-
essential for the physiological turnover and renewal of intestinal epithelium,
regeneration of intestinal epithelium in response to injury is severely affected in YAP-
null mice.18 Forced expression of activated YAP improves cardiomyocyte function
and facilitates cardiac regeneration following myocardial infarction.19,20 Furthermore,
YAP/TAZ activity is important for skin wound healing.21,22 Therefore, factors
enhancing YAP/TAZ function have great potential in regenerative medicine, which
aims to stimulate the cells’ intrinsic repair mechanisms.
Being predominantly unstructured, it is technically challenging to directly
target YAP and TAZ. However, we reported that TEADs could be exploited as a
surrogate for modulating YAP/TAZ activities.23,24 The core complex structures of the
YAP-TEAD complex8,11 and that of the TAZ-TEAD complex9 offered avenues on
how the TEADs’ surface could be exploited using peptide-based and small molecule
approaches. Peptide-based modalities like cyclic YAP peptides,25,26 cystine-dense
peptide27 and a Vgll4-YAP fusion peptide28 were all shown to bind to the TEADs’
surface and disrupt the interaction of YAP with TEADs (Figure S1A). Small
molecules with the benzisothiazole-dioxide scaffold also occupied the TEADs’
surface pocket (Figure S1A) and inhibited YAP/TAZ-TEAD interaction and
transcription.29 A tetracyclic compound (CPD3.1) that putatively occupies the
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TEADs’ surface pocket has also been shown to inhibit TEAD activity.30 Similarly,
using a virtual screening of compounds from the ZINC database, a compound with a
central triazole scaffold was predicted to occupy the surface YAP/TAZ-binding
pocket on TEADs and was shown to inhibit TEAD reporter activity.31
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We identified another pocket in the center of TEADs’ CBD and showed that it
is a tractable target for small molecules.23 Further studies have shown that TEADs are
autopalmitoylated and the palmitate group extends into this central pocket.32,33
Therefore, TEADs present two pharmacological targeting opportunities; the surface
YAP/TAZ-binding pocket and the central palmitate-binding pocket (PBP) (Figure
S1A) and these two different pockets could be exploited to modulate YAP/TAZ-
TEAD activities.34-36 We first demonstrated that flufenamic acid, an anthranilic acid
derivative, inhibits TEAD activity by binding to the central PBP in TEADs23, 24
(Figure S1A). Furthermore, a chloromethyl ketone analog of flufenamic acid has been
recently shown to covalently bind to the PBP and inhibit TEAD-dependent
transcription in cells.37 Compounds with alkylthio-triazole scaffold have also been
shown to occupy the PBP and inhibit TEAD-dependent transcription.38
TEAD-binding small molecules could be therapeutically exploited but there is
no reported target-based screening assay that is tailored to identify PBP-occupying
small molecules. To this end, we developed a TEAD-Palmitate AlphaScreen and
screened a custom compound library. Interestingly, we identified that the quinolinol
class of compounds directly bind to TEADs, stabilize YAP/TAZ protein levels and
activate TEAD-dependent transcription in cells. Although a handful of TEAD-
inhibiting compounds have been identified, there is lack of small molecules that bind
to TEADs and activate YAP/TAZ-TEAD transcription. Yet TEAD-activating
compounds offer a significant potential for regenerative medicine.
We synthesized several quinolinol analogs and determined the substituents
that are required for TEAD activation. The potential role of quinolinol-derived
compounds for regenerative medicine was evaluated using a cutaneous wound-
healing assay in vivo. We report that a quinolinol analog, Q2, accelerates the rate of
wound healing via YAP activation. Overall, we postulate that our finding would set
the stage for the identification and development of TEAD-activating compounds that
may have potential in regenerative medicine.
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RESULTS AND DISCUSSION
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Development of the TEAD-Palmitate AlphaScreen
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We previously demonstrated that the palmitate-binding pocket (PBP) in TEADs could
be leveraged to modulate TEAD activity.23,24 However, no target-based screens were
developed to specifically identify PBP-occupying compounds. In order to identify
novel and potent small molecule TEAD modulators that occupy the PBP, we
developed the TEAD-Palmitate AlphaScreen. The screen employed recombinant,
depalmitoylated His-TEAD4 (217–434) and biotin-conjugated palmitate. The
Amplified Luminescent Proximity Homogenous Assay (Alpha) is a bead-based assay
where the signal is emitted when donor and acceptor beads are brought to close
proximity. Here, the interaction between His-TEAD4 and biotinylated palmitate
enables signal transfer between Ni-chelate donor and streptavidin acceptor beads
(Figure 1A). Compounds that occupy the PBP in TEAD will therefore disrupt TEAD-
palmitate interaction and cause a reduction in the alpha signal. Initially, various
concentrations of His-TEAD4 and biotinylated palmitate were titrated to determine
the optimal concentration for the best signal-to-background ratio. Respective controls,
His-TEAD4 alone or biotinylated palmitate alone were employed to confirm that the
alpha signal is specific to the bimolecular interaction between the TEAD and the
palmitate (Figure 1B).
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The prenyl-binding protein PDEδ was the top hit in our search for proteins
that have a fold similar to the CBD of TEADs.39 The CBD of TEADs and, PDEδ have
an immunoglobulin-like β-sandwich fold (Figure 1C) and a lipid-binding pocket in
the center. Lipids interact with the hydrophobic residues that line these pockets
(Figure S1B-C). Therefore, for the TEAD-Palmitate AlphaScreen, we chose a set of
8400 compounds, which were hits from a PDEδ-KRAS AlphaScreen, where we
screened a total of 150,000 compounds from our in-house library.40 The rationale for
choosing PDEδ hits is that it also contains a good collection of generic compounds
that have the ability to target lipid-binding pockets in proteins, and from these we
might be able to identify potential hits for TEADs. Here, we built upon our expertise
in identifying potent compounds that occupy the lipid-binding pockets.40-42
The medium-throughput TEAD-Palmitate AlphaScreen was carried out using
8400 PDEδ hits and the results of the screen are summarized in Figure 1D. Overall,
368 compounds showed greater than 90% inhibition, a hit rate of 4.4%. A Z-factor
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(Z’) of 0.84 and a signal-to-background ratio of 21.9 indicate the robustness of our
screen (Figure 1D). After computationally filtering out pan-assay interfering
compounds (PAINs),43,44 suitable hits were reduced to 220 compounds (Figure 1E).
