Dishevelled (Dvl) proteins are important regulators of the Wnt signalling
pathway, interacting through their PDZ domains with the Wnt receptor
Frizzled. Blocking the Dvl PDZ–Frizzled interaction represents a potential
approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and
Ky-02327. Aiming to develop tighter binding compounds without side effects,
we investigated structure–activity relationships of sulfonamides. X-ray
crystallography showed high complementarity of anthranilic acid derivatives
in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC
Dishevelled (Dvl) proteins comprise 500 to 600 amino acids and contain three conserved domains: an N-terminal DIX (Dishevelled/Axin) domain
(Schwarz-Romond et al., 2007; Madrzak et al., 2015), a central PDZ (PSD95/Dlg1/ZO-1) domain (Doyle et al., 1996; Ponting et al., 1997), and a C-terminal DEP (Dishevelled/Egl-10/Pleckstrin) domain (Wong et al., 2000; Wallingford and Raymond, 2005). Dvl transduces Wnt signals from the membrane receptor Frizzled to downstream components via the interaction between Dvl PDZ and Frizzled (Wong et al., 2003); thus, it has been proposed as a drug target (Klaus and Birchmeier, 2008; Holland et al., 2013; Polakis, 2012). Several studies identified internal peptides of the type KTXXXW as well as C-terminal peptides of the type
PDZ domains appear in 440 copies spread over more than 260 proteins of the
human proteome (Ponting et al., 1997). They maintain relatively specific
protein–protein interactions and are involved, for example, in signalling
pathways, membrane trafficking, and in the formation of cell–cell junctions
(Zhang and Wang, 2003; Fanning and Anderson, 1996; Kurakin et al., 2007). Hence, they are potentially attractive drug targets (Rimbault et al., 2019; Christensen et al., 2020). PDZ domains consist of about 90 amino acids which fold into two
PDZ domains are divided into at least three main classes on the basis of
their amino acid preferences at these two sites: class I PDZ domains
recognize the motif S/T-X-
Due to their occurrence in important proteins, PDZ domains received early
attention as drug targets, which has been nicely summarized in Christensen et al. (2019). There are several examples of Dvl PDZ inhibitors of a peptide or peptidomimetic nature (e.g. Hammond et al., 2006; Haugaard-Kedstrom et al., 2021), including peptide conjugates (e.g. Qin et al., 2021; Hegedüs et al., 2021) and on an organic small-molecule basis. The
latter approach is considered most beneficial in long-term medical
treatments of conditions like cancer or neurological disorders. NSC668036
(Shan et al., 2005; Wang et al., 2015) is a peptide-mimic compound which interferes
with Wnt signalling at the Dvl level. Based on a computational pharmacophore
model of NCS668036, additional compounds were later reported (Shan et al., 2012).
Known as the first non-peptide inhibitor, the 1H-indole-5-carboxylic acid
derivative FJ9 (Fujii et al., 2007) showed therapeutic potential. Further examples including Sulindac (Lee et al., 2009b), 2-((3-(2-phenylacetyl)amino)benzoyl)amino)benzoic acid (3289-8625, also
called CalBioChem(CBC)-322338) (Grandy et al., 2009; Hori et al., 2018),
N-benzoyl-2-amino-benzoic acid analogues (Hori et al., 2018), phenoxyacetic acid analogue (Choi et al., 2016), and ethyl 5-hydroxy-1-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)-1H-indole-2-carboxylate
(KY-02327) (Kim et al., 2016) have been reported, with the last one showing the
highest in vitro affinity (8.3
The PDZ targeted library was designed to cover all PDZ domains with available structure. For this, all X-ray- and NMR-derived PDZ structures were retrieved from the PDB, clustered, and six selected centroids were subjected to the virtual screening routine. The area considered is shown in Fig. 1a, with the blue sphere indicating the geometrical centre. The clustering of the PDZ domains was performed according to the shapes of their binding sites rather than backbone conformation. This approach accounts for the importance of surface complementarity of protein–small-molecule interactions and the critical contribution of van der Waals interactions to the binding free energy. On the other hand, PDZ domains have evolved to recognize a carboxyl group that is mostly derived from the C-terminus of natively binding proteins. Finally, the fact that PDZ can recognize internal motifs (Hillier et al., 1999), including KTXXXW of Frizzled-7 recognized by Dvl PDZ (Wong et al., 2003; Chandanamali et al., 2009), raises the question of what are key binding contributions with PDZ domains: negative charge, hydrogen bonding, or shape complementarity (Harris et al., 2003). For this reason, tangible compounds were preselected to have extensive hydrophobic contacts as well as chemical groups that mimic the carboxylic group.
