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| Targets |
INX-315 is a potent and selective inhibitor of cyclin-dependent kinase 2 (CDK2). Biochemical IC₅₀ values (mean ± SEM, n ≥ 3) determined by Nanosyn assay are: CDK2/cyclin E1 0.6 ± 0.2 nmol/L, CDK2/cyclin A2 2.5 ± 0.7 nmol/L, CDK1/cyclin B1 2.5 ± 0.7 nmol/L, CDK3/cyclin E1 4 ± 1 nmol/L, CDK5 1 ± 0.2 nmol/L, CDK5/p25 4 ± 1 nmol/L, CDK7 4640 ± 390 nmol/L, CDK9/cyclin T1 30 ± 8.3 nmol/L, CDK4/cyclin D1 1240 ± 40 nmol/L, CDK6/cyclin D3 1710 ± 480 nmol/L. It also inhibits CSF1R with an IC₅₀ of 2.29 nmol/L in follow-up assays. Intracellular target engagement IC₅₀ measured by NanoBRET: CDK2/cyclin E1 2.3 ± 0.07 nmol/L, CDK2/cyclin A1 71 ± 0.5 nmol/L, CDK1/cyclin B1 374 ± 63.5 nmol/L, CDK9/cyclin T1 2950 ± 314 nmol/L. The selectivity for CDK2 over CDK1 is approximately 50-fold in biochemical assays and 163-fold in NanoBRET assays [1].
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| ln Vitro |
INX-315 potently inhibits proliferation of CCNE1-amplified cancer cell lines with a mean IC₅₀ of 36 nmol/L (range 10–64 nmol/L) in ovarian and gastric cancer cell lines (OVCAR3, COV318, Kuramochi, MKN1, etc.), whereas non-amplified lines are much less sensitive (mean IC₅₀ 1435 nmol/L). In CDK4/6i-resistant breast cancer cells (MCF7 and T47D resistant to abemaciclib or abemaciclib/fulvestrant), INX-315 shows enhanced sensitivity (IC₅₀ values significantly lower than in parental cells). In contrast, the normal fibroblast line Hs68 is insensitive to INX-315 (IC₅₀ > 1 μmol/L) [1].
Cell cycle analysis in OVCAR3 and MKN1 cells reveals a potent G₁ arrest after 24 h treatment with 30–100 nmol/L INX-315; at higher concentrations (≥300 nmol/L) a combined G₁/G₂ arrest appears. The G₁ arrest is accompanied by reduced phosphorylation of retinoblastoma protein (Rb) at Ser807/811 and Thr821, decreased levels of CDK2 substrates such as CDC6 and nucleolin, and downregulation of the E2F target cyclin A2, as shown by Western blot [1]. INX-315 induces therapy-induced senescence (TIS) in CCNE1-amplified cells. After 7-day treatment (100–1000 nmol/L), OVCAR3 and MKN1 cells show increased senescence-associated β-galactosidase activity, enlarged nuclear size, and upregulation of senescence gene signatures (RNA-seq). In CDK4/6i-resistant breast cancer cells, addition of INX-315 reinstates a senescence phenotype similar to that induced by CDK4/6i in parental cells, including chromatin remodeling and activation of AP-1 target genes [1]. Combination of INX-315 with CDK4/6 inhibitors (abemaciclib, palbociclib, ribociclib) in resistant cells results in synergistic growth inhibition and deeper suppression of E2F target genes compared to monotherapy [1]. Long-term clonogenic assays show that combined CDK4/6i plus INX-315 prevents the emergence of resistance to CDK4/6 inhibition over 10 weeks, whereas CDK4/6i alone leads to resistant outgrowth [1]. |
| ln Vivo |
In CCNE1-amplified tumor models, oral administration of INX-315 induces dose-dependent tumor growth inhibition. In gastric adenocarcinoma PDX GA0103 (CCNE1 ~10 copies), INX-315 at 25, 50, or 100 mg/kg twice daily (BID) for 56 days significantly inhibits tumor growth, with 100 mg/kg BID causing regression. In another gastric PDX GA0114 (CCNE1 ~3 copies), 100 mg/kg BID for 5 weeks inhibits growth (P < 0.0001). In ovarian carcinoma PDX OV5398 (CCNE1 ~9 copies), 100 mg/kg BID for 8 weeks inhibits growth (P < 0.0001). In OVCAR3 CDX, 100 mg/kg BID similarly inhibits growth. No significant body weight loss (>5%) or signs of toxicity are observed [1].
