KRAS(G12D)[DC50 = 37 nM]; Von Hippel-Lindau（VHL
Setidegrasib (Example 8) is a PROTAC-type KRAS degrader targeting the G12D-mutated KRAS protein with a DC50 of 37 nM. The compound recruits the VHL E3 ubiquitin ligase to induce proteasomal degradation of mutant KRAS.

Setidegrasib has an excellent activity of inducing the degradation of G12D-mutation KRAS protein and an activity of inhibiting a G12D-mutation KRAS, and can be used as a therapeutic agent for pancreatic cancer.
In the metastatic pancreatic cancer cell line AsPC-1, Setidegrasib demonstrated exceptional degradation activity with DC50 and IC50 values mostly below 10 nM. The compound showed high selectivity for G12D-mutated KRAS over wild-type KRAS in cellular assays

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- ASP3082 effectively induces KRAS(G12D) protein degradation with remarkable selectivity. In AsPC-1 cells (KRAS(G12D)-mutated), it degraded KRAS(G12D) with a DC50 of 38 nM (95% CI, 14-108 nM). [2]
- ASP3082 inhibited ERK phosphorylation in AsPC-1 cells with an IC50 of 14 nM (95% CI, 8-23 nM) and inhibited cell proliferation with an IC50 of 19 nM (95% CI, 13-30 nM). [2]
- Selectivity profiling showed that ASP3082 degraded KRAS(G12D) in cancer cells harboring the mutation (AsPC-1, HPAC, PK-59), while only partial degradation of wild-type KRAS in A375 cells was observed at higher concentrations (1 µM). [2]
- In a 3D cell viability assay, ASP3082 exhibited growth-inhibitory effects against various KRAS(G12D)-mutated cancer cell lines (PK-59, HPAC, GP2d, GP5d) with IC50 values ranging from 3.6 to 29 nM. In contrast, its effects on KRAS wild-type cancer cell lines (A375, HT-29, BxPC-3, COLO-320) were observed only at higher concentrations (IC50 > 10 µM). The order of response for different KRAS mutations was 12D > 12C, 12V > 13D > 12R, wild-type. [2]
- Proteomic analysis in AsPC-1 cells treated with ASP3082 showed selective reduction of KRAS(G12D) and the MAPK-dependent gene DUSP4, with no significant reduction observed for over 9000 other non-KRAS proteins. [2]
- Mechanistic studies confirmed that ASP3082-induced KRAS(G12D) degradation is mediated by the proteasome (as degradation was rescued by the proteasome inhibitor MG-132) and by the E3 ligase VHL (as degradation was blocked by an epi-ASP3082 that does not bind VHL, and partially suppressed under VHL knockdown conditions). [2]
- Comparison with a GDP-bound KRAS(G12D) inhibitor (compound 6) showed that ASP3082 produced a more durable reduction of GTP-bound KRAS, p-ERK, p-AKT, and p-S6 over time, and resulted in near-complete degradation of total KRAS(G12D) protein in cell lines after 1-3 days of treatment with a single addition of the compound. [2]

- In a PK-59 pancreatic cancer xenograft model (KRAS(G12D)-mutated), a single intravenous dose of ASP3082 resulted in dose-dependent KRAS(G12D) degradation in tumors. Maximum degradation was observed at 24 hours post-dose (82% degradation at 10 mg/kg, and 93% degradation at 30 mg/kg), with sustained degradation for at least 48 hours. [2]
- In the same model, ASP3082 administered intravenously once weekly at doses of 10 and 30 mg/kg induced significant tumor regression (18% and 63% regression on day 21, respectively). The compound was well tolerated with no body weight loss or other overt toxicity at the highest dose (30 mg/kg). [2]
- In multiple additional CDX (cell line-derived xenograft) models of pancreatic (AsPC-1, HPAC, PK-1), colorectal (GP2d, GP5d), and lung (NCI-H358) cancer harboring the KRAS(G12D) mutation, as well as in PDX (patient-derived xenograft) models (LXFA 1125, LXFA 2204) of lung cancer, ASP3082 administered once or twice weekly demonstrated potent anti-tumor activity and induced tumor regression. [2]

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In the PK-59 mouse xenograft model of pancreatic cancer, Setidegrasib achieved 98% tumor growth inhibition after two intravenous administrations (dosing frequency and formulation details not available in public sources).

