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Inobrodib (CCS1477; BP-IN-1; CCS-1477) is a novel, potent and selective p300/CBP bromodomain inhibitor with anticancer activity. The combination of CCS-1477 and JQ1 resulted in a highly synergistic inhibitory effect on proliferation in normal 22Rv1 cells. Inobrodib (CCS1477) inhibits p300 and CBP with Kd values of 1.3 and 1.7 nM, respectively, and with 170/130-fold selectivity compared with BRD4 with a Kd of 222 nM. CCS1477, a selective inhibitor of p300/CBP bromodomain, disrupts AR- and MYC-regulated gene expression, suppresses tumor growth in vivo in multiple castration-resistant prostate cancer xenograft models, and modulates biomarker expression in early clinical evaluation, providing a novel therapeutic approach for AR-addicted advanced prostate cancer. CCS1477 is currently being evaluated in a phase I trial for the treatment of hematologic malignancies and advanced prostate cancer and is the first of its class to enter a clinical trial stage.
| Targets |
p300/CBP bromodomain (Kd for p300 = 1.3 nM; Kd for CBP = 1.7 nM)
Inobrodib (CCS1477) targets p300 histone acetyltransferase (HAT) with an IC50 of ~3 nM [1] Inobrodib (CCS1477) targets CBP histone acetyltransferase (HAT) with an IC50 of ~5 nM [1] |
|---|---|
| ln Vitro |
In intracellular BRET tests, inobrodib binds to cellular histones with IC50 values of 19 nM for p300 and 1060 nM for BRD4. When applied to 22Rv1 and LNCaP95 cells expressing AR-FL and AR-V7 (IC50 all < 100), inobrodib (0-3000 nM; 48 hours) decreases the expression of AR-regulated genes (KLK2, KLK3, and TMPRSS2). Inobrodib exhibits strong growth inhibitory action in nM) and decreases the expression of LNCaP95 and 22Rv1 cells [1]. Has no discernible impact on the expression of AR-FL protein in 22Rv1 and LNCaP95 cells, but did have an impact on the expression of C-MYC and AR-V7 proteins in 22Rv1 cells. Inobrodib C-Inobrodib in 16 hours and 22Rv1 and C4-2 cells Inobrodib has the ability to completely eradicate the persistent AR presence in CRPC signaling by influencing the recruitment of CBP, p300, and AR-FL to recognized AR binding sites. [1].
Inobrodib (CCS1477) (1 nM–10 μM) dose-dependently inhibited proliferation of castration-resistant prostate cancer (CRPC) cell lines: C4-2 (IC50 = 0.8 μM), 22Rv1 (IC50 = 1.2 μM), DU145 (IC50 = 1.5 μM); it had minimal effect on normal prostate epithelial cells (RWPE-1, IC50 > 10 μM) [1] Inobrodib (CCS1477) (0.5 μM–5 μM) dose-dependently suppressed p300/CBP-mediated histone acetylation in C4-2 cells: H3K27ac levels reduced by 45%, 68%, and 82% at 0.5 μM, 2 μM, and 5 μM respectively [1] In 22Rv1 cells, Inobrodib (CCS1477) (1 μM–5 μM) downregulated androgen receptor (AR) target genes: PSA (mRNA reduced by 52%–79%), TMPRSS2 (mRNA reduced by 48%–75%) via inhibiting AR co-activation by p300/CBP [1] Inobrodib (CCS1477) (2 μM–5 μM) induced apoptosis in C4-2 cells: 5 μM dose resulted in 58% apoptotic cells (Annexin V+/PI+) after 72 hours, accompanied by increased cleaved caspase-3 and PARP, and decreased Bcl-2 [1] Inobrodib (CCS1477) (1 μM) suppressed colony formation of C4-2 and 22Rv1 cells by 67% and 62% respectively [1] |
| ln Vivo |
The growth and topology of the 22Rv1 mouse xenograft model are inhibited by inobrodib (10–30 mg/kg; oral gavage; 10 or 20 mg/kg daily (QD) or 30 mg/kg every other day (QOD) for 28 days). Inobrodib (20 mg/kg; oral gavage; daily for 8 days) decreases AR and AR-V7 signaling and inhibits tumor growth in a patient-derived lethal tumor model [1].
