s961  (TargetMol)

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a-b) Cartoon of (a) the apo/inactive FL-IR illustrating the physical separation of the TK domains, and (b) the insulin-bound/active FL-IR showing dimerization of the TK domains. Cartoon generated using PDBs: 6PXV, 4ZXB, Alphafold3 , and BioRender.com. The ectodomain (ECD) is boxed in (a-b). (c) Domain organization and disulfide linkages of FL-IR drawn to scale using illustrator of biological sequences . The α and β chains are indicated and both protomers are shown. (d) Structure of the apo IR ECD in the inverted-V conformation (PDB: 4ZXB). (e) Structure of the IR showing only the ECD bound to one site 1 insulin in an asymmetric, active conformation (PDB: 7STI). (f) Structure of the IR showing only the ECD bound to four insulin molecules in the activated T-shape conformation (PDB: 6PXV). (g) Structure of the IR showing only the ECD bound to two S597 molecules (PDB: 8DTL), where IR adopts a variation of the active T shape conformation. For structures shown in panels (d-g), one IR protomer is shown in grey and the other color coded by domain according to the color scheme shown in panel (c): leucine-rich 1 domain (L1, light blue), cysteine-rich domain (CR, red), leucine-rich 2 domain (L2, green), fibronectin type-III 1 domain (FnIII-1, dark purple), fibronectin type-III 2 domain (FnIII-2, coral), alpha C-terminal helix (αCT, pink), insert domain (ID, pink), and fibronectin type-III 3 domain (FnIII-3, dark blue). (h) Sequences of human insulin and IR ligands used in this study with site 1- and site 2-binding segments indicated. Human insulin A and B chains are colored magenta and gold, respectively. S597 and S961 site 1 and site 2 segments are shown in purple and salmon, respectively. The S961 flexible linker is shown in grey. The Ins-AC-S2 A chain contains binding segments for site 1 and site 2 (colored in magenta and salmon, respectively), with the linker shown in grey. The Ins-AC-S2 B chain is shown in gold. Disulfide bonds are indicated.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Generated, Binding Assay

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a-b) Cryo-EM density map determined to 3.68 Å from 378,182 particles showing (a) front view and (b) top view of the receptor. Protomers 1 and 2 are colored in dark and light blue, respectively. The density for S961 site 1- and site 2-binding segments are colored in purple and salmon, respectively, and the linker density is shown in grey. The region with missing density corresponding to the FnIII-3 domain of protomer 2 is circled. (c) Model of the IR/S961 complex showing the separation distance of the fibronectin stalks (distance measured from L909 to L909’ on the opposite protomer). (d-f) Representative map and model of (d) S961 site 1 helix, (e) β-strand from L1 belonging to IR site 1, and (f) S961 site 2 helix. (g-h) Side views of IR/S961 density map and model from (a) showing (g) the better reconstructed, and (h) the less well reconstructed receptor halves with contour levels adjusted to see S961 at both sets of binding sites. Zoomed in views of the map and model overlayed at both sets of binding sites are shown with binding sites labeled.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Cryo-EM Sample Prep, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a) IR/S961 model showing the site 1 helix overlayed with S519C16 (site 1 component, grey, PDB: 5J3H) and S597 (white, PDB: 8DTL) aligned to L1. (b) IR/S961 model showing the site 2 helix overlayed with isolated site 2 peptide (tan, PDB: 8DTM) and S597 (grey, PDB: 8DTL) aligned to FnIII-1. (c) Side view showing half of the IR/S961 complex (color) overlayed with apo IR (white, PDB: 4ZXB). (d) Published structure of the IR/S597 complex (PDB: 8DTL). (e-f) Side view showing one half of (e) the IR/S961 complex and (f) the IR/S597 complex highlighting the N- to C-terminal orientation of S961 and S597 with arrows. Site 1 and site 2 are labeled in purple and salmon, respectively.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Isolation, Labeling

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a) Overlay of density maps obtained from the two most different subsets of particles obtained by 3DVA shown in tan and steel blue. The dynamic receptor arm containing site 1 is boxed. (b-c) Zoomed in view of S961 at site 1 and site 2 fit into the density maps shown in (a). (b) Map showing strong density for S961 at site 1 determined to 3.99 Å resolution from 343,705 particles. (c) Map showing weak density for S961 at site 1 determined to 3.97 Å from 386,871 particles. Site 1 and site 2 helices of S961 are labeled. (d) IR/S961 site 1 helix overlayed with apo IR aCT (white, PDB: 4ZXB) showing sidechains pointed towards the L1 domain and aligned to the L1 domain.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Labeling