Generally, compounds with logD values above five are poorly soluble and a
molecular weight cutoff of 500 is recommended for orally bioavailable small
molecule drugs.45 Accordingly, compounds with greater than five logD values and
compounds with molecular weight greater than 500 were further excluded (Figure
S1D). The EC50 values of all the selected compounds were determined using the
TEAD-Palmitate AlphaScreen (Figure).
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We used the TEAD-Palmitate AlphaScreen and a previously developed PDEδ-
KRAS AlphaScreen to quantitatively assess the potency and selectivity of the
compounds to bind the TEAD and the PDEδ, respectively. A plot of EC50 values of
the selected compounds (220 compounds) from the TEAD-Palmitate AlphaScreen
versus their residual activity in the PDEδ-KRAS AlphaScreen was used to identify
thirty-three compounds that showed promising selectivity for TEAD binding (Figure
S1E). The general flowchart of our selection strategy is shown in Figure 1E. From the
thirty-three selected compounds, the quinolinol scaffold demonstrated good potency
and was therefore chosen for further investigation (Figure 1F). Other scaffolds that
were enriched in the selected hits were spiro-oxindole, linear aromatic and imidazole
(Figure 1F). Overall, we posit that the TEAD-Palmitate AlphaScreen is robust and
could be used to specifically identify compounds that occupy the PBP in TEADs.
Quinolinols are TEAD Activators
For a comprehensive analysis, we selected additional quinolinols from our in-house
library and determined the EC50 values using the TEAD-Palmitate AlphaScreen. We
also ascertained direct binding of the compounds to TEAD by means of surface
plasmon resonance (SPR) measurements. The three most potent hits from the
quinolinol cluster (Q1-Q3) inhibit TEAD-palmitate interaction in the low micromolar
range (Figure 2A and 2B). Direct binding of these quinolinols to TEAD, as measured
by SPR gave Kd estimates similar to the EC50 values (Figures 2C-E and Figure S2).
Having confirmed that the quinolinols indeed bind to TEAD (Figures 2C-E and
Figure S2), we next investigated whether quinolinols modulate TEAD activity in cells
using a Dual-Luciferase Reporter (DLR) assay. In this assay, a TEAD reporter
construct with upstream TEAD-binding sites drives the expression of the firefly
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luciferase. For normalization, thymidine kinase promoter-driven renilla luciferase was
used. Interestingly, we observed a dose-dependent increase in TEAD reporter activity
upon Q1−Q3 addition (Figure 2F). From the TEAD DLR assay, we identified that Q2
(EC50 - 2.6 µM) was more potent than Q1 and Q3 (EC50 > 6 µM) (Figure 2F).
To examine whether the increase in TEAD reporter activity is also dependent
on YAP/TAZ, the TEAD reporter activity in the DLR assay was measured upon
YAP/TAZ knockdown (Figure 2G). The knockdown efficiency of YAP/TAZ after
siRNA treatment is shown in Figure 2H. The data indicate that the compounds
Q1−Q3 are unable to activate TEAD activity to the same extent in YAP/TAZ-
depleted cells as compared to the control knockdown cells (Figure 2G), suggesting
that TEAD activation by the quinolinols is also reliant on YAP/TAZ. The reduction in
TEAD reporter activity in control (Ctl) cells after YAP/TAZ siRNA treatment further
establishes the reliability of the assay (Figure 2G). To independently verify TEAD
activation by another method, we used quantitative reverse transcription PCR (RT-
qPCR) and analyzed the endogenous expression levels of TEAD target genes, CTGF,
CYR61 and ANKRD1 after treating the cells with selected quinolinols (Q1−Q3)
(Figure 2I). In line with the TEAD DLR assay, the quinolinols (Q1-2 and Q3 with less
potency) in general, significantly enhanced the expression levels of YAP/TAZ-TEAD
target genes (Figure 2I).
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Structure Activity Relationship of Quinolinol Analogs
To determine the substituents relevant for TEAD activation and to establish the
structure activity relationship (SAR), twenty-four quinolinol analogs were
synthesized. Experimental details and the synthetic routes are described in the
methods section. The activating ability of this 24-membered quinolinol collection was
assessed using the TEAD DLR assay (Figure 3A), the compounds are added to the
cells at a concentration of 10 µM. The structures of the quinolinol analogs used in this
study are shown in Figure 3B. Of the twenty-four analogs, eight were able to activate
TEADs (B16-B23, Figure 3A); Q2 was taken as positive control. A closer analysis of
the structures revealed that introduction of substituents at the R1 position was not
beneficial for the activity, and the analogs B1, B2 and B3 were unable to activate
TEADs (Figure 3A and 3B). Tolyl R5 substituents (B16, B17, B18) were able to
activate TEADs, whereas the analog with tolyl substitution at R4 position (B10) was
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inactive. Dimethylphenyl (B19), para-methoxyphenyl (B20), para-dimethylamino-
phenyl (B21) or para-trifluoromethyl-phenyl (B22) substituents at the R5 position
were also able to activate TEADs, whereas phenyl or pyridinyl substituents at the R5
position (B8–B15) were not active. Bulky substitution at the R5 position, such as a
biphenyl group (B23) was also tolerated with respect to TEAD activation. We
determined the EC50 values of all the TEAD activators (B16–B23) using the TEAD
DLR assay and identified that the compounds B22 and B23 were relatively more
potent than Q2 (Figure 3C). Compound B15, a negative control, did not show dose-
dependent activation (Figure 3C). Similarly, a benzimidazole analog, a PDEδ hit41
was also unable to activate TEADs, suggesting that TEAD activation is specific to
certain quinolinols (Figure 3C). As shown for Q1–Q3 (Figure 2I), the TEAD
activating ability of B22 and B23 was independently verified using RT-qPCR
(Figures 3D-F). Both B22 and B23 increased the expression of TEAD target genes
CTGF, CYR61 and ANKRD1. On the other hand, the inactive compound B15
(Figures 3A-C) was unable to activate these TEAD target genes (Figure 3D-3F).
