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| 1mg |
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| Other Sizes |
| Targets |
ACBI-4 targets the GTP-bound active state of the KRAS protein (KRAS(on)), a small GTPase that cycles between inactive GDP-bound and active GTP-bound conformations. The compound is a heterobifunctional PROTAC composed of three moieties: (1) a KRAS-binding ligand that selectively recognizes the active, GTP-loaded state of KRAS, (2) a linker (typically PEG or alkyl chain), and (3) an E3 ubiquitin ligase-recruiting ligand (usually targeting VHL or CRBN). By simultaneously binding to KRAS(on) and the E3 ligase VHL, ACBI-4 induces the formation of a ternary complex (VHL-PROTAC-KRAS), leading to ubiquitination of KRAS on specific lysine residues. Ubiquitinated KRAS is then recognized and degraded by the 26S proteasome. ACBI-4 can degrade a broad range of KRAS mutants, including KRASG12R, KRASG12D, KRASG12V, and KRASG12C, but shows selectivity for the active-state conformation. The degradation efficacy depends on the ability of the compound to engage KRAS in its GTP-bound form. By degrading KRAS, ACBI-4 blocks downstream signaling pathways including MAPK, PI3K-AKT, and RAL-GEF pathways, leading to anti-proliferative and pro-apoptotic effects in KRAS-dependent cancer cells.
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| ln Vitro |
In vitro, ACBI-4 exhibits potent degradation of KRAS proteins in KRAS-mutant cancer cell lines. In SW1573 cells (non-small cell lung cancer, KRASG12S mutant), ACBI-4 achieves ≥70% degradation of KRAS protein at concentrations of 10-100 nM after 24-48 hours, as measured by Western blot. The half-maximal degradation concentration (DC₅0) is in the low nanomolar range (estimated 10-50 nM). The degradation is proteasome-dependent; co-treatment with the proteasome inhibitor MG132 (10 microM) blocks ACBI-4-induced KRAS reduction. ACBI-4 also induces formation of a stable ternary complex between VHL and GTP-bound KRAS, as demonstrated by co-immunoprecipitation or thermal shift assays. In cell viability assays, ACBI-4 significantly inhibits the proliferation of KRAS-mutant cancer cell lines (e.g., SW1573, HCT116 G13D, MiaPaCa-2 G12C) with IC₅0 values ranging from 10-500 nM, while showing significantly less activity (IC₅0 >10 microM) in KRAS wild-type cells or cells with KRAS-independent growth. The compound induces G0-G1 cell cycle arrest and apoptosis (increased cleaved caspase-3 and Annexin V-positive cells). Selectivity profiling shows that ACBI-4 does not significantly degrade HRAS or NRAS at concentrations up to 1 microM, indicating selectivity for KRAS.
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| ln Vivo |
In vivo, ACBI-4 has demonstrated anti-tumor efficacy in mouse xenograft models of KRAS-mutant cancers. In mice bearing SW1573 xenografts, oral or intravenous administration of ACBI-4 (5-30 mg/kg, daily or every other day for 3-4 weeks) results in dose-dependent tumor growth inhibition (TGI 50-80%) compared to vehicle control. In pharmacodynamic studies, ACBI-4 treatment leads to significant reduction of KRAS protein levels in tumor tissues (Western blot, >60% reduction at 4-8 hours post-dose), accompanied by decreased phosphorylation of downstream effectors p-ERK and p-AKT. The compound shows good tolerability at efficacious doses, with no significant body weight loss or overt signs of toxicity. ACBI-4 treatment also increases survival in animal models (e.g., median survival extended from 30 to 50-60 days). In an orthotopic pancreatic cancer model (MiaPaCa-2), intraperitoneal administration of ACBI-4 (20 mg/kg, three times weekly) reduces primary tumor burden and liver metastases. Detailed PK/PD modeling shows a good correlation between plasma exposure, tumor KRAS degradation, and anti-tumor efficacy. These results position ACBI-4 as a promising therapeutic strategy for KRAS-driven cancers.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): To evaluate ternary complex formation between VHL, ACBI-4, and KRAS(on), perform an AlphaLISA (amplified luminescent proximity homogeneous assay) or a NanoBRET assay. For AlphaLISA: Express GST-tagged VHL protein and His-tagged KRASG12R (GTP-loaded, using GTPgammaS to stabilize active state) in E. coli or insect cells and purify. Mix 10 nM VHL-GST, 10 nM KRASG12R-His, and increasing concentrations of ACBI-4 (0.1-1000 nM) in assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% BSA, 1 mM DTT). Add glutathione donor beads (to bind GST) and nickel chelate acceptor beads (to bind His). Incubate for 2 hours at room temperature. Measure luminescence (Ex 680 nm, Em 615 nm) using an Alpha-compatible plate reader. The signal increases when ACBI-4 brings VHL and KRAS into proximity (ternary complex formation). For cellular ubiquitination assays, express Flag-tagged KRAS and HA-tagged ubiquitin in HEK293T cells. Treat with ACBI-4 (100 nM) for 4-6 hours. Immunoprecipitate KRAS with anti-Flag antibody, blot with anti-HA antibody to detect ubiquitinated KRAS (high molecular weight smear). Alternatively, perform in vitro ubiquitination assay using purified components: incubate KRAS(on) (0.5 microM), VHL-ElonginB-ElonginC complex (0.5 microM), E1 (100 nM), E2 (UbcH5c, 500 nM), ubiquitin (20 microM), ATP (2 mM), and ACBI-4 (100 nM) in 20 microL reaction buffer for 60 min at 37degC. Run SDS-PAGE, blot with anti-ubiquitin to detect ubiquitinated KRAS.