Journal: Stem Cell Research & Therapy

Article Title: hucMSC-derived exosomes targeting macrophage polarization attenuate systemic inflammation in T1DM via INS/SOD1 delivery

doi: 10.1186/s13287-025-04521-0

Figure Lengend Snippet: Anti-inflammatory protein components in hucMSC-EXOs. ( A-F ) qPCR was performed to compare the effects of heat-inactivated hucMSC-EXOs and untreated hucMSC-EXOs on the expression levels of inflammatory cytokines in macrophages. ( G ) Topological analysis of hucMSC-EXOs proteins (top 200 by IBAQ value) via the STRING database, identifying the top 40 hub proteins ranked by interaction degree. Their degrees of interaction are visualized as a concentric circle plot. ( H-K ) ELISA was used to detect the INS ( H-I ) content and SOD1 ( J-K ) content of the hucMSCs culture supernatant and hucMSC-EXOs. (L‒O) qPCR analysis the effects of EXO alone, EXO combined with S961, and EXO combined with ATN-224 on the expression of inflammatory factors in macrophages. ( P ) WB experiment analysis the influence of EXO alone, EXO combined with S961, and EXO combined with ATN-224 treatment on PI3K/AKT and P65 phosphorylation in macrophages. * P < 0.05; ** P < 0.01 ; *** P < 0.001

Article Snippet: THP-1 cells were differentiated into M0 macrophages with 100 nM phorbol myristate acetate (PMA; HY-18739, MCE, US) for 24 h, and then divided into six experimental groups: PMA control, lipopolysaccharide (LPS; 50 ng/mL; L2880, Sigma, US), LPS + 5 nM INS (GC2646, Genxion, China), LPS + 1 μg SOD1 (HY-P71048A, MCE, US), LPS + hucMSC-EXOs (8.5 × 10^5 particles), LPS + heat-inactivated hucMSC-EXOs (8.5 × 10^5 particles), LPS + hucMSC-EXOs + 5 nM INS receptor antagonist S961 (HY-P2093B, MCE, US), and LPS + hucMSC-EXOs + 20 nM SOD1 inhibitor ATN-224 (HY-16074, MCE, US).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Stem Cell Research & Therapy

Article Title: hucMSC-derived exosomes targeting macrophage polarization attenuate systemic inflammation in T1DM via INS/SOD1 delivery

doi: 10.1186/s13287-025-04521-0

Figure Lengend Snippet: S961 and ATN-224 partially reversed the therapeutic effect of hucMSC-EXOs on T1DM mice. ( A ) Schematic diagram shows the treatment plan for T1DM mice with S961, ATN-224 and hucMSC-EXOs. ( B ) Images of T1DM, EXO, EXO + S961, EXO + ATN-224 mice. ( C-F ) Physiological and metabolic indices, including food intake ( C ), water intake ( D ), body weight ( E ), and blood glucose levels ( F ), of the mice in the four groups. ( n = 6). ( G-H ) Glucose metabolism was evaluated by the OGTT ( G ) and ITT ( H ) in four groups of mice ( n = 5). ( I ) HE histopathological staining of the pancreas, spleen, liver, kidney and heart of the four groups of mice ( n = 3). ( J-N ) Concentration monitoring of multiple organ function impairment markers in the serum of the four groups of mice ( n = 6). * P < 0.05 ; ** P < 0.01; *** P < 0.001; ns ( P > 0.05) . Scale bars: Pancreas, 400 μm; other tissues, 200 μm

Article Snippet: THP-1 cells were differentiated into M0 macrophages with 100 nM phorbol myristate acetate (PMA; HY-18739, MCE, US) for 24 h, and then divided into six experimental groups: PMA control, lipopolysaccharide (LPS; 50 ng/mL; L2880, Sigma, US), LPS + 5 nM INS (GC2646, Genxion, China), LPS + 1 μg SOD1 (HY-P71048A, MCE, US), LPS + hucMSC-EXOs (8.5 × 10^5 particles), LPS + heat-inactivated hucMSC-EXOs (8.5 × 10^5 particles), LPS + hucMSC-EXOs + 5 nM INS receptor antagonist S961 (HY-P2093B, MCE, US), and LPS + hucMSC-EXOs + 20 nM SOD1 inhibitor ATN-224 (HY-16074, MCE, US).