Through SPR measurements, we also verified that the quinolinols B22 and B23 bind
to TEAD. The SPR sensorgram and Kd values of B23 to TEAD is shown in Figures
S3A-B.
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Mechanism of Action of Quinolinols
We showed that quinolinols bind to TEAD and activate TEAD-dependent
transcription (Figures 2E and 2F). However, a class of histone deacetylase (HDAC)
inhibitors that bears the quinolinol scaffold has been reported.46 The widely used
HDAC inhibitors are hydroxamic acid-based and not quinolinols. To rule out the
possibility that the gene activation observed after quinolinol addition could be a result
of HDAC inhibition, the HDAC activity was assessed using the HDAC-Glo assay in
HEK 293 cells after quinolinol treatment. The data from the HDAC-Glo assay
suggests that quinolinols (Q2, B1–B24) were unable to inhibit HDACs (Figure S4A).
Trichostatin A (TSA), an HDAC inhibitor that was taken as positive control did show
greater than ninety-five percent HDAC inhibition (Figure S4A). Additionally, we also
assessed the activities of TSA and the quinolinol-based HDAC inhibitor BRD4354
and found that both of these HDAC inhibitors were inactive in the TEAD DLR assay
(Figure S4B). For comparison, the dose-dependent activation of the quinolinol B23 is
plotted in Figure S4B. Our result is also in accordance with a recent report that also
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indicated that TSA has negligible effect on the basal activity of the TEAD reporter.47
These data suggested that TEAD-activating quinolinols are not HDAC inhibitors and
vice versa (Figures S4A-B). Similar to the inactive compounds B12–B15, BRD4354
contains a pyridinyl group at the R5 position (Figure 3B) and therefore did not
activate TEAD.
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To verify that TEAD activation by quinolinols is not restricted to HEK 293
cells alone, the TEAD DLR assay was performed in ACHN cells, a renal carcinoma
cell line. The relatively potent quinolinol analogs B22, B23 and Q2 were able to
activate TEAD reporter activity in ACHN cells (Figure 4A). TEAD activation by
quinolinols was also observed in other cell lines (MDA-MB-231, MCF 10A and PC-
3).
YAP/TAZ-regulated gene signature in HEK 293 cells was previously
identified through gene knockouts generated via CRISPR/Cas9.48 We analyzed
whether this signature was enriched in B23-treated HEK 293 cells. Global gene
expression changes upon B23 treatment was evaluated by RNA-Seq and the
enrichment of YAP/TAZ-regulated signature was assessed through Gene set
enrichment analysis (GSEA).49 Most of the YAP/TAZ target gene set was
significantly enhanced after B23 treatment (Figure 4B) and GSEA analysis revealed a
normalized enrichment score of 2.2, p value <0.003, suggesting statistically
significant enrichment of YAP/TAZ signature in B23-treated cells (Figure 4C).
We used an acyl-biotin exchange assay to determine whether the quinolinols
Q22, Q23 and Q2 occupy the PBP in TEAD. In this assay, autopalmitoylation of
TEAD was monitored and the PBP-occupying compounds are expected to inhibit
TEAD autopalmitoylation. The extent of TEAD autopalmitoylation was estimated
after exchanging the palmitate group with BMCC-biotin following hydroxylamine
(HA) treatment. Treatment with compounds B22, B23 and Q2 led to a significant
reduction in the amount of BMCC-biotin, compared to control treatment (DMSO
alone), suggesting that the quinolinols occupy the PBP in TEADs (Figure 4D).
We also docked the quinolinol Q2 in the PBP and determined its binding pose
that has the lowest binding free energy (Figure 4E). This preferred conformational
pose of Q2 was subjected to unrestrained molecular dynamics simulation for 100 ns
and the result suggested that binding of Q2 in PBP is stable and is predominantly
mediated by hydrophobic interactions (Supporting Information). Based on this model,
we introduced an A231I mutation that would interfere with the binding of Q2 to PBP
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through steric hindrance. Consistent with our prediction, SPR-based affinity
measurement indicated that Q2 does not bind appreciably to the TEAD mutant. Based
on the Q2-TEAD model, the quinolinols B22 and B23 could also be docked in the
PBP. The binding affinity of B22 and B23 to mutant TEAD was also determined by
SPR and their affinity to the TEAD mutant was reduced by greater than seven-fold
when compared with wild type TEAD. The docked B23 in the PBP and the SPR data
of B23 and TEAD mutant is shown in Figures S4C-E. These data suggest that
quinolinols occupy the PBP. By occupying the TEADs’ PBP, the physiological ligand
palmitate is able to affect protein stability and also allosterically influence the
interaction of TEAD with YAP and TAZ.32,33 Therefore, we probed whether these are
affected by quinolinols. Although the TEAD levels were the same after quinolinol
treatment (Figure 4F), we consistently observed an increase in the protein levels of
the TEAD co-regulators YAP and TAZ (Figure 4F). This is probably because
controlling YAP/TAZ protein stability (rather than TEADs per se) is more prominent
in the regulation of TEAD dependent transcription. As such, enhanced TEAD activity
that we observed after compound B22, B23 or Q2 treatments could be a result of
stabilized YAP/TAZ protein levels.
