Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: A-B) Comparison of transcriptional PEM effects in intestinal epithelial cells (IEC, blue) and organoids (yellow) isolated of mice from two separate dietary PEM-recovery intervention experiments during (A) acute PEM and (B) after recovery. The dot plot depicts the Log 2 [fold-changes PEM_4w/10w vs Ctrl_4w/10w] whereas the Venn diagram depicts the overlap in up- and downregulated DEGs. C) Number of differentially (hypo- and hyper-) methylated positions (DMPs) detected by BeadCHiP array in small intestinal epithelial cells from mice during acute PEM (4 weeks) and after recovery (10 weeks) and respective controls. D) Shared DMPs between acute PEM (4 weeks) and recovery (10 weeks). E) Predicted transcription factor binding sites enriched in observed DMPs. Dot size is proportional to the odds ratio and color corresponds to the p-value of enrichment. Top selected transcription factors are visualized. F) Schematic depiction of gene expression - DNA methylation integration analysis. Spearman’s rank correlation coefficient between the methylation intensity and gene expression values was calculated between the DMPs located within a range of 5kb before or after the transcription start site of the respective DEG. G) Venn Diagram depicting the overlaps between shared DEGs between PEM_4w and Rec_10w and DEGs correlated with their nearby DMPs, identifying 31 candidate DEGs linked with DMPs including GDF15 that has been linked to body mass regulation.

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Comparison, Isolation, Methylation, Binding Assay, Gene Expression, DNA Methylation Assay

Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: A) Expression of Gdf15 , Pparg and Pparbd were assessed in small intestinal tissue of both CONVR and GF mice subjected to PEM and after recovery. *p < 0.05, ***p < 0.001 and ****p < 0.0001, ns = non-significant. Significance testing was performed using Wilcoxon-Mann-Whitney-Test. B) Schematic drawing of organoid intervention experiment. Murine intestinal organoids were first cultured in ENR-CV stem cell organoid medium and then Paneth cell differentiation was induced using ENR-CD or PEM-CD medium. Stimulants (recombinant GDF15 [1 µg/ml], pan-PPAR-agonist Lanifibranor [20 µM] or the lipid 9-HODE [1 µM] were added during initial ENR-CD and PEM-CD culture and kept throughout the experimental duration. All experiments were performed independently at least twice in triplicates. C-E) Relative Lyz1 , Defa5 and Gdf15 mRNA expression in organoids during Paneth cell differentiation and stimulated with (C) recombinant GDF15, (D) the Pan-PPAR-agonist Lanifibranor or the lipid (E) 9-HODE. Note that PPAR-activation boosts GDF15 levels thereby blocking Paneth cell differentiation and the bacterial PEM metabolite 9-HODE also suppresses Paneth cell differentiation. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 and ns = not significant using Mann-Whitney U test.

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Expressing, MANN-WHITNEY, Cell Culture, Cell Differentiation, Recombinant, Activation Assay, Blocking Assay

Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: Expression of Lyz1 and Gdf15 were assessed in intestinal organoids stimulated with the (A) pan-PPAR-agonist Lanifibranor [20 µM] or the (B) lipid 9-HODE [1 µM] during Paneth cell differentiation. Note that stimulation with Lanifibranor or 9-HODE did not alter Lyz1 or Gdf15 expression in contrast to stimulation during PEM as shown in .

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Expressing, Cell Differentiation

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Cryo-EM structure of the extracellular GDF15–GFRAL–RET complex (PDB: 6Q2J) showing a 2:2:2 stoichiometry, wherein the dimeric GDF15 bridges two GFRAL co-receptors and two RET receptors. b. Binding interfaces of GDF15 with GFRAL (top) and RET (bottom). Key interacting residues are shown as sticks. Hydrophobic hotspot residues used for binder design are highlighted in pink. c. Target sites for GDF15 binder design. The convex surface engaging GFRAL (site A, red) and the concave surface contacting RET (site B, light blue) are highlighted. Insets show electrostatic surface potentials of each site (white, hydrophobic; blue, positive charge; red, negative charge). d. Workflow of binder design using three scaffold generation strategies: Scaffold Grafting (SG), Diffusion-based de novo Design, and Scaffold-Search and Grafting (SSG).