Virtual screening was performed with QXP (McMartin and Bohacek, 1997), and the generated complexes were sequentially filtered with a self-designed MultiFilter algorithm. From the resulting 1119 compounds a randomly selected set of 250 compounds was subjected to NMR validation.
The results of virtual screening were checked experimentally by comparing 2D
Upon NMR titration experiments for compound
Compounds
By comparing the binding of several sulfonamide compounds in a secondary
screening event and making use of our in-house library, four new compounds
(
Basic fragment for further synthesis.
In order to assess the importance of the aryl group at R
Binding constants K
nd: not determined.
To further explore the importance of the fluorine site inside the
hydrophobic pocket, substitutions by bromine, chlorine, methyl, and
trifluoromethyl were chosen. In fact, the methyl group has a similar van der Waals (vdW)
radius as the CF
The 1.85 Å crystal structure of the Dvl-3 PDZ domain with compound
Following the analysis of the complex involving compound
The group-3 compounds (
Possible cytotoxic effects of compounds
Isothermal titration calorimetric data for the reaction between
Dvl-3 PDZ; Dvl-1 PDZ; and our compounds
Our compounds bind to Dvl-3 with a K
Compounds
Taking advantage of a lentivirus that encodes GFP in a
DVL inhibitors antagonize Wnt signalling and Wnt-related
properties of cancer cells induced by Wnt3a.
To further evaluate the specificity of our Dvl inhibitors, the conventional
TOPflash (Molenaar et al., 1996) and other luciferase reporter assays were
performed. In HeLa cells, 20 inhibited TOP-luciferase signals stimulated by
Wnt3a but not by CHIR99021 (Sineva and Pospelov, 2010), a compound that activates Wnt
signalling downstream of Dvl (Fig. 2a, c). Compound
Increased
Canonical Wnt signalling contributes to cancer progression by inducing high
motility and invasion of cancer cells while retaining the self-renewal
property of cancer-initiating cells (Fritzmann et al., 2009; Sack et al., 2011; Vermeulen et al., 2010; Malanchi et al., 2008). In particular, cancer-initiating cells are propagated and enriched in non-adherent sphere culture, demonstrating the self-renewal capacity of the stem cells (Kanwar et al., 2010; Fan et al., 2011). To investigate the
potential value of the Dvl inhibitors for interfering with these Wnt-related
properties of cancer cells, the sub-line SW480WL was derived from the SW480
colon cancer cell line, which exhibits a low level of endogenous Wnt
activity (Fang et al., 2012). The cell migration and self-renewal properties of SW480WL cells were enhanced by Wnt3a treatment, as revealed by trans-well and sphere formation assays (Fig. 2g, h). Compounds
Three-dimensional structures of PDZ domains were retrieved from the PDB (Berman et al., 2000). At the time of the study, from a total of 266 PDB files 126 were NMR solution structures and 140 were derived from X-ray diffraction studies. The structures belong to 163 PDZ domains of 117 different proteins from 11 organisms. Files which contain more than one 3D conformation for a domain (up to 50 for NMR-derived data) were split into separate structures and were considered independently. The total number of unique 3D structures was 2708.
Amino acid sequences of PDZ domains were aligned using Clustal Omega
software (Sievers et al., 2011). Based on the alignment, for each structure,
residues which form the binding site (strand
The triangulated solvent-accessible surface for each PDZ structure was built
using MSMS software (Sanner et al., 1996) with a spherical probe radius of 1.4 Å and vertex density 10 Å
Screening collection by Enamine Ltd. (Chuprina et al., 2010), containing a total of 1 195 395 drug-like compounds, was used as the primary source of small molecules. The natural ligand of PDZ is the C-terminus of a peptide with
carboxylic group making an extensive hydrogen-bond network with the “
Processing of docking poses started with filtering by contact term
Two-dimensional
All reagents and starting materials were purchased from Sigma-Aldrich Chemie, ABCR, Alfa Aesar, or Acros Organics and
used without further purification. All air or moisture-sensitive reactions
were carried out under dry nitrogen using standard Schlenk techniques.