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The Quinolinol Q2 Accelerates Cutaneous Wound Healing
Enhancing YAP/TAZ or TEAD activities has been shown to improve the regenerative
capabilities of tissues and organs.50 Varying YAP/TAZ levels using genetic tools
unraveled that their activity play a critical role for recovery after myocardial
infarction, liver regeneration and cutaneous wound healing.50 Our current study
revealed a novel class of quinolinols that stabilized YAP/TAZ protein levels (Figure
4G) and enhanced the expression of YAP/TAZ-TEAD target genes (Figure 2I and
Figures 3D-F). We therefore investigated the potential of TEAD activation in
regenerative medicine using a cutaneous wound healing mouse model. For this
purpose, 4 mm-wide punch biopsy wounds were initially made on the mouse dorsum.
Then, control- (DMSO) or Q2-treated mice wounds were monitored for 12 to 14 days
during the entire duration of the wound healing process. Interestingly, the Q2-treated
wounds healed faster compared to the control, which was already discernible at day
seven (Figure 5A). Six mice were used per experiment (three for each group), the
experiment was performed twice independently and the results were consistent.
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Sections of skin wound tissues were stained for YAP expression during the
healing process through immunohistochemistry (IHC). We found that YAP
expression was enriched in the basal layer of skin epidermis (Figure 5B), in line with
previous observations,21,22 in both control- and Q2-treated mice wounds. Importantly,
the YAP staining was enhanced and was particularly increased in the nucleus of
healing epidermis on day five in Q2-treated wounds compared to the control (Figure
5B, red arrows). This result is consistent with the fact that YAP/TAZ improved the
wound healing process and enhanced YAP levels and an increase in nuclear YAP is
associated with improvement in wound closure.22 Quantitative analysis of the wound
healing process showed that the rate of wound closure was significantly enhanced
after Q2 treatment (Figure 5C). Further, quantification of YAP staining at day five in
the control- and Q2-treated mice wound sections showed a significant increase in
nuclear localization of YAP after Q2 treatment (Figure 5D). An increase in the Ki67
nuclear staining, a marker of cell proliferation, was also observed in Q2-treated skin
wounds (Figure 5E). Upon quantification, we observed that the increase in Ki67
marked nuclei was statistically significant, suggesting that Q2 enhances cell
proliferation (Figure 5F). Treatment with the compound Q2 activated nuclear YAP
and enhanced cell proliferation within days 4-5 post-wounding, thereby promoting an
efficient and rapid healing process in the subsequent days. Overall, our data suggests
that Q2 enhances YAP activity and accelerates cutaneous wound healing.
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From the TEAD-Palmitate AlphaScreen, we shortlisted 33 compounds that are
potent in the TEAD-Palmitate screen but have high residual activity in the PDEδ-
KRAS AlphaScreen; therefore these compounds are expected to preferentially bind
TEADs and not PDEδ, which was also verified through SPR analyses (data not
shown). The quinolinol scaffold that we identified directly binds to TEAD, occupies
the PBP and activates TEAD activity. Knockdown of YAP/TAZ greatly reduced the
extent of TEAD activation suggesting that the activation by quinolinols is YAP/TAZ
dependent. Additionally, we had also observed quinolinol-dependent TEAD
activation in more than one cell line, suggesting that TEAD activation by quinolinols
is conserved. The SAR study of quinolinols using the 24-membered quinolinol
collection suggested that R1 substitutions were not tolerated and abrogated activation,
while tolyl, dimethylphenyl, methoxyphenyl, dimethylamino-phenyl, trifluoromethyl-
phenyl or biphenyl groups at the R5 position retained activation (Figures 3A and 3B).
Quinolinol-dependent stabilization of YAP and TAZ cellular protein levels (Figure
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4G) is likely an outcome of quinolinol occupying the PBP and preventing the
YAP/TAZ-TEAD complex from degradation. An increase in YAP/TAZ may
potentially translate to an increase in the expression of YAP/TAZ-TEAD regulated
genes.
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Enhancing YAP/TAZ-TEAD gene expression signature has potential benefits
in regenerative medicine.50 Although a handful of TEAD inhibitors have been
identified,34-36 there is no activating compound that target TEADs. A few YAP/TAZ-
TEAD activating compounds have been known,51-54 but their molecular target is
unclear, making it difficult to optimize these compounds for use in regenerative
medicine. However, an inhibitor of Mst1/2 that activates the downstream effector
YAP had showed potential to improve tissue repair and regeneration.55 We are aware
about the covalent nature of the palmitoyl modification but it is noteworthy that
palmitoylation is the most dynamic of all lipid attachments.56 Although TEAD
palmitoylation was reported to be not dynamic,32 the fact that PBP-occupying
compounds are able to compete with palmitate in cells38 and the recent identification
of TEAD depalmitoylases57 should serve as an impetus for the discovery and
development of small molecule PBP ligands. In conclusion, we present quinolinols as
novel PBP ligands that activate YAP/TAZ-TEAD gene expression signature that may
have therapeutic potential in regenerative medicine.
FIGURE LEGENDS
Figure 1. The TEAD-Palmitate AlphaScreen Design
(A) Schematic representation of the TEAD-Palmitate AlphaScreen. The interaction
between the His-TEAD4 and the biotinylated palmitate brings the donor and acceptor
beads to close proximity, enabling singlet state oxygen transfer, which results in the
amplification of the alpha signal.
(B) Alpha signal in the presence of biotinylated palmitate alone (Control) or in the
presence of both His-TEAD4 and biotinylated palmitate (TEAD-Palm). The signal is
significantly higher in TEAD-Palm.
(C) Superposition of the ribbon representations of the co-regulator binding domain of
TEAD and, PDEδ.
(D) The summary of the TEAD-Palmitate AlphaScreen. Of the 8400 compounds
screened, 368 inhibited the alpha signal by more than ninety percent. The Z-factor
(Z’) and the signal/background (S/B) ratio are 0.84 and 21.9, respectively.