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Cryo-EM Sample Prep, Binding Assay, Diffusion-based Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of binder design via scaffold grafting. The GFRAL D2 domain was extracted as the initial scaffold to generate SG A 1 and SG A 2. SG A 2 was further optimized by scaffold-guided partial diffusion, resulting in five variants (SG A 2-1 to SG A 2-5). b. Extracted GFRAL D2 scaffold (residues 129∼211) from the GFRAL extracellular domain. The N- and C-termini, helices, and disulfide bonds are indicated. c. Backbone RMSD distribution of 100 ProteinMPNN-designed variants relative to the parental GFRAL D2 scaffold (mean RMSD = 0.85 Å). d. Structural alignment of the initial scaffold GFRAL D2 with SG A 1 (left) and SG A 2 (right). RMSD with AF3-predicted structure and AF3 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. e. Structural comparison of GFRAL-D2, SG A 1, and SG A 2 at the binding interface. Residues that enhance binding, commonly observed in both SG A 1 and SG A 2, are indicated. The rightmost panel shows the superposition of three scaffolds, highlighting α5 displacement. f. Superposition of SG A 1 and SG A 2, showing a unique electrostatic interaction in SG A 2. g. Structural alignment of SG A 2 with its partial diffusion-derived variants. RMSD with AF2-predicted structure and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. h. Backbone RMSD distribution of partial diffusion–derived variants relative to the SG A 2 scaffold (mean RMSD = 4.6 Å). i. Structural model of SG A 2-4 at the binding interface. Conserved binding residues in α5 (D69, Q72, L73, Q76), retained in SG A 2-4 variants compared to SG A 2, are highlighted. j. Binding interface comparison of SG A 2 and SG A 2-4, highlighting the shifted α1 position and distinct interacting residues on this helix.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Diffusion-based Assay, Labeling, Comparison, Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet:

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of de novo binder design using RF diffusion. A total of 1,728 initial scaffolds (50∼90 a.a) were generated, sequence-designed (three sequences per each backbone), and computationally filtered using in silico evaluation metrics. The top five binders (DE A 1–DE A 5) were structurally analyzed and experimentally validated by expression, purification, and binding analysis. The best-performing DE A 3 was further optimized by scaffold-guided partial diffusion, resulting in seven variants (DE A 3-1 to DE A 3-7). b. Distribution of helix counts in RFdiffusion-generated scaffolds to analyze structural diversity. c. AF2-predicted structural models of the five selected de novo binder candidates in complex with the GDF15 dimer. Binder lengths, pAE_interaction, and pLDDT values are indicated, with the N- and C-termini labeled. d. SDS-PAGE analysis of binders (DE A 1–5, left; DE A 3-1 to DE A 3-7, right) after E. coli expression and affinity purification. e. Binding interface comparison of DE A 3 (left) and DE A 3-5 (right) with GDF15. Key interacting residues are shown as sticks and labeled.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Diffusion-based Assay, Generated, Sequencing, In Silico, Expressing, Purification, Binding Assay, Labeling, SDS Page, Affinity Purification, Comparison

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. The RET domain segment (residues 586–622) was tested as an initial scaffold for site B binders design. b. AF2-predicted structures of 100 RET-derived variants by sequence design. Only disulfide-constrained β-strands remained folded. c. In silico filtering of 500 de novo backbones generated by RFdiffusion with site B hotspot constraints (W225, W228, M253, and Y297). No candidates satisfied filtering thresholds (pLDDT > 85 and pAE_interaction < 10; red box). d. Workflow of the scaffold search and grafting (SSG) strategy. The GDF15 structure was used as a query in the DALI server to search the Protein Data Bank (PDB) for natural scaffolds with similar topology and surface geometry. Candidate scaffolds were then subjected to scaffold-guided partial diffusion and sequence design. e. Representative scaffold candidates identified from the DALI server search: Follistatin/Activin A (PDB 2B0U), BMP9 pro-complex (mature domain + prodomain) (PDB 4YCI), BMP2/RGMA (PDB 4UHY), TGF-β3/GC-1008 antibody (PDB 3EO1), and BMP2/BMP2 receptor A (PDB 1ES7). RMSD relative to GDF15 is indicated. f. Structural comparison of GDF15/RET (green/pink) and BMP2/RGMA (olive/purple) complexes. GDF15 and BMP2 show overall similarity (RMSD = 2.5 Å), but interacting partners differ topologically. g. Structural alignment of RGMA with RGMA-derived binder variants (SSG B 1 to SSG B 5). RMSD with AF2-predicted structures and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated. h. SDS-PAGE analysis of binders (SSG B 1-SSG B 5) after E. coli expression and affinity purification.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Derivative Assay, Sequencing, In Silico, Generated, Diffusion-based Assay, Comparison, SDS Page, Expressing, Affinity Purification