Techniques: Staining, Concentration Assay

Journal: Stem Cell Research & Therapy

Article Title: hucMSC-derived exosomes targeting macrophage polarization attenuate systemic inflammation in T1DM via INS/SOD1 delivery

doi: 10.1186/s13287-025-04521-0

Figure Lengend Snippet: S961 and ATN-224 reversed the hucMSC-EXOs-mediated anti-inflammatory in multiple organs. ( A-D ) IHC staining of F4/80 in the pancreas ( A ), spleen ( B ), liver ( C ), and heart ( D ) of CON, T1DM, EXO, EXO + S961, EXO + ATN-224 group mice ( n = 3). ( E-G ) Flow cytometry detects the F4/80-positive macrophages in the pancreas ( E ), spleen ( F ), and liver ( G ) via flow cytometry ( n = 4). ( H-P ) Flow cytometry analysis the protein levels of IL-6, CCL-2, and TNF-α in infiltrating macrophages from the pancreas ( H-J ), spleen ( K-M ), and liver ( N-P ), ( n = 4) . * P < 0.05 ; ** P < 0.01 ; *** P < 0.001 . Scale bars: Pancreas, 400 μm; other tissues, 200 μm

Article Snippet: THP-1 cells were differentiated into M0 macrophages with 100 nM phorbol myristate acetate (PMA; HY-18739, MCE, US) for 24 h, and then divided into six experimental groups: PMA control, lipopolysaccharide (LPS; 50 ng/mL; L2880, Sigma, US), LPS + 5 nM INS (GC2646, Genxion, China), LPS + 1 μg SOD1 (HY-P71048A, MCE, US), LPS + hucMSC-EXOs (8.5 × 10^5 particles), LPS + heat-inactivated hucMSC-EXOs (8.5 × 10^5 particles), LPS + hucMSC-EXOs + 5 nM INS receptor antagonist S961 (HY-P2093B, MCE, US), and LPS + hucMSC-EXOs + 20 nM SOD1 inhibitor ATN-224 (HY-16074, MCE, US).

Techniques: Immunohistochemistry, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Regulation of LEAP2 by insulin and glucagon in mice and humans

doi: 10.1016/j.xcrm.2025.101996

Figure Lengend Snippet:

Article Snippet: S961 , Novo Nordisk , NNC0069-0961.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Amplified Luminescent Proximity Homogenous Assay, RNA Sequencing, Gene Expression, Expressing, Derivative Assay, Software

Journal: Cell Reports Medicine

Article Title: Regulation of LEAP2 by insulin and glucagon in mice and humans

doi: 10.1016/j.xcrm.2025.101996

Figure Lengend Snippet: Insulin is required for the meal-induced upregulation of hepatic Leap2 mRNA expression and plasma LEAP2 levels in lean mice (A) Schematic of the experimental in vivo study for examining the role of insulin in the LEAP2 response after administration of a mixed meal. The three different groups of mice were those that received an s.c. vehicle dosage 2 h prior to an oral water challenge (gray), those that received an s.c. vehicle dosage prior to an oral mixed meal challenge (orange), and those that received an s.c. insulin receptor antagonist (S961) prior to a mixed meal challenge (yellow). (B and C) Blood glucose profiles (B) and plasma insulin levels (C) during the intervention. (D and E) Plasma LEAP2 levels 1 (D) and 2 h (E) post oral gavage. (F) Liver (left), jejunal (middle), and duodenal (right) Leap2 mRNA levels in response to the intervention. (G–L) Hepatic glucose (G), glycogen (H), fatty acid (I), triglyceride (J), ALAT protein (K), and ASAT protein (L) levels in response to the intervention. (M and N) Hepatic Pck1 (M) and Insr (N) mRNA levels in response to the intervention. Data are presented as mean +/- SEM. Statistics in (B) and (C) by mixed-effects models. Statistics in (D), (E), (F), (K), (M), and (N) by Kruskal-Wallis and uncorrected Dunn’s test. Statistics in (G), (H), (J), and (L) one-way ANOVA and uncorrected Fisher’s LSD test. p < 0.05, ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001, ∗∗∗∗.

Article Snippet: For the insulin receptor antagonism study, all mice were fasted for 14 h prior to subcutaneous (s.c.) dosing with the insulin receptor antagonist, S961 (NNC0069-0961, 400 nmol/kg, Novo Nordisk, 2760 Måløv, Denmark) ( n = 8) or vehicle (10mM sodium phosphate, 140mM sodium chloride, 0.007% polysorbate 20, pH 7.4) ( n = 16).