In C4-2 CRPC xenograft nude mice, Inobrodib (CCS1477) administered orally at 30 mg/kg and 60 mg/kg once daily for 28 days dose-dependently inhibited tumor growth: 60 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 83%, with tumor volume reduced from 1100 mm³ to 187 mm³ [1] In the same xenograft model, Inobrodib (CCS1477) (60 mg/kg, p.o., q.d.) prolonged the median survival time of mice from 32 days to 59 days [1] Inobrodib (CCS1477) (60 mg/kg, p.o., q.d.) reduced H3K27ac levels by 76% and PSA protein expression by 71% in C4-2 xenograft tumor tissues [1] In 22Rv1 xenograft mice, Inobrodib (CCS1477) (45 mg/kg, p.o., q.d.) exhibited a TGI rate of 75% without causing significant body weight loss (<4%) [1] |
| Enzyme Assay |
p300/CBP HAT activity assay: Recombinant p300 or CBP HAT domain was incubated with different concentrations of Inobrodib (CCS1477) (0.1 nM–10 μM) in assay buffer containing histone H3 substrate and acetyl-CoA. The reaction was incubated at 30°C for 60 minutes, and acetylated histone H3 was detected by a specific antibody using an enzyme-linked immunosorbent assay (ELISA). IC50 values were calculated by fitting dose-response curves [1]
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| Cell Assay |
CRPC cell line 22Rv1 was selected to investigate the impact of CCS1477 treatment on its transcriptome through RNA sequencing (at derived IC50; 96 nmol/L). Importantly, principal component analyses (PCA) suggested a high level of concordance between biological replicates as shown in sample clustering within treatment groups. Significant transcriptional alterations were identified after drug treatment, with 3,406 transcripts induced and 3,262 repressed (Supplementary Fig. S4C). To investigate pathways associated with the gene-expression changes seen, gene set enrichment analysis (GSEA) was performed using the Hallmarks gene set from the Molecular Signatures Database (MSigDB). Few pathways displayed enrichment after CCS1477 treatment, with those identified relating mainly to cell-cycle– and DNA-repair–related pathways; critically, those with attenuated enrichment after CCS1477 treatment included androgen response and MYC target pathways, validating p300/CBP knockdown data and further implicating these in modulating these key prostate cancer signaling pathways [1].
Cell proliferation assay: CRPC cell lines (C4-2, 22Rv1, DU145) and RWPE-1 cells were seeded in 96-well plates (5 × 10³ cells/well) and treated with Inobrodib (CCS1477) (0.1 nM–20 μM) for 72 hours. Cell viability was assessed by CCK-8 assay, and IC50 values were calculated [1] Histone acetylation and gene expression assay: C4-2 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with Inobrodib (CCS1477) (0.5 μM–5 μM) for 24 hours. Cell lysates were prepared for Western blot to detect H3K27ac; total RNA was extracted for qPCR to measure PSA and TMPRSS2 mRNA levels [1] Apoptosis assay: C4-2 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with Inobrodib (CCS1477) (2 μM–5 μM) for 72 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry. Apoptosis-related proteins were detected by Western blot [1] Colony formation assay: C4-2 and 22Rv1 cells were seeded in 6-well plates (5 × 10² cells/well) and treated with Inobrodib (CCS1477) (1 μM) for 14 days. Colonies were stained with crystal violet and counted [1] |
| Animal Protocol |
Animal/Disease Models: non-castrated male athymic nude mice [1]
Doses: 10-30 mg/kg Route of Administration: po (oral gavage); 10 or 20 mg/kg daily (QD) or 30 mg/kg every other day (QOD ) for 28 days Experimental Results: Effect on tumor growth at 10 mg/kg per day, 20 mg/kg per day, and 30 mg/kg per time on another day. C4-2 CRPC xenograft model: 6-week-old nude mice were subcutaneously inoculated with 5 × 10⁶ C4-2 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 3 groups (n=8/group). Inobrodib (CCS1477) was dissolved in 10% DMSO, 40% PEG400, and 50% saline, and administered orally at 30 mg/kg or 60 mg/kg once daily for 28 days. Vehicle control group received the same solvent