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, 11 (1), 414

Structural Insight Into Small Molecule Action on Frizzleds

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Structural Insight Into Small Molecule Action on Frizzleds

Paweł Kozielewicz et al. Nat Commun.

Abstract

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The binding pocket of FZD6 accommodates SAG1.3.
a Sequence alignments of the binding pockets of the human SMO, FZD6, and FZD4 (Supplementary Fig. 1 and Supplementary Data file 34). Red squares indicate residues in close proximity (<4 Å) between SAG1.3 and the receptor from the SMO and FZD6 molecular dynamics (MD) simulations. b Structure of SAG1.3. The bold nitrogen represents the N2 referred to in the MD simulations below. c Comparison of the SAG1.3 binding sites of SMO and FZD6 (inactive model; upper panel), and SMO and FZD4 (lower panel) underlining the inability of FZD4 to accommodate a ligand-like SAG1.3 in this binding space because of the short TM6 (red arrow). d The last frames from the selected MD simulations of the SAG1.3–SMO (left panel) and the SAG1.3–FZD6 complexes (inactive model: middle panel, active-like model: right panel) with the important residues of the binding site depicted as sticks. Different positions of SAG1.3 throughout the time of simulation are indicated by transparent SAG1.3 molecules in the binding pocket. e Distance plots over simulation time between SMO D4736.54–SAG1.3, SMO E5187.38–SAG1.3, FZD6 E4386.54–SAG1.3, and R4426.58–SAG1.3 (inactive and active-like models), which are predicted to form H-bonding interactions important for stabilizing the SAG1.3 binding conformation. The dotted line (red) indicates the maximum distance (4 Å) that is still likely to allow polar interactions. Thick traces indicate the moving average smoothed over a 2 ns window and thin traces represent raw data. The origin of the y-axis for all graphs e is 0 Å.
Fig. 2
Fig. 2. BODIPY–cyclopamine and SAG1.3 bind to FZD6.
a The scheme depicts the experimental set up of NanoBRET analysis between the nanoluciferase-tagged FZD6 and the BODIPY–cyclopamine. b BODIPY–cyclopamine induces a saturable concentration-dependent increase of BRET ratio. The graph presents raw NanoBRET values obtained following 90 min ligand exposure to living ΔSMO HEK293 cells. Data are presented as mean ± s.e.m. of total n = 4 individual experiments. c SAG1.3 displaces bound BODIPY–cyclopamine (300 nM) in a concentration-dependent manner. Data are presented as mean ± s.e.m. of total n = 4 individual experiments. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. SAG1.3 induces conformational changes in FZD6.
a The model of the active-like FZD6 (blue) showing a pronounced outward-motion (Δ) of the TM6 as compared to the inactive model (gray), justifying positioning of FRET acceptor and donor in the ICL3 and C-terminus, respectively. b The scheme depicts the FZD6–FlAsH–TFP construct. The FlAsH-binding motif (FLNCCPGCCMEP) was inserted into the ICL3, between G404 and R405. Receptor activation is predicted to result in a loss of FRET due to conformational rearrangement in accordance to previous data obtained for FZD5. c WNT-5A induced a concentration-dependent decrease of the FRET ratio (FlAsH/TFP) in HEK293 cells overexpressing FZD6–FlAsH–TFP. The FRET ratio change induced by each concentration was normalized to basal FRET ratio. Data are represented as mean ± s.e.m. of total n = 5 individual experiments. d SAG1.3 induces a concentration-dependent decrease of the FRET ratio (FlAsH/TFP) in HEK293 cells overexpressing FZD6–FlAsH–TFP. The FRET ratio change induced by each concentration was normalized to basal FRET ratio. Data are presented as mean ± s.e.m. of total n = 7 individual experiments. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. SAG1.3 mediates recruitment of mGsi proteins to FZD6.