- Surface Plasmon Resonance (SPR) Biosensor Analysis: - Binary Binding to KRAS(G12D): Biotinylated Avi-KRAS(G12D) protein was immobilized on a sensor chip. Increasing concentrations of test compounds were injected over the chip surface in a single-cycle or multicycle format. The association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) were calculated. For ASP3082, the binary binding KD to KRAS(G12D) was 56 nM (95% CI, 39-81 nM). [2]
- Binary Binding to VHL: His-tagged VHL/ElonginC/ElonginB (VCB) complex was immobilized on an NTA sensor chip. Test compounds were injected to assess binding. For ASP3082, the binary binding KD to VHL was 7.5 nM (95% CI, 3.5-16 nM), with a slow dissociation rate constant (kd) of 2.1 × 10⁻³ s⁻¹ (95% CI, 1.6-2.7 × 10⁻³ s⁻¹). [2]
- Ternary Complex Formation: A series of test compound dilutions were pre-mixed with VCB and injected over the KRAS(G12D)-immobilized chip in single-cycle mode. This allowed for the measurement of ternary complex affinity (KD). For ASP3082, the ternary binding KD was 0.95 nM (95% CI, 0.61-1.5 nM), with a kd of 4.4 × 10⁻⁴ s⁻¹ (95% CI, 1.6-12 × 10⁻⁴ s⁻¹). [2]
- Cell-Free TR-FRET Assay (KRAS-RAF PPI Inhibition): A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to measure the inhibition of the protein-protein interaction between KRAS(G12D) and cRAF. The assay involved incubating GDP-bound KRAS(G12D) with SOS to load GTP, followed by addition of GST-tagged cRAF, ULight-anti-GST antibody, and LANCE Eu-W1024 Streptavidin. The TR-FRET ratio was measured to determine the inhibitory activity of test compounds. [2]

- In-Cell ELISA KRAS(G12D) Degradation Assay: AsPC-1 cells were seeded in 384-well plates and treated with various concentrations of test compounds for 24 hours. Cells were fixed, permeabilized, and stained with anti-RAS(G12D) and anti-β-actin antibodies, followed by fluorescent secondary antibodies. Fluorescence signals were quantified, and the KRAS(G12D) signal was normalized to β-actin. DC50 values were calculated from the concentration-response curves. [2]
- In-Cell ELISA ERK1/2 Phosphorylation (p-ERK) Assay: AsPC-1 cells were treated with test compounds for 2 or 24 hours. Cells were fixed, permeabilized, and stained with anti-p-ERK1/2 antibody. Fluorescence signals were quantified, and inhibition was calculated relative to a MEK inhibitor (trametinib) control. IC50 values were calculated. [2]
- In Vitro Cell Growth Assay: Cells were seeded in low-attachment 96- or 384-well round-bottom white plates and treated with test compounds the following day. After 6 days of incubation, CellTiter-Glo 2.0 Reagent was added to measure luminescence, which is proportional to cell viability. IC50 values for growth inhibition were calculated. The assay was performed in 3D format to assess anchorage-independent growth. [2]
- Immunoblot Analyses (In Vitro): Cells were treated with test compounds for indicated times. Cell lysates were prepared, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were probed with primary antibodies (e.g., anti-KRAS(G12D), anti-p-ERK, anti-total ERK, anti-p-AKT, anti-p-S6) and then with HRP-conjugated secondary antibodies. Chemiluminescent signals were detected to assess protein levels. [2]
- RAF-RBD Pulldown Assay: To detect the active GTP-bound form of KRAS, cell lysates were incubated with GST-tagged Raf-RBD protein and glutathione particles. After washing, bound proteins were eluted and subjected to immunoblot analysis to detect KRAS-GTP. [2]
- Proteomic Analysis: AsPC-1 cells were treated with DMSO or 1 µM ASP3082 for 4 and 24 hours. Cell pellets were collected, and protein samples were prepared for LC-MS/MS analysis. Protein abundance was quantified, and the percentage of protein abundance in ASP3082-treated samples relative to DMSO-treated controls was calculated to assess degradation selectivity. [2]
- VHL Knockdown Assay with siRNA: PK-59 cells were transfected with anti-human VHL siRNA or control siRNA using lipofectamine. After 2-3 days, cells were treated with ASP3082 for 24 hours. KRAS(G12D) degradation was assessed by In-Cell ELISA to evaluate the dependency of degradation on VHL. [2]