Techniques: Expressing, Clinical Proteomics, In Vivo

Journal: Cell Reports Medicine

Article Title: Regulation of LEAP2 by insulin and glucagon in mice and humans

doi: 10.1016/j.xcrm.2025.101996

Figure Lengend Snippet: LEAP2 mRNA levels decrease in mouse primary hepatocytes and HepG2-IGF1R-KO cells upon insulin stimulation in vitro (A) Representative graph of phosphorylated S473 (pS473) Akt1/2/3 signals after surefire analysis of a mouse primary hepatocyte batch stimulated with a 2-fold titration of 100 nM human insulin (orange) or a 2-fold titration of 1000 nM S961 and a fixed human insulin concentration of 10 nM (yellow) for 15 min to test for insulin responsiveness. Data points represent technical replicates. (B) Pck1 (left) and Leap2 (right) and mRNA levels from mouse primary hepatocytes that were fasted overnight and stimulated for 4 h with either 3 nM human insulin, 20 mM glucose, 30 nM S961, in combination or alone, or with a control solution of starvation medium containing 1% human serum albumin as indicated. Data from four pooled technical replicates from two independent biological replicates. (C) Supervised principal component analysis plot of normalized counts from bulk RNA-seq-yielded transcriptomes of HepG2-IGF1R-KO cells incubated in DMEM with 10% FBS (normal medium, control) with no stimulation (gray) and with 18 h 3 nM insulin stimulation (orange). Overlay of cluster designations from a k -means clustering with k = 2. Data from normalized counts of three independent experiments with averages from three technical repeats per experiment. (D) Volcano plot of same samples as in (C) with each gene color-coded according to the adjusted p value < 0.05 (orange) or ≥0.05 (gray). Black data points indicate genes that are independently filtered during differential gene expression analysis. (E) Normalized counts of LEAP2 from same samples as in (C) and (D). Data in (A), (B), and (E) are presented as mean +/- SEM. Statistics in (B) by ANOVA. Statistics in (E) from differential gene expression analysis (please refer to STAR Methods). p < 0.0001, ∗∗∗∗.

Article Snippet: For the insulin receptor antagonism study, all mice were fasted for 14 h prior to subcutaneous (s.c.) dosing with the insulin receptor antagonist, S961 (NNC0069-0961, 400 nmol/kg, Novo Nordisk, 2760 Måløv, Denmark) ( n = 8) or vehicle (10mM sodium phosphate, 140mM sodium chloride, 0.007% polysorbate 20, pH 7.4) ( n = 16).

Techniques: In Vitro, Titration, Concentration Assay, Control, RNA Sequencing, Incubation, Gene Expression

Journal: Cell Reports Medicine

Article Title: Regulation of LEAP2 by insulin and glucagon in mice and humans

doi: 10.1016/j.xcrm.2025.101996

Figure Lengend Snippet:

Article Snippet: For the insulin receptor antagonism study, all mice were fasted for 14 h prior to subcutaneous (s.c.) dosing with the insulin receptor antagonist, S961 (NNC0069-0961, 400 nmol/kg, Novo Nordisk, 2760 Måløv, Denmark) ( n = 8) or vehicle (10mM sodium phosphate, 140mM sodium chloride, 0.007% polysorbate 20, pH 7.4) ( n = 16).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Amplified Luminescent Proximity Homogenous Assay, RNA Sequencing, Gene Expression, Expressing, Derivative Assay, Software

Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: The course of the carried experiments. PCNA- proliferating cell nuclear antigen, P53- tumor protein P53, BAX- bcl-2-like protein 4, BCL2- B-cell lymphoma 2, CASP’s- caspases, INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription, AMPKα- 5'AMP-activated kinase, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques:

Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: Immunolocalization of visfatin and INSR (A), visfatin effect at a concentration of 10 ng/mL on mRNA and protein expression of INSR (B), and protein kinases phosphorylation (C) along with the molecular mechanism of proliferation (D) in JEG-3 cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD multiple range test (MEAN ± SEM, P < 0.05); image magnification × 40, scale bar 100 μm. The effect of visfatin on INSR expression was examined after 48 and 72 h of incubation, kinase phosphorylation after 1, 5, 15, 30, 45, 60 min, and molecular mechanism after 48 h. C- control, V10- visfatin (10 ng/mL), INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription 3, AMPKα- 5'AMP-activated kinase, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor, ACTB- β-actin, AU- arbitrary units, RFU- relative fluorescence units.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques: Concentration Assay, Expressing, Incubation, Control, Fluorescence

Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: Immunolocalization of visfatin and INSR (A), visfatin effect at a concentration of 10 ng/mL on mRNA and protein expression of INSR (B), and protein kinases phosphorylation (C) along with molecular mechanism of apoptosis (D) in BeWo cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD multiple range test (MEAN ± SEM, P < 0.05); image magnification × 40, scale bar 100 μm. The effect of visfatin on INSR expression was examined after 48 and 72 h of incubation, kinase phosphorylation after 1, 5, 15, 30, 45, 60 min, and molecular mechanism after 72 h. C- control, V10- visfatin (10 ng/mL), INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription 3, AMPKα- 5'AMP-activated kinase, ACTB- β-actin, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor, AU- arbitrary units, RFU- relative fluorescence units.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques: Concentration Assay, Expressing, Incubation, Control, Fluorescence