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a, b. Schematic illustration (a) and structural model (b) of the BAT biosensor (SmBiT-GDF15 binder-LgBiT) for GDF15 detection in the “OFF” and “ON” states. The BAT biosensor consists of a designed GDF15 binder (red) flanked by SmBiT at the N-terminus (cyan, 1, SmBiT) and LgBiT at the C-terminus (blue, 2, LgBiT). In the absence of GDF15, two split luciferase fragments remain apart (“OFF” state with only background activity). Upon GDF15 binding, steric constraints bring two split luciferase fragments into proximity, enabling fragment complementation and restoring NanoLuc activity (“ON” state). The lengths of linker1 and linker2 (b, left) are key determinants for background signals in the absence of GDF15. c. SDS-PAGE analysis of SmBiT-DE A 3-LgBiT with various linker combinations after E.coli expression and affinity purification. d. Screening of linker combinations for SmBiT-DE A 3-LgBiT using a luminescence assay. Signal-to-noise ratios (luminescence intensity of each construct divided by that of the control without GDF15) are shown, with optimal linker combinations highlighted in red. e, g. Luminescent signals of SmBiT-DE A 3-LgBiT with 0-10 linkers (e) and of SmBiT-DE A 3-5-LgBiT with 5-5 linkers (g). Luminescence (arbitrary units, AU) is plotted against various concentrations of human or mouse GDF15 (n = 3). The linear detection range is indicated with a red box (0–10 nM). f. Sequence alignment of human and mouse GDF15. Conserved residues at site A are marked with black circles; species-specific substitutions at the interface are highlighted with green circles. h. AF3-predicted structures of DE A 3 or DE A 3-5 bound to human or mouse GDF15. Per-residue pLDDT values are color-coded according to the scale bar.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Luciferase, Activity Assay, Binding Assay, SDS Page, Expressing, Affinity Purification, Luminescence Assay, Construct, Control, Sequencing, Residue

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Schematic diagram of Fc-fused SG A 2-4 binder (SG A 2-4-Fc). b. SEC profile of SG A 2-4-Fc on a Superdex® 200 Increase 10/300 GL column (left) and SDS-PAGE analysis of elution fractions (right). c. SPR analysis of SG A 2-4-Fc and ponsegromab binding to immobilized GDF15. Sensorgrams are shown for analytes ranging from 5 to 50 nM (SG A 2-4-Fc or ponsegromab). d. Inhibition of GDF15-induced RET, AKT, and ERK phosphorylation in HEK293T cells stably expressing GFRAL and RET. Cells were co-treated with GDF15 (10 nM, 246 ng/ml) and either SG A 2-4-Fc or ponsegromab (100 nM; 7.8 μg/ml for SG A 2-4-Fc or 14.6 μg/ml for ponsegromab) for 30 mins. Phosphorylation was quantified relative to total protein (RET, AKT, and ERK each), normalized to the GDF15-only condition (n = 3). Statistical significance was determined using unpaired t-test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant). e. Dose-dependent inhibition of GDF15-induced SRE-luciferase activity by SG A 2-4-Fc or ponsegromab in HEK293T cells co-expressing GFRAL, RET, and an SRE-Luc2 reporter. Data were normalized to GDF15-induced luminescence (100%), and IC 50 values were determined by non-linear regression fitting in GraphPad Prism.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: SDS Page, Binding Assay, Inhibition, Phospho-proteomics, Stable Transfection, Expressing, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Cryo-EM structure of the extracellular GDF15–GFRAL–RET complex (PDB: 6Q2J) showing a 2:2:2 stoichiometry, wherein the dimeric GDF15 bridges two GFRAL co-receptors and two RET receptors. b. Binding interfaces of GDF15 with GFRAL (top) and RET (bottom). Key interacting residues are shown as sticks. Hydrophobic hotspot residues used for binder design are highlighted in pink. c. Target sites for GDF15 binder design. The convex surface engaging GFRAL (site A, red) and the concave surface contacting RET (site B, light blue) are highlighted. Insets show electrostatic surface potentials of each site (white, hydrophobic; blue, positive charge; red, negative charge). d. Workflow of binder design using three scaffold generation strategies: Scaffold Grafting (SG), Diffusion-based de novo Design, and Scaffold-Search and Grafting (SSG).