In a transgenic MMTV-rtTA/tetO-HER2 breast cancer model, mice with acquired resistance to abemaciclib were treated with vehicle, continued abemaciclib, INX-315 (50 mg/kg BID), or the combination. Combination significantly slowed tumor growth over 5 weeks, whereas monotherapies had modest effects. Analysis of tumors showed deeper suppression of E2F target genes and reinstatement of senescence markers with combination [1]. In a CDK4/6i-resistant breast cancer PDX (ST4316B), combination of ribociclib (50 mg/kg QD) and INX-315 (50 mg/kg BID) significantly inhibited tumor growth compared to either monotherapy, with good tolerability [1]. Pharmacodynamic analysis in OVCAR3 and OV5398 tumors shows sustained reduction of pRb and cyclin A2 levels up to 16 h after final dose, confirming target engagement in vivo [1]. |
| Enzyme Assay |
Biochemical kinase assays were performed using the Nanosyn Caliper Assay Platform with microfluidic detection technology. Compounds were tested in 12-point dose-response format in singlicate at the Km for ATP, with a phosphoacceptor substrate peptide concentration of 1 μmol/L. Staurosporine was used as reference compound. IC₅₀ values were determined for various CDK/cyclin complexes. For selectivity profiling, INX-315 was tested at 100 nmol/L in the LanthaScreen Eu Kinase Binding Assay and Z'-Lyte Kinase Assay (Thermo Fisher Scientific). Kinases showing ≥80% inhibition were retested in 10-point dose-response curves (1 μmol/L to 49.5 pmol/L) to determine IC₅₀ values [1].
Microsomal stability assay: INX-315 (1 μmol/L) was incubated with liver microsomes from human, rat, mouse, and dog in the presence of NADPH at 37°C. Aliquots were taken at 0, 10, 20, 30, and 60 min, and analyzed by LC-MS/MS. Percent remaining was calculated, and half-life and clearance were determined using GraphPad software [1]. |
| Cell Assay |
Cells seeded in 96-well plates (1,000–5,000 cells/well) were treated with serial dilutions of INX-315 for 6 days (or 2 doubling times for some lines). Viability was measured using CellTiter-Glo (Promega) according to manufacturer's instructions. Luminescence was read on a plate reader, and IC₅₀ values were calculated [1].
Cell cycle analysis: Cells treated with INX-315 for 24 h were fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Data were processed using FlowJo software [1]. Western blotting: Cell or tumor lysates were prepared in RIPA or Cell Lysis Buffer with protease/phosphatase inhibitors. Proteins were separated by SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and probed with antibodies against pRb, Rb, cyclin A2, p-CDC6, nucleolin, GAPDH, etc. Detection used HRP-conjugated secondary antibodies and chemiluminescence [1]. Senescence-associated β-galactosidase staining: Cells were fixed and stained using a β-galactosidase staining kit. Images were acquired, and integrated signal per cell was quantified. Nuclei were stained with DAPI and cytoskeleton with phalloidin to assess nuclear and cell size [1]. RNA-seq and ATAC-seq: RNA was extracted using RNeasy kits, libraries prepared with TruSeq Stranded mRNA, and sequenced on Illumina NextSeq 500. ATAC-seq was performed on 50,000 nuclei using Omni-ATAC protocol with Illumina Tagment DNA Enzyme, sequenced on NextSeq 500. Data were analyzed with standard pipelines [1]. |
| Animal Protocol |
For PDX studies, tumor fragments were implanted subcutaneously into immunocompromised mice (NOD/SCID for OV5398; BALB/c nude for GA0103 and GA0114; athymic nude for OVCAR3 and ST4316B). When tumors reached ~150–300 mm³, mice were randomized to treatment groups (n = 8–10 per group). INX-315 was formulated weekly in 100% PEG400 and administered by oral gavage at doses of 25–100 mg/kg BID or 100 mg/kg QD. Abemaciclib was prepared as previously described and given at 50–75 mg/kg; ribociclib was prepared in 0.5% methylcellulose and given at 50 mg/kg QD. Tumor volume was measured twice weekly with calipers, and body weight was monitored. Animals were euthanized at experimental endpoints or when tumor burden exceeded limits [1].