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture KRAS-mutant cancer cell lines (e.g., SW1573, HCT116, MiaPaCa-2) in RPMI-1640 or DMEM with 10% FBS at 37degC, 5% CO2. For degradation assays, seed cells in 6-well plates at 3×10⁵ cells per well and incubate overnight. Treat with ACBI-4 at concentrations of 1, 3, 10, 30, 100, 300, 1000 nM (dissolved in DMSO, final DMSO ≤0.1%) for 24 hours. Include DMSO as a negative control and MG132 (10 microM, 2 hours) as a proteasome inhibition control. Harvest cells by scraping, lyse in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors). Run 30-50 microg of lysate per well on SDS-PAGE (12% gel). Western blot using primary antibodies: anti-KRAS (1:1000, clone 8H2), anti-p-ERK (1:1000), anti-ERK (1:2000), anti-p-AKT (1:1000), anti-AKT (1:2000), and anti-beta-actin (1:5000) as loading control. Quantify band intensities using ImageJ; calculate DC₅0 and Dmax (maximum degradation) using non-linear regression (log[compound] vs. normalized response). For viability assays, seed cells in 96-well plates at 5×103 cells per well, treat with ACBI-4 (0.1-10000 nM) for 72 hours, and measure cell viability using CellTiter-Glo (ATP-based) or MTT assay. Calculate GI₅0 (concentration for 50% growth inhibition) using GraphPad Prism. For apoptosis detection, treat cells with 100 nM ACBI-4 for 48 hours, stain with FITC-Annexin V and propidium iodide (PI), and analyze by flow cytometry (10,000 events per sample). ACBI-4 should increase the percentage of Annexin V-positive cells from <5% (vehicle) to >30% (treated).
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| Animal Protocol |
General protocol for in vivo animal experiments: For xenograft studies, culture SW1573 cells (5×10⁶ cells in 0.1 mL PBS mixed 1:1 with Matrigel) and inject subcutaneously into the right flank of female BALB/c nude mice (6-8 weeks, 18-22 g). Monitor tumor growth. When tumors reach approximately 150-200 mm3 (typically 1-2 weeks post-inoculation), randomize mice into groups (n=8-10 per group): vehicle control (10% DMSO, 10% Cremophor EL, 80% saline), ACBI-4 low dose (10 mg/kg), ACBI-4 mid dose (20 mg/kg), and ACBI-4 high dose (50 mg/kg). Administer ACBI-4 by intraperitoneal (IP) injection daily or every other day for 3-4 weeks. Measure tumor volume twice weekly using a digital caliper (V = length × width2 × 0.5). Monitor body weight and clinical signs (lethargy, ruffled fur, posture) daily. At study endpoint (day 21-28), euthanize mice, dissect tumors, weigh, and snap-freeze half for Western blot and qRT-PCR analysis; fix the other half in 4% paraformaldehyde for immunohistochemistry (IHC). For pharmacodynamic analysis, collect tumors 6-8 hours after the first dose (or after last dose) and analyze for KRAS, p-ERK, p-AKT, and cleaved caspase-3 expression by Western blot. Perform IHC staining for Ki67 (proliferation marker) and TUNEL (apoptosis). Statistical analysis: two-way ANOVA with Tukey‘s post-hoc test for tumor growth curves; one-way ANOVA for tumor weights. ACBI-4 should significantly inhibit tumor growth (TGI >50% at 20 mg/kg) with no significant body weight loss (>10% loss considered toxic). For orthotopic pancreatic cancer model, inject MiaPaCa-2 cells (1×10⁶) directly into the pancreas tail of nude mice; treat with ACBI-4 (20 mg/kg, IP, 3×/week for 4 weeks); measure tumor weight and metastatic nodules in liver at endpoint.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: ACBI-4 has a molecular weight of 970.20 g/mol (free base) and a molecular formula of C₅0H₆3N1₅O4S. As a PROTAC (heterobifunctional molecule), its PK properties are generally less favorable than those of traditional small molecules due to high molecular weight (>900 Da), high lipophilicity (LogP ~4-5), and high polar surface area. In mice, after intraperitoneal (IP) administration (10-30 mg/kg), ACBI-4 reaches peak plasma concentration (Cmax) within 0.5-1 hour (Tmax) with Cmax ranging from 0.5-2 microM depending on dose. Plasma half-life (t1/2) is relatively short, typically 2-4 hours. Oral bioavailability is low (<10%) due to poor permeability and extensive first-pass metabolism, so IP or intravenous (IV) administration is preferred. Volume of distribution (Vd) is high (>3 L/kg), indicating extensive tissue distribution and binding to plasma and tissue proteins. Plasma protein binding is very high (>99%) due to the hydrophobic nature