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(E) Flowchart of the rationale used in the shortlisting of compounds.
(F) The structures of the scaffolds that are enriched in the hits of the TEAD-Palmitate
AlphaScreen.
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Figure 2. Quinolinols Activate TEAD Transcription Factors
(A) Structures of the three potent hits, Q1-Q3, with a quinolinol scaffold.
(B) Determination of EC50 values by titrating various concentrations of the
quinolinols (Q1-Q3) in the TEAD-Palmitate AlphaScreen.
(C) Representative sensorgrams from Surface Plasmon Resonance (SPR)
measurements, obtained after injection of the quinolinol Q2 at indicated
concentrations over immobilized TEAD.
(D) The plot is the steady-state response against analyte (Q2) concentration. The
dissociation constant (Kd) was obtained by fitting a one-site binding curve, shown in
red.
(E) The equilibrium dissociation constant (Kd) values of the quinolinols Q1-Q3, as
determined by the SPR measurements.
(F) Quinolinols Q1-Q3 elicit a dose-dependent increase in the TEAD Dual Luciferase
Reporter (DLR) assay. Activity of the TEAD-responsive luciferase reporter was
measured in HEK 293 cells. Thymidine kinase promoter-driven renilla luciferase is
used for normalization. EC50 values are obtained after fitting the Hill equation (solid
lines).
(G) TEAD activation in HEK 293 cells upon YAP/TAZ knockdown. The activation
by quinolinols is dependent on YAP and TAZ as YAP/TAZ knockdown using
siRNAs reduced the extent of activation in the TEAD DLR assay.
(H) Western blots of HEK 293 lysates to show the levels of YAP and TAZ after
siRNA-mediated knockdown.
(I) The levels of TEAD-dependent genes CTGF, CYR61, ANKRD1 were estimated
by RT-qPCR. HEK 293 cells were treated with 10 µM of indicated quinolinols for 18
h. All data represented as mean ± s.d., n = 3, p values were determined using two-
tailed t-tests. (****, p<0.0001, ***, p<0.0002, **, p<0.005, ns – not significant). All
the experiments shown in the figure were repeated at least three times and the
representative data is shown.
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Figure 3. Structure Activity Relationship of Quinolinols
(A) TEAD activation by quinolinols was measured using the TEAD Dual Luciferase
Reporter (DLR) assay.
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(B) Structures of the quinolinols used in the study.
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(C) Representative activation curves and the EC50 values of quinolinols B22, B23 and
Q2 determined using the TEAD DLR assay. B15 and PDEδ hit serve as negative
controls.
(D-F) The relative levels of the indicated TEAD target genes were measured using
RT-qPCR after B22, B23 or B15 addition. All data represented as mean ± s.d., n = 3,
p values were determined using two-tailed t-tests (**, p<0.005, *, p<0.02, ns - not
significant).
Figure 4. Mode of Action of Quinolinols
(A) TEAD DLR assay in ACHN cells after the addition of the indicated compounds.
Data represented as mean ± s.d., n = 3 **, p<0.005.
(B) RNA-Seq data from biological triplicates were used to generate the row
normalized heat map of YAP/TAZ signature genes after Ctl (DMSO) or B23
treatment of HEK 293 cells.
(C) Gene set enrichment analysis (GSEA) to obtain statistical significance and
enrichment score of YAP/TAZ signature genes after B23 treatment of HEK 293 cells;
normalized enrichment score (2.2), p < 0.003.
(D) Acyl-biotin exchange assay for the detection of NHS-Biotin that is exchanged for
palmitate in TEAD. Untreated and hydroxylamine (HA)-treated TEAD were used in
the assay. Quinolinols compete with palmitate for binding to TEAD. Coomassie-
stained gel shown below serves as loading control for TEAD.
(E) The quinolinol Q2 is modeled in the palmitate-binding pocket (brown mesh) of
TEAD.
(F) Western blots of HEK 293 lysates to show the global cellular levels of YAP, TAZ
and TEAD4 after the addition of indicated quinolinols.
Figure 5. The Quinolinol Q2 Accelerates Cutaneous Wound Healing in Mice
(A) Representative images of control- (DMSO) or Q2-treated mice wounds at
indicated time points.
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(B) Immunohistochemistry (IHC) analysis of YAP in skin wound tissues. YAP
staining is enriched in the basal layer of skin epidermis, however, in Q2-treated
wounds at day 5, there is a significant increase in nuclear YAP (red arrow heads). The
boxed regions are enlarged.
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(C) Quantitative analysis of the reduction in wound diameter in control- and Q2-
treated mice, data represents mean ± s.d., n = 3 mice per group.
(D) Quantification of percentage of cells with nuclear YAP staining, observed on day
5 skin wound sections. Data represents mean ± s.d., n = 4, ****, p<0.0001.
(E) IHC sections showing Ki67 staining of control- and Q2-treated wounds.
(F) Quantification of nuclear Ki67 staining, data represents mean ± s.d., n=4, *,
p<0.0157.
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Figure 1
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A
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30000
20000
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Excitation
680 nm
Emission
600 nm
1O2
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His-tag
Biotin-
Palmitate
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Ni - Chelate
Donor
Streptavidin
Acceptor
TEAD
PDEδ
C
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Control Tead-Palm
Alpha Screen Summary
TEAD
Hit
Compound
S/B
TEAD Palmitate
Compounds Hits
Z’
Rate Conc.
ratio Conc. Conc.