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Cryo-EM Sample Prep, Binding Assay, Diffusion-based Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of binder design via scaffold grafting. The GFRAL D2 domain was extracted as the initial scaffold to generate SG A 1 and SG A 2. SG A 2 was further optimized by scaffold-guided partial diffusion, resulting in five variants (SG A 2-1 to SG A 2-5). b. Extracted GFRAL D2 scaffold (residues 129∼211) from the GFRAL extracellular domain. The N- and C-termini, helices, and disulfide bonds are indicated. c. Backbone RMSD distribution of 100 ProteinMPNN-designed variants relative to the parental GFRAL D2 scaffold (mean RMSD = 0.85 Å). d. Structural alignment of the initial scaffold GFRAL D2 with SG A 1 (left) and SG A 2 (right). RMSD with AF3-predicted structure and AF3 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. e. Structural comparison of GFRAL-D2, SG A 1, and SG A 2 at the binding interface. Residues that enhance binding, commonly observed in both SG A 1 and SG A 2, are indicated. The rightmost panel shows the superposition of three scaffolds, highlighting α5 displacement. f. Superposition of SG A 1 and SG A 2, showing a unique electrostatic interaction in SG A 2. g. Structural alignment of SG A 2 with its partial diffusion-derived variants. RMSD with AF2-predicted structure and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. h. Backbone RMSD distribution of partial diffusion–derived variants relative to the SG A 2 scaffold (mean RMSD = 4.6 Å). i. Structural model of SG A 2-4 at the binding interface. Conserved binding residues in α5 (D69, Q72, L73, Q76), retained in SG A 2-4 variants compared to SG A 2, are highlighted. j. Binding interface comparison of SG A 2 and SG A 2-4, highlighting the shifted α1 position and distinct interacting residues on this helix.

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Diffusion-based Assay, Labeling, Comparison, Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet:

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of de novo binder design using RF diffusion. A total of 1,728 initial scaffolds (50∼90 a.a) were generated, sequence-designed (three sequences per each backbone), and computationally filtered using in silico evaluation metrics. The top five binders (DE A 1–DE A 5) were structurally analyzed and experimentally validated by expression, purification, and binding analysis. The best-performing DE A 3 was further optimized by scaffold-guided partial diffusion, resulting in seven variants (DE A 3-1 to DE A 3-7). b. Distribution of helix counts in RFdiffusion-generated scaffolds to analyze structural diversity. c. AF2-predicted structural models of the five selected de novo binder candidates in complex with the GDF15 dimer. Binder lengths, pAE_interaction, and pLDDT values are indicated, with the N- and C-termini labeled. d. SDS-PAGE analysis of binders (DE A 1–5, left; DE A 3-1 to DE A 3-7, right) after E. coli expression and affinity purification. e. Binding interface comparison of DE A 3 (left) and DE A 3-5 (right) with GDF15. Key interacting residues are shown as sticks and labeled.

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Diffusion-based Assay, Generated, Sequencing, In Silico, Expressing, Purification, Binding Assay, Labeling, SDS Page, Affinity Purification, Comparison

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. The RET domain segment (residues 586–622) was tested as an initial scaffold for site B binders design. b. AF2-predicted structures of 100 RET-derived variants by sequence design. Only disulfide-constrained β-strands remained folded. c. In silico filtering of 500 de novo backbones generated by RFdiffusion with site B hotspot constraints (W225, W228, M253, and Y297). No candidates satisfied filtering thresholds (pLDDT > 85 and pAE_interaction < 10; red box). d. Workflow of the scaffold search and grafting (SSG) strategy. The GDF15 structure was used as a query in the DALI server to search the Protein Data Bank (PDB) for natural scaffolds with similar topology and surface geometry. Candidate scaffolds were then subjected to scaffold-guided partial diffusion and sequence design. e. Representative scaffold candidates identified from the DALI server search: Follistatin/Activin A (PDB 2B0U), BMP9 pro-complex (mature domain + prodomain) (PDB 4YCI), BMP2/RGMA (PDB 4UHY), TGF-β3/GC-1008 antibody (PDB 3EO1), and BMP2/BMP2 receptor A (PDB 1ES7). RMSD relative to GDF15 is indicated. f. Structural comparison of GDF15/RET (green/pink) and BMP2/RGMA (olive/purple) complexes. GDF15 and BMP2 show overall similarity (RMSD = 2.5 Å), but interacting partners differ topologically. g. Structural alignment of RGMA with RGMA-derived binder variants (SSG B 1 to SSG B 5). RMSD with AF2-predicted structures and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated. h. SDS-PAGE analysis of binders (SSG B 1-SSG B 5) after E. coli expression and affinity purification.