- Formulation and Administration: ASP3082 was formulated as a solution in a vehicle composed of 5% glucose solution with 4% ethanol, 0.5% (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), and 9% hydrogenated castor oil 40 (HCO 40). The compound was administered intravenously (i.v.) at various doses, with dosing schedules including once weekly or twice weekly. [2]
- PK/PD Study in PK-59 Xenograft Model: PK-59 cells were subcutaneously implanted into male nude mice. When tumors reached a suitable size, mice were administered a single i.v. dose of vehicle, 10 mg/kg, or 30 mg/kg ASP3082. Blood, tumors, and pancreas were collected from three mice per group at 2, 6, 24, 48, 72, and 144 hours post-dose for pharmacokinetic and pharmacodynamic analysis. [2]
- Anti-Tumor Efficacy Studies in CDX Models: Various cancer cell lines (e.g., PK-59, AsPC-1, HPAC, GP2d) were subcutaneously implanted into male nude mice. Mice were randomized based on tumor volume and treated i.v. with vehicle or ASP3082 once weekly (e.g., on days 1, 8, 15) or twice weekly. Tumor volume and body weight were measured twice weekly to assess efficacy and tolerability. [2]
- Anti-Tumor Efficacy Studies in PDX Models: Patient-derived xenograft (PDX) tumors (e.g., LXFA 1125, LXFA 2204) were subcutaneously implanted into nude mice. Mice were treated i.v. with vehicle or ASP3082 once weekly. Tumor growth inhibition and regression were assessed, and tumor samples were collected for immunohistochemical analysis of KRAS(G12D) protein levels. [2]

- In Vivo Pharmacokinetics in PK-59 Xenograft Model: After a single intravenous administration of ASP3082 at 10 and 30 mg/kg in tumor-bearing mice, compound concentrations in plasma decreased rapidly. In contrast, concentrations in tumors were maintained at a certain level up to 144 hours post-dose, indicating prolonged retention in tumor tissue. Concentrations in normal pancreas decreased rapidly, similar to plasma. [2]
- In Vitro Plasma Protein Binding: The mean unbound fraction ratio of ASP3082 in mouse plasma was determined to be 0.00784 using the ultracentrifugation method at concentrations of 10, 50, and 200 µg/mL. [2]

- In Vivo Tolerability: In anti-tumor efficacy studies in mice, ASP3082 was well tolerated. At the highest tested dose (30 mg/kg, once weekly), no body weight loss or other overt toxicity was observed. [2]

[1]. Preparation of quinazoline-linked (4R)-4-hydroxy-L-prolinamide compounds for inducing degradation of G12D-mutation KRAS protein: World Intellectual Property Organization, WO2022173032. 2022-08-18.

[2]. Discovery of KRAS(G12D) selective degrader ASP3082. Commun Chem. 2025 Aug 23;8(1):254.

Setidegrasib, developed by Astellas Pharma, is a quinazoline-linked (4R)-4-hydroxy-L-proline amide (PROTAC) compound. Its chemical structure consists of three modular parts: a KRAS G12D targeting active group (blue), a linking group (black), and a VHL recruiting group (pink). The patent emphasizes that subtle variations in the red-boxed regions of the molecule (Figures 8 and 39) can significantly affect in vivo efficacy, likely due to pharmacokinetic differences.

C60H65FN12O7S

1117.30

1116.48

C, 64.50; H, 5.86; F, 1.70; N, 15.04; O, 10.02; S, 2.87

2821793-99-9

2821793-99-9

164875418

White to off-white solid

7.7

5

17

17

81

2130

6

O(C1C2N=C(N=C(N3C[C@@]4([H])NC[C@]3([H])C4)C=2C=C(C2CC2)C=1C1C(=C(C=C2NN=CC=12)F)C)OC1CCOCC1)CC1C=CC(C2N=NN(C=2)[C@@H](C(C)C)C(N2C[C@@H](C[C@H]2C(=O)N[C@H](C2C=CC(C3SC=NC=3C)=CC=2)CO)O)=O)=CC=1

XXWMEQMGQNHRAF-WFJRNIGCSA-N

InChI=1S/C60H65FN12O7S/c1-31(2)54(59(77)72-26-41(75)20-50(72)58(76)65-49(28-74)37-11-13-38(14-12-37)56-33(4)63-30-81-56)73-27-48(69-70-73)36-7-5-34(6-8-36)29-79-55-52(51-32(3)46(61)22-47-45(51)24-64-68-47)43(35-9-10-35)21-44-53(55)66-60(80-42-15-17-78-18-16-42)67-57(44)71-25-39-19-40(71)23-62-39/h5-8,11-14,21-22,24,27,30-31,35,39-42,49-50,54,62,74-75H,9-10,15-20,23,25-26,28-29H2,1-4H3,(H,64,68)(H,65,76)/t39-,40-,41+,49-,50-,54-/m0/s1

(2S,4R)-1-((2S)-2-(4-(4-(((4-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-6-cyclopropyl-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-2-((tetrahydro-2H-pyran-4-yl)oxy)quinazolin-8-yl)oxy)methyl)phenyl)-1H-1,2,3-triazol-1-yl)-3-methylbutanoyl)-4-hydroxy-N-((R)-2-hydroxy-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

KRAS G12D inhibitor 17; ASP-3082; Setidegrasib; 2821793-99-9; ASP3082; 3NQ4ME292X; RefChem:1098491; setidegrasib [INN]; SCHEMBL26963151;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (112 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)