For the transgenic MMTV-rtTA/tetO-HER2 model, tumor formation was induced, and mice with established tumors were treated continuously with abemaciclib (75 mg/kg) until acquired resistance (tumor regrowth). Resistant mice were then randomized to vehicle, continued abemaciclib (50 mg/kg), INX-315 (50 mg/kg BID), or combination. Treatments continued for up to 5 weeks with tumor monitoring [1]. |
| ADME/Pharmacokinetics |
INX-315 exhibited high metabolic stability in liver microsomes. After 60 minutes of incubation, the parent compound was recovered in the following ways: 94% in humans, 100% in rats, 100% in mice, and 100% in dogs (data from Supplementary Figure S1B; specific values are not provided in the text, but this indicates high stability). Half-life and intrinsic clearance were calculated (data not shown) [1].
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| Toxicity/Toxicokinetics |
In all in vivo efficacy studies, INX-315 was well tolerated. During treatment, the weight loss in all mice did not exceed 5%. No adverse clinical signs (changes in body condition, respiratory rate, hair condition, posture, or behavior) were observed at doses up to 100 mg/kg twice daily for 8 weeks. No additional toxicities other than those observed when used in combination with CDK4/6 inhibitors were observed when used in combination with CDK4/6 inhibitors alone [1].
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| References | |
| Additional Infomation |
INX-315 is a novel, highly effective and selective CDK2 inhibitor developed by optimizing the structure-activity relationship of the CDK4/6 inhibitor trilaciclib. It is designed to target cancers with high CDK2 activity, such as CCNE1-amplified tumors (e.g., ovarian cancer, gastric cancer) and breast cancers that have developed resistance to CDK4/6 inhibitors. Preclinical data show that INX-315 can induce cell cycle arrest and treatment-induced senescence, thereby achieving durable tumor control in xenograft and transgenic models. It has a synergistic effect with CDK4/6 inhibitors, which can overcome resistance and delay its onset. INX-315 is currently being evaluated in a phase I clinical trial in patients with advanced solid tumors (NCT05735080) [1]. The CDK2 inhibitor INX-315 is a small molecule cyclin-dependent kinase 2 (CDK2) inhibitor with high oral bioavailability and potential antitumor activity. After oral administration, the CDK2 inhibitor INX-315 selectively targets, binds to, and inhibits CDK2 activity. This may lead to cell cycle arrest, induction of apoptosis, and inhibition of tumor cell proliferation. CDKs are a class of serine/threonine kinases and are important regulators of cell cycle progression and cell proliferation, frequently overexpressed in tumor cells.
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| Molecular Formula |
C19H21N7O3S
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|---|---|
| Molecular Weight |
427.480141401291
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| Exact Mass |
427.142
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| Elemental Analysis |
C, 53.38; H, 4.95; N, 22.94; O, 11.23; S, 7.50
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| CAS # |
2745060-92-6
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| PubChem CID |
162360412
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| Appearance |
White to off-white solid powder
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| LogP |
2.4
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
30
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| Complexity |
759
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1C=CC(=CC=1)NC1=NC=C2C=C3NNC(C4(CCCCC4)N3C2=N1)=O)(N)(=O)=O
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| InChi Key |
KGJVKYSVVOQPDL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H21N7O3S/c20-30(28,29)14-6-4-13(5-7-14)22-18-21-11-12-10-15-24-25-17(27)19(8-2-1-3-9-19)26(15)16(12)23-18/h4-7,10-11,24H,1-3,8-9H2,(H,25,27)(H2,20,28,29)(H,21,22,23)
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| Chemical Name |
4-[(12-oxospiro[1,3,5,10,11-pentazatricyclo[7.4.0.02,7]trideca-2,4,6,8-tetraene-13,1'-cyclohexane]-4-yl)amino]benzenesulfonamide
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| Synonyms |
INX-315; 2745060-92-6; INX315; 2QD8YT8RWZ; orb2565961;
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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 |
| 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 :~100 mg/mL (~233.93 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution.
For example, if 1 mL of working solution is to be prepared,you can add 100 μL of 25.0 mg/mL clear DMSO stock solution and add it to 400 μL PEG300 and mix well. Then add 50 μL Tween-80 to the above system and mix well. Then continue to add 450 μL of physiological saline to make up to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one),clear solution. For example, if 1 mL of working solution is to be prepared,you can add 100 μL of 25.0 mg/mL clear DMSO stock solution and add it to 900 μL of 20% SBE-β-CD saline solution and mix well. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one),clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3393 mL | 11.6965 mL | 23.3929 mL | |
| 5 mM | 0.4679 mL | 2.3393 mL | 4.6786 mL | |
| 10 mM | 0.2339 mL | 1.1696 mL | 2.3393 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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