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Derivative Assay, Sequencing, In Silico, Generated, Diffusion-based Assay, Comparison, SDS Page, Expressing, Affinity Purification

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a, b. Schematic illustration (a) and structural model (b) of the BAT biosensor (SmBiT-GDF15 binder-LgBiT) for GDF15 detection in the “OFF” and “ON” states. The BAT biosensor consists of a designed GDF15 binder (red) flanked by SmBiT at the N-terminus (cyan, 1, SmBiT) and LgBiT at the C-terminus (blue, 2, LgBiT). In the absence of GDF15, two split luciferase fragments remain apart (“OFF” state with only background activity). Upon GDF15 binding, steric constraints bring two split luciferase fragments into proximity, enabling fragment complementation and restoring NanoLuc activity (“ON” state). The lengths of linker1 and linker2 (b, left) are key determinants for background signals in the absence of GDF15. c. SDS-PAGE analysis of SmBiT-DE A 3-LgBiT with various linker combinations after E.coli expression and affinity purification. d. Screening of linker combinations for SmBiT-DE A 3-LgBiT using a luminescence assay. Signal-to-noise ratios (luminescence intensity of each construct divided by that of the control without GDF15) are shown, with optimal linker combinations highlighted in red. e, g. Luminescent signals of SmBiT-DE A 3-LgBiT with 0-10 linkers (e) and of SmBiT-DE A 3-5-LgBiT with 5-5 linkers (g). Luminescence (arbitrary units, AU) is plotted against various concentrations of human or mouse GDF15 (n = 3). The linear detection range is indicated with a red box (0–10 nM). f. Sequence alignment of human and mouse GDF15. Conserved residues at site A are marked with black circles; species-specific substitutions at the interface are highlighted with green circles. h. AF3-predicted structures of DE A 3 or DE A 3-5 bound to human or mouse GDF15. Per-residue pLDDT values are color-coded according to the scale bar.

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: Luciferase, Activity Assay, Binding Assay, SDS Page, Expressing, Affinity Purification, Luminescence Assay, Construct, Control, Sequencing, Residue

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Schematic diagram of Fc-fused SG A 2-4 binder (SG A 2-4-Fc). b. SEC profile of SG A 2-4-Fc on a Superdex® 200 Increase 10/300 GL column (left) and SDS-PAGE analysis of elution fractions (right). c. SPR analysis of SG A 2-4-Fc and ponsegromab binding to immobilized GDF15. Sensorgrams are shown for analytes ranging from 5 to 50 nM (SG A 2-4-Fc or ponsegromab). d. Inhibition of GDF15-induced RET, AKT, and ERK phosphorylation in HEK293T cells stably expressing GFRAL and RET. Cells were co-treated with GDF15 (10 nM, 246 ng/ml) and either SG A 2-4-Fc or ponsegromab (100 nM; 7.8 μg/ml for SG A 2-4-Fc or 14.6 μg/ml for ponsegromab) for 30 mins. Phosphorylation was quantified relative to total protein (RET, AKT, and ERK each), normalized to the GDF15-only condition (n = 3). Statistical significance was determined using unpaired t-test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant). e. Dose-dependent inhibition of GDF15-induced SRE-luciferase activity by SG A 2-4-Fc or ponsegromab in HEK293T cells co-expressing GFRAL, RET, and an SRE-Luc2 reporter. Data were normalized to GDF15-induced luminescence (100%), and IC 50 values were determined by non-linear regression fitting in GraphPad Prism.

Article Snippet: Cells were treated with recombinant human GDF15 (10 nM; #9279-GD, R&D Systems) in the presence or absence of SG A 2-4-Fc or ponsegromab (100 nM) for 30 min. After washing with cold PBS, cells were lysed in RIPA buffer (#RC2002-050-00, Biosesang) supplemented with phosphatase inhibitor (#4906845001, Roche) and protease inhibitor cocktail (#11836170001, Roche) for 60 min at 4°C.

Techniques: SDS Page, Binding Assay, Inhibition, Phospho-proteomics, Stable Transfection, Expressing, Luciferase, Activity Assay