Solvents were removed by evaporation on a Heidolph Laborota 4000 with vacuum
provided by a PC 3001 Vaccubrand pump. Thin-layer chromatography (TLC) was
performed on plastic-backed plates pre-coated with silica gel 60 F
To a solution of anthranilic acid substituted with the appropriate R
Synthesis of compounds
(0.52 g, 74 % yield)
(0.13 g, 67 % yield)
(0.07 g, 42 % yield)
(0.6 g, 29 % yield)
(0.6 g, 78 % yield)
(0.4 g, 63 % yield)
(0.4 g, 65 % yield)
(0.38 g, 62 % yield)
50
Binding was deduced if the resonance position of a cross peak was significantly shifted compared to the reference spectrum. The active compound was obtained through successive deconvolution. Experiments were recorded on a Bruker DRX600 spectrometer equipped with a triple-resonance cryoprobe. The preparation of samples was done automatically by a Tecan Genesis RSP 150 pipetting robot. Spectra were analysed using the programs TOPSPIN and SPARKY.
Chemical shift perturbations were obtained by comparing the
Isothermal titration calorimetry (ITC) experiments were performed using a
VP-ITC system (MicroCal). Protein in 20 mM HEPES buffer and 50 mM NaCl (pH 7.4) was centrifuged and degassed before the experiment. A 200
PDZ domains of human AF6 (P55196-2, residues 985–1086) and murine
The production of Dvl-3 (Q92997 residues 243–336), mShank3 (Q4ACU6, residues 637–744) PDZ domains and the three PDZ domains of PSD95 was described by Saupe et al. (2011).
The His-tagged cleaved human Dvl-3 PDZ domain was concentrated to 12–20 mg mL
Phases for the Dvl-3 PDZ domain in complex with compound
HEK293 cells were plated on a 96-well plate and treated with different
concentrations of Dvl inhibitors. After 24 h treatment, 20
The lentivirus particle (CCS-018L, SABiosciences) encoding GFP under the
control of a basal promoter element (TATA box) joined to tandem repeats of a
consensus TCF/LEF binding site was transfected into HEK293 cells. Stable
cells were selected by puromycin (2
Plasmids encoding a firefly luciferase reporter gene under the control of
different responsive elements were transfected into HeLa cells with a
pRL-SV40 normalization reporter plasmid using the Lipofectamine 2000
(Invitrogen). After desired treatment, cells were harvested in the passive
lysis buffer (Promega), and 15
To assess the
To measure the Wnt target accumulation at mRNA level, HeLa cells were
treated with Wnt3a in the presence of Dvl inhibitors for 24 h. mRNA was
extracted according to the standard TRIzol® protocol (Invitrogen) and reverse-transcribed using random primers (Invitrogen) and M-MLV reverse transcriptase (Promega). The qRT-PCR was performed in a iQ5 multi-colour real-time PCR detection system (Bio-Rad) using
SYBR® Green (Thermo Scientific) and gene-specific primer pairs of Bmp2, Axin2, LEF1, and
Cell motility was assessed using 24-well trans-well assay (pore diameter: 8
SW480WL cells were trypsinized into single cells, seeded on 24-well cell
culture plates, pre-coated with 250
In the present work, small molecules that bind to Dvl PDZ in the one-digit micromolar range with considerable selectivity have been developed by an extensive structure-based design approach. With regards to the affinity
determined by ITC, compound
Accession codes, atomic coordinates, and structure factor amplitudes for Dvl-3 PDZ domain in complex with compounds
The underlying research data are available from the authors upon request by email.
The supplement related to this article is available online at:
HO, WB, UH, and JR initiated and planned the project. NK conducted the NMR screening and the NMR titration, and YR conducted crystallization, X-ray data collection, structure determination, and refinement. YR and NK performed isothermal shift experiments, NK and JS conducted chemical synthesis, PS developed pulse programs and set up NMR experiments, DK, MOP, and OB conducted virtual screening, AD conducted protein expression, AK and GK studied structure activity relationships, LF conducted biological experiments, HO, NK, YR and DK wrote the first versions of the manuscript.
The authors declare that they have no conflict of interest.
This article is part of the special issue “Robert Kaptein Festschrift”. It is not associated with a conference.
We thank Martina Leidert and Silke Radetzki for protein preparation and Brigitte Schlegel for NMR assistance. We also thank Edgar Specker for compound analysis.
This research has been supported by the European Commission, Horizon 2020 (iNEXT (grant no. 653706)) and the Deutsche Forschungsgemeinschaft (DFG), Research Group 806.The publication of this article was funded by the Open Access Fund of the Leibniz Association.
This paper was edited by Rolf Boelens and reviewed by Mingjie Zhang and one anonymous referee.