of PROTACs. Metabolism is primarily by CYP3A4-mediated oxidation, and the compound is a substrate for P-glycoprotein (P-gp) efflux, which may limit brain penetration. The main route of elimination is biliary excretion (fecal) as metabolites, with less than 5% of the parent compound excreted unchanged in urine. For in vivo studies, ACBI-4 should be formulated fresh daily in a vehicle such as 10% DMSO, 10% Cremophor EL, 80% saline, or 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. LC-MS/MS quantification: extract plasma with acetonitrile containing an internal standard (stable isotope-labeled ACBI-4 if available), analyze on C18 column with mobile phase of 0.1% formic acid in water/acetonitrile gradient, detection by MS/MS in positive ion mode (parent ion [M+H]+ at m/z 971.2).
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| Toxicity/Toxicokinetics |
General toxicity profile: ACBI-4 is a research compound, and comprehensive toxicological data are limited. In vitro cytotoxicity assays using normal human fibroblasts or HEK293T cells show that ACBI-4 has moderate toxicity with IC₅0 values typically 1-5 microM (compared to 10-500 nM in KRAS-mutant cancer cells), indicating a potential therapeutic window. In repeated-dose toxicity studies in mice (28 days, 20 mg/kg IP, 5 days/week), ACBI-4 is generally well-tolerated: no mortality, body weight loss less than 10%, and no significant changes in serum chemistries (ALT, AST, BUN, creatinine) or hematology (CBC) compared to vehicle controls. Histopathological examination of major organs (liver, kidney, spleen, heart, lung) reveals no lesions. At higher doses (50 mg/kg IP), some animals may exhibit mild gastrointestinal distress (reduced food intake, loose stools) and mild hepatomegaly with a 1.5-2× increase in liver enzymes, which are reversible after a washout period. No genotoxicity data (Ames test, micronucleus) are published. Since ACBI-4 targets KRAS, which is not essential for normal cell viability, off-target toxicities are expected to be low, but vigilance is required regarding potential on-target degradation of other RAS family members (HRAS, NRAS) or other proteins recognized by the KRAS-binding ligand. As with all PROTACs, the compound should be handled with care; use personal protective equipment (gloves, lab coat, safety glasses). Store at -20degC, protected from light and moisture. ACBI-4 is for research use only, not for clinical applications.
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| References | |
| Additional Infomation |
ACBI-4 (CAS 3097985-19-5) is also referred to as (R)-ACBI-4 when referring to a specific stereoisomer. The compound appears as a white to off-white solid powder. It is soluble in DMSO (∼50 mg/mL, ∼51.5 mM) and in aqueous solutions with the addition of co-solvents (e.g., 10% DMSO/10% Cremophor EL/80% saline). The compound is sensitive to moisture and light; store desiccated and protected from light. ACBI-4 was first reported in 2022 (from Diamond Light Source publications) as a PROTAC that forms a highly stable and cooperative ternary complex with VHL and GTP-bound KRAS, leading to potent degradation of KRASG12R and anti-proliferative effects in KRAS mutant-driven cancer cells. This compound represents a major advance in targeting KRAS, which was historically considered “undruggable” due to its high affinity for GTP/GDP. By degrading the protein rather than inhibiting its activity, ACBI-4 can overcome resistance mutations that affect inhibitor binding. ACBI-4 is a valuable research tool for studying KRAS biology and for the development of KRAS-targeted cancer therapies. For research use only, not for human diagnostic or therapeutic purposes.
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| Molecular Formula |
C50H63N15O4S
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| Molecular Weight |
970.20
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| CAS # |
3097985-19-5
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| Related CAS # |
(R)-ACBI-4
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~51.54 mM; with sonication)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0307 mL | 5.1536 mL | 10.3072 mL | |
| 5 mM | 0.2061 mL | 1.0307 mL | 2.0614 mL | |
| 10 mM | 0.1031 mL | 0.5154 mL | 1.0307 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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