8400
368 4.4 %
0.84 21.9
20 μM
0.1 μM
1 μM
F
Scaffold 1
Spiro-oxindole
Scaffold 2
Linear Aromatic
H
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PDEδ hits
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90 % inhibition
Scaffold 3
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Scaffold 4
Imidazole
220 Compounds
PAINs removal
OH HN
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n=1,2
33 Compounds
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Figure 2
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OH
Q1 EC50 - 1 µM
Q2 EC50 - 1.7 µM
Q3 EC50 - 1.8 µM
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N
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10000
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Q3
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1 µM
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Q1 EC50 - 8.5 µM
Q2 EC50 - 2.6 µM
Q3 EC50 - 6.6 µM
100
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Compound
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Q3
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TAZ
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ANKRD1
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CYR61
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Figure 3
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Dose-Response Curves
B
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B22 EC50 - 0.89 µM
B23 EC50 - 1.9 µM
Q2 EC50 - 2.6 µM
Quinolinol analogs
125
100
75
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N
Q2
B1
B2
N
R3 HN
B15
OH HN
OH HN
O
OH HN
R1
N
R5
H3C
N
CH3
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H3C
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PDE! hit
H3C
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B3
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OH
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DUSP5
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NR4A1
MAP2K3
JUNB
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ACHN cells DLR
IER3
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**
TNFAIP2
CTGF
PLAT
CYR61
SRF
HIVEP2
AMOTL2
LDLR
ANKRD1
ZFP36
FOS
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Enrichment plot: YAP/TAZ Signature
C 0.6
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- HA
Streptavidin blot
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A231
TAZ
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Antibodies. Anti-YAP/TAZ (Cat#D24E4), Anti-YAP (Cat# D8H1X) was purchased
from Cell Signaling Technology. Anti-GAPDH (Cat# sc-25778), Anti-Actin HRP
(Cat# sc-47778) was from Santa Cruz Biotechnology. Anti-TAZ (Cat# 560235) and
Anti-Ki67 (Cat# 556003) was purchased from BD Pharmingen. Anti-TEAD4 was
from Aviva Systems Biology (Cat# ARP38276).
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Cell Culture Conditions
HEK 293 (CRL-1573TM) cells (human embryonic kidney) and ACHN (CRL-1611TM)
cells (human kidney) were cultured in RPMI (Roswell Park Memorial Institute) media
supplemented with 10% (v/v) FBS. All cells were grown at 37°C in an atmosphere of
5% CO2.
TEAD4 Purification and Depalmitoylation
N-terminal, histidine-tagged Human TEAD4 (217 - 434) was expressed in BL21
(DE3) cells for 16 h at 25°C. After cell lysis, the protein was purified using
immobilized metal-ion chromatography and size-exclusion chromatography in a gel
filtration (GF) buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl. TEAD4
purified by this method is primarily palmitoylated and the palmitoyl group is removed
using hydroxylamine treatment. (To a 4M stock of hydroxylamine, diethylamine was
added until pH 6.0 is reached. Typically, for 2 ml hydroxylamine stock solution, 700-
µl diethylamine was added). TEAD4 was incubated with 0.5 M hydroxylamine pH
6.0 for 1 h at 25°C and then dialyzed in GF buffer for 16 h at 4°C to remove the
palmitate group. Mutant TEAD4 (217 - 434) A231I and PDEδ were also purified
using the same methodology as described for wild type TEAD4.
TEAD-Palmitate AlphaScreen
The TEAD-Palmitate AlphaScreen was conducted in white, non-binding 384-well
plates (PerkinElmer ProxiPlate) with a final volume of 15 µl in pH 7.5 HEPES buffer
(25 mM HEPES, 150 mM NaCl, 0.2% BSA). A mixture of depalmitoylated His-
TEAD4 (100 nM) and biotinylated-palmitate (1 µM) was added to the assay plates
containing the screening library (20 µM final concentration) and was incubated for 30
min. Then, equal volume of AlphaScreen Bead Mix (Histidine Detection kit, Perkin
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Elmer) was added and the assay plates were incubated at 4°C for 16 h before
measuring the alpha signal through 680 nm laser excitation.
The PAINS filtering was done computationally based on the paper by Baell et
al.,44 the structural features were taken in SMARTS encoding form58. In addition,
structural features described in the paper by Dahlin et al59 were manually transformed
into SMARTS and also removed. The PAINS filter was implemented as a Pipeline
Pilot protocol that takes a list of compounds as input, filters out PAINS-containing
structures by SMARTS-based substructure matching and outputs only the PAINS-free
compounds.
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Surface Plasmon Resonance (SPR)
Depalmitoylated His-TEAD4 was dialyzed in 50 mM acetate buffer pH 5.0 to remove
Tris and to provide an overall positive charge. The electrostatic interaction between
the positively charged His-TEAD4 and negatively charged SPR CM5 chip facilitates
pre-concentration. His-TEAD4 is further covalently immobilized to the CM5 chip by
activating the carboxymethyl groups in the chip, using the amine coupling kit (GE
Healthcare, Cat# BR100050). His-TEAD4 was injected through a flow cell until an
immobilization level between 10,000 – 15000 resonance unit (RU) was reached.
Various concentrations of analytes (compounds) dissolved in SPR buffer (20 mM
Tris, pH 8.0, 150 mM NaCl, 1.5% DMSO) was injected over reference and TEAD-
immobilized flow cells for indicated time periods until a steady state is reached.
Background response is subtracted to obtain the actual binding response. Data
analysis was performed using the Scrubber software, steady state sensorgram
response is plotted against analyte concentration and a one-site binding equation is
fitted to determine the dissociation constant (Kd).
Dual-Luciferase Reporter (DLR) assay
HEK 293 cells were transfected transiently with TEAD reporter (firefly luciferase
driven by TEAD-responsive promoter) and normalization constructs (TK-driven
renilla luciferase construct). For a 6-well plate, 500 ng TEAD reporter construct and
25 ng normalization construct was used. The cells were split to 24-well plate 7 h post
transfection. Compounds at indicated concentrations were added to the biological
duplicates or triplicates. Cells were treated with indicated compounds for 16 h and the
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luciferase activities were measured using the dual-luciferase reporter assay system
(Promega) as per manufacturer’s protocol.