a The scheme depicts the experimental set up of BRET analysis between the luciferase-tagged FZD6 and the Venus-tagged mGsi. Ligand stimulation initiates the mG protein recruitment to the receptor resulting in the increase of BRET. WNT-5A induced a concentration-dependent recruitment of the Venus–mGsi to SNAP–FZD6Rluc8 (b; total n = 4 individual experiments) and FLAG–FZD6–Nluc (c; total n = 3 individual experiments) in transiently transfected HEK293 cells. SAG1.3 induced a bell-shaped, concentration-dependent recruitment of the Venus–mGsi to SNAP–FZD6Rluc8 (d; n = 10 individual experiments), FLAG–FZD6–Nluc or ΔCRD FLAG–FZD6–Nluc (e; total n = 11 individual experiments for FLAG–FZD6–Nluc, and total n = 8 individual experiments for ΔCRD FLAG–FZD6–Nluc) in transiently transfected HEK293 cells. f Similar experiments were performed in HEK293 lacking endogenous SMO (ΔSMO HEK293 cells). SAG1.3 showed concentration-dependent effects on SNAP–FZD6Rluc8 (f; total n = 11 individual experiments), FLAG–FZD6–Nluc (g; total n = 10 individual experiments) and ΔCRD FLAG–FZD6–Nluc-transfected (g; total n = 8 individual experiments) ΔSMO HEK293 cells. h SAG1.3 did not evoke Venus–mG13 recruitment to FZD4–Nluc, which is consistent with the in silico prediction (total n = 4 individual experiments). All BRET data are presented as mean ± s.e.m. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. SAG1.3 induces FZD6-dependent dissociation of heterotrimeric Gi and phosphorylation of ERK1/2.
a Schematic view of the split NanoBiT luciferase assay. Ligand stimulation of a GPCR results in dissociation of the heterotrimeric G protein and a decrease in the Nluc luminescence. b SAG1.3 stimulation of SNAP–FZD6 transiently overexpressed in ΔSMO HEK293 cells resulted in a concentration-dependent, biphasic decrease in basal luminescence as a measure of the dissociation of the Gαi1–LgBiT, SmBiT–Gβ5, and Gγ2 complex (filled black circles). pcDNA served as no-receptor-control (open gray circles). Data are represented as mean ± s.e.m. of n = 6 individual experiments. c SAG1.3 (10 min) induced phosphorylation of ERK1/2 (P-ERK1/2) in a biphasic manner only in SNAP–FZD6-transfected ΔSMO HEK293 cells. Serum starved cells were pretreated with C59 (5 nM; overnight). Representative immunoblots are shown. Data are presented as mean ± s.e.m. of n = 4 individual experiments; F(4,14) = 3.141. *P < 0.05, **P < 0.01 (one-way ANOVA). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. SAG1.3 modifies the interactions between FZD6 and DVL2.
a Schematic illustration of the bystander BRET setup to detect SNAP–FZD6-induced recruitment of Nluc–DVL2 to membrane bound Venus–KRas. b WNT-5A stimulation of SNAP–FZD6-transfected ΔFZD1–10 HEK293 cells increased the bystander BRET ratio in a concentration-dependent manner (filled black circles; total n = 5 individual experiments). WNT-5A did not affect BRET in cells transfected with pcDNA (open gray circles; total n = three individual experiments). c Bystander BRET ratio changes (Nluc–DVL2 and Venus–KRas) assessed in ΔSMO HEK293 cells in response to increasing concentrations of SAG1.3 in the presence of SNAP–FZD6 (filled black circles; total n = 9 individual experiments) or pcDNA (open gray circles; n = 5 individual experiments). d Bystander BRET ratio changes (Nluc–DVL2 and Venus–KRas) assessed in ΔSMO HEK293 cells in response to increasing concentrations of SAG1.3 in the presence of FLAG–FZD6–His (total n = 6 individual experiments) or ΔCRD FLAG–FZD6–His (total n = 6 individual experiments) in the ΔSMO HEK293 cells. e The scheme illustrating the direct BRET setup in which the signal is detected between FLAG–FZD6–Venus and Nluc–DVL2. f WNT-5A induced BRET ratio indicative of closer interactions between FLAG–FZD6–Venus and Nluc–DVL2 (total n = 6 individual experiments) in the ΔFZD1–10 HEK293 cells. g SAG1.3 induced BRET indicative of closer interactions between FLAG–FZD6–Venus (total n = 11 individual experiments) or ΔCRD FLAG–FZD6–Venus (total n = 6 individual experiments) and Nluc–DVL2 in the ΔSMO HEK293 cells. h SAG1.3 did not induce the bystander BRET (n = 4 individual experiments) or i TOPFlash reporter activity in the ΔSMO HEK293 cells with transiently overexpressed SNAP–FZD4 (WNT-3A used as a positive control; n = 3 individual experiments; F(2,12) = 88.69. ****P < 0.0001, two-way ANOVA). Data are presented as mean ± s.e.m. Source data are provided as a Source Data file.

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