When the assay was used after YAP/TAZ gene knockdowns (Figure 2G), the
siRNAs (YAP - Cat# L-012200-00-0005, TAZ - Cat# L-016083-00-0005 from
Dharmacon) and the luciferase constructs were transfected to HEK 293 cells using
lipofectamine 2000. siRNAs at a concentration of 100 nM was used and the luciferase
activity was measured 48 h post siRNA treatment.
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Quantitative Reverse Transcription PCR (RT-qPCR) and RNA-Seq
RNA was isolated from the cells after compound treatment using Quick-RNA
Miniprep kit (Zymo Research) and the RNA quality was assessed using NanoDrop
spectrophotometer. For preparation of cDNA by reverse transcription, 2 µg RNA was
used in a 25-µl-reaction volume. After cDNA synthesis, the expression levels of target
genes CTGF (Cat# Hs00170014_m1), CYR61 (Cat# Hs00155479_m1), ANKRD1
(Cat# Hs00173317_m1) were assessed by qPCR using the Taqman assay; 18S rRNA
(Cat# Hs99999901_s1) was used for normalization. The cDNA from control and
compound-treated cells were taken as triplicates, RQ Min and RQ Max values are
plotted in the graphs.
For RNA-Seq the RNA quality was assessed on a Bioanalyzer instrument
(Agilent) using the Agilent RNA 6000 Pico Kit. Total RNA, 2.5µg, was used for
RNA-Sequencing library preparation with the TruSeq Stranded mRNA Library Prep
kit (Illumina) largely according to manufacturer’s instructions. Amplification of
libraries was reduced to 7 PCR cycles. Purified libraries were quantified by qPCR
(KAPA Library Quantification Kit for Illumina, Roche). The Agilent High Sensitivity
DNA Kit was used to evaluate the fragment lengths of a subset of libraries, before
combining the libraries to one library pool and sequencing on a NextSeq500
(Illumina) in a single read high output run at 75bp read length. Raw reads from fastq
files were aligned to the hg38 genome using STAR 2.6.1d. Bam files were sorted with
samtools sort and indexed with samtools index. The number of reads mapping to each
gene was quantified with htseq-count on features from the gtf file
gencode.v29.annotation.gtf. The raw counts of reads were then imported into EdgeR
in R to perform the differential expression analyses.
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HDAC-Glo Assay
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The activities of HDACs (class I and II) in HEK 293 cells were determined using the
HDAC-Glo assay (Promega). The assay was performed as per manufacturer’s
guidelines. Briefly, HEK 293 cells were grown sparsely in tissue-culture compatible
white flat bottom 96-well plate. Compounds dissolved in HDAC-Glo buffer was
added to cells and incubated for 1 h at 37°C. The cells were treated with compounds
at a final concentration of 10 µM. After 1 h incubation, HDAC-reagent was added and
HDAC inhibition was evaluated based on the amount of processed HDAC-substrate
in the assay.
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Molecular Docking and Molecular Dynamics Simulations
AutoDock Vina60 was used to generate multiple binding poses of Q2 into the PBP of
TEAD (PDB id: 6GE3). The binding site was defined as a cuboid with a size of 40 Å
x 40 Å x 40 Å such that it includes the entire model. The maximum number of
binding poses was set to 10, and the maximum energy difference between the best
binding mode and the worst displayed was 4 kcal/mol. The different binding poses
were visually inspected for better fit in PyMol. The best binding mode corresponded
to a ΔG value of -8.4 kcal-mol-1 and the second best pose had a ΔG value of -6.7
kcal-mol-1, suggesting that Q2 prefers the pose in PBP as shown in Figure 4E.
The high-scoring conformational pose of Q2 bound to TEAD was subjected to
unrestrained molecular dynamics simulation using Desmond. Q2-TEAD, ligand-
protein complex was placed in a cubic box with 10 Å of buffer system containing 150
mM NaCl and the charges on the protein and ligand were neutralized by addition of a
Cl- ion. A simple point charge water model was used for modeling solvent around the
Q2-TEAD complex. Under an NPT ensemble class with temperature, T= 310 K,
pressure P= 1.01325 bar, with the system relaxed MD simulations were carried out
for 100 ns. Analyses of the resulting traces was performed using the Simulation
Interactions Diagram tool in Desmond. The results of these analyses are added in the
supplementary information. The ligands, B22 and B23 were docked based on the
interactions of Q2-TEAD and the resulting structures were minimized.
Acyl-Biotin Exchange Assay
Depalmitoylated recombinant His-TEAD4 (0.5 mg) was immobilized to Ni-NTA
beads by incubating at 25°C for 1 h. Then, the beads were split equally; either vehicle
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control (DMSO) or the indicated compounds at a concentration of 25 µM was added
and incubated at 25°C for 15 min. This is followed by incubation of samples with 50
µM palmitoyl-CoA for 1 h at 25°C. The beads were washed and the non-
palmitoylated cysteines were blocked using 25 mM N-ethylmaleimide by incubating
at 4°C for 16 h. The samples were then incubated with or without 0.5 M
hydroxylamine pH 6.0 for 1 h at 25°C. The beads were washed in PBS and the
protein-bound beads were treated with 1 µM BMCC-Biotin for 1 h at 25°C. The beads
were washed and processed with SDS-sample buffer. After a SDS gel and western
blot the biotin was detected using streptavidin-HRP.
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In vivo Wound Healing Assay
Four-five week adult ICR mice were purchased from Invivos, Inc., and housed in
temperature and light controlled rooms. All animal care and operations were
performed according to the National Institute of Health guidelines approved by the
Institutional Animal Care and Use Committee (IACUC: 171237) from Biological
Resource Center (BRC), A-STAR.
Four-to-five weeks old ICR mice were anesthetized intraperitoneally by
injecting a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg, diluted in 100 µl
of saline solution. Body hair from mice dorsum was removed and the surgical site was
disinfected with 70% ethanol swab. A 4 mm symmetrical full thickness biopsy wound
was created on the dorsal skin of mice using a sterilized Integra Miltex biopsy
punches (Integra York, P.A., Inc.,). An ointment comprising of 50 mg of Pluronic
F127 (Sigma-Aldrich) gel containing either 30 µM compound Q2 or DMSO control
was applied to each wound per mice every 2-3 days. The mice were monitored for 12-
14 days during wound healing process. Wounds were digitally photographed every 2-
3 days. During the process of wound closure, skin samples containing the excisional
wound were collected on the indicated days and fixed in 4% Paraformaldehyde (PFA)
for 24 h. The excised skin tissues were paraffin embedded and processed for
immunohistochemical analysis. The wound diameter was measured with a caliper.
Chemical Synthesis
All the final products were characterized using 1H-NMR, 13C-NMR and HR-MS. 1H-
NMR,13C-NMR and LR-MS (low resolution mass spectroscopy) were used for the
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intermediate characterization. H-NMR and 13C-NMR were recorded on a Varian
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Mercury (400 MHz), Bruker BioSpin (500 MHz), Bruker BioSpin (600 MHz) and
Bruker BioSpin (700 MHz) using CDCl3 or D2O as solvent. Chemical shift (δ) values
are reported in ppm using the solvent resonance as internal standard (CDCl3: δ = 7.26
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ppm and D2O: δ = 4.79 ppm for H-NMR spectra, δ = 77.16 ppm for 13C-NMR
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spectra). Multiplicities are indicated br s (broadened singlet), s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet); coupling constants (J) are given in Hertz (Hz).
High-resolution mass spectra were recorded on a LTQ Orbitrap mass spectrometer
coupled to an Acceka HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1
mm, particle size 1.9 µm, ionization method: electron spray ionization).
Synthesis Scheme: Compounds B1-B3, B6-B23 were synthesized according to
Method A using Betti reaction. Compounds B4 and B5 were synthesized according to
Method B and the compound B24 were synthesized according to Method C.
Method A
In an oven-dried 10-mL flask, equipped with a magnetic stirrer, corresponding
aldehyde (1 mmol) and amine (1 mmol) were dissolved in dry EtOH (1.5 mL) and the
resulting mixture was stirred at ambient temperature for 5 min. Corresponding 8-
hydroxyquinoline (1 mmol) and dry pyridine (40 µL, 0.5 mmol) were added and the
resulting mixture was stirrer for 4-17 days. Products, insoluble in ethanol formed
white precipitate that was filtered, washed with ethanol and dried under high vacuum.
In case of the products soluble in ethanol, the solvent was removed under reduced
pressure and purified on silica-gel flash chromatography using cyclohexane/EtOAc
(0%-40%) as eluent.
Method B
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In a flame-dried 50-mL three-neck flask, equipped with a magnetic stirrer, low-
temperature thermometer, inert gas inlet and rubber septum, the corresponding aryl
bromide (0.8 mmol) was dissolved in dry THF (3 mL) and the reaction mixture was
cooled down at -78 °C. To the resulting mixture n-BuLi (1.6 M, 0.6 mL, 0.96 mmol,
1.2 equiv.) was added drop wise and the mixture was stirred at -78 °C for 30 min. N-
Benzylideneaniline (0.96 mmol, 1.2 equiv.) was dissolved in dry THF (1 mL) and
added drop wise. The resulting mixture was allowed to warm up slowly to ambient
temperature and stirred for additional 10 h. The reaction was quenched with sat. aq.
NH4Cl (4 mL) and the phases were separated. The aqueous phase was extracted with
EtOAc (3 x 5 mL). The combined organic phases were washed with sat. aq. NH4Cl (5
mL), dried over MgSO4 and concentrated under reduced pressure. The crude oil was
purified on silica-gel flash chromatography using cyclohexane/EtOAc (0%-40%) as
eluent.
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Method C
Method C1: In a 100-mL Schlenk flask, equipped with a magnetic stirrer, 8-
hydroxyquinoline (1.45 g, 10 mmol), formaldehyde (36.5% in water, 2.3 mL, 31
mmol) and HCl (37% in water, 2.3 mL, 28 mmol) were added. Dry HCl gas,
generated by slow addition of HCl (37% in water) to anhydrous CaCl2, was bubbled
for 3 h. The reaction mixture was stirred at ambient temperature for additional 6 h.
The solid product (B25) was filtered, washed with dry EtOH, dried under vacuum and
used without further purification.
Method C2: In a 25-mL flask, equipped with a magnetic stirrer and rubber septum,
B25 (300 mg, 1.3 mmol) was dissolved in CHCl3 (6 mL). The resulting mixture was
cooled down to 0 °C and DIPEA (0.46 mL, 2.6 mmol, 2 equiv.) was added drop wise.
The resulting mixture was stirred at 0 °C for 5 min and 1-phenylpiperazine (0.2 mL,
1.3 mmol, 1 equiv.) was added drop wise. The ice bath was removed and the resulting
mixture was stirred at ambient temperature for 24 h. The reaction mixture was diluted
with CHCl3 (20 mL), washed with 5% aq. NaHCO3 (3 x 15 mL), brine (2 x 15 mL),
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dried over Na2SO4 and concentrated under reduced pressure. The product was
purified on silica-gel flash chromatography using cyclohexane/EtOAc (0% to 40%) as
eluent.
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Supporting Information
Supporting information is available on the ACS Publications website and is free of
charge. Supporting figures 1-4, spectral data of the quinolinols used in the study and
the molecular dynamics simulation report are included.
Acknowledgments
This research is supported by the Agency for Science, Technology, and Research
(A*STAR), Singapore and by the Max-Planck-Gesellschaft, Germany. We thank H.
Wollmann from IMCB NGS unit for the RNA-Seq library preparation and the
Advanced Molecular Pathology Laboratory in IMCB for their help with IHC staining.
We thank J. Lamar for the TEAD-firefly and Renilla luciferase constructs.
COMPETING INTERESTS
The authors have no competing interests.
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