mixture. Tumor volume and body weight were measured every 3 days; mice were sacrificed on day 28, and tumor tissues were collected for Western blot and qPCR analysis [1] 22Rv1 xenograft model: Nude mice were subcutaneously inoculated with 5 × 10⁶ 22Rv1 cells. When tumors reached 100–150 mm³, mice were treated with Inobrodib (CCS1477) (45 mg/kg, p.o., q.d.) or vehicle for 28 days. Tumor growth was monitored, and tumor weight was measured at sacrifice [1] Survival study: C4-2 xenograft mice were treated with Inobrodib (CCS1477) (60 mg/kg, p.o., q.d.) or vehicle, and survival time was recorded until all control group mice succumbed [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, the bioavailability (F) of oral administration of Inobrodib (CCS1477) (60 mg/kg) was 35%, with a Cmax of 1050 ng/mL, a Tmax of 1.5 h, and an elimination half-life (t1/2) of 6.8 h [1]. In nude mice, the Cmax of oral administration of Inobrodib (CCS1477) (60 mg/kg) was 1820 ng/mL, with a Tmax of 1.0 h and a t1/2 of 5.9 h [1]. Inobrodib (CCS1477) showed moderate stability in human liver microsomes (t1/2 = 5.2 h) [1]. The plasma protein binding of Inobrodib (CCS1477) was 88% (human plasma) [1].
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| Toxicity/Toxicokinetics |
Acute toxicity studies in ICR mice: Oral administration of Inobrodib (CCS1477) at doses up to 200 mg/kg did not cause death or significant toxic symptoms (e.g., weight loss, behavioral abnormalities) within 14 days [1]. Subchronic toxicity studies in Sprague-Dawley rats (oral administration of 30 mg/kg, 60 mg/kg, and 120 mg/kg daily for 28 days): No significant changes were observed in body weight, food intake, hematological parameters (white blood cells, red blood cells, platelets), or biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of the liver, kidneys, heart, lungs, and spleen revealed no drug-related lesions [1].
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| References | |
| Additional Infomation |
Inobrodib is a small molecule inhibitor with high oral bioavailability that inhibits the highly conserved bromodomains of histone acetyltransferase (HAT) homolog p300 (E1A-associated protein p300; p300 HAT) and CREB-binding protein (CBP), exhibiting potential antitumor activity. After oral administration, inobrodib selectively and reversibly binds to the bromodomains of p300 and CBP. This disrupts the acetylation of histones and other proteins and prevents the co-activation of key transcription factors, including androgen receptor (AR), androgen receptor splice variant (AR-SV), hypoxia-inducible factor 1-α (HIF-1-α), and Myc proto-oncogene protein (c-Myc), which contribute to tumor progression. Histone acetyltransferase (HAT) homologs p300 and CBP are key transcriptional coactivators essential for various cellular processes and are associated with progression and treatment resistance in some cancers.
Inobrodib (CCS1477) is a selective small molecule p300/CBP histone acetyltransferase inhibitor designed to target the HAT domain of these enzymes[1]. The mechanism of action of Inobrodib (CCS1477) in treating castration-resistant prostate cancer (CRPC) includes inhibition of p300/CBP-mediated histone acetylation, blocking androgen receptor (AR) coactivation, downregulation of AR target gene expression, and induction of CRPC cell apoptosis[1]. Inobrodib (CCS1477) is being evaluated for its efficacy in treating lethal castration-resistant prostate cancer (CRPC), particularly for patients resistant to conventional therapies. Androgen receptor targeted therapy[1] Inobrodib (CCS1477) selectively inhibits p300/CBP, avoiding the off-target effects associated with non-selective HAT inhibitors, thus exhibiting good toxicity characteristics[1] |
| Molecular Formula |
C30H32F2N4O3
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|---|---|
| Molecular Weight |
534.5969
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| Exact Mass |
534.24
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| Elemental Analysis |
C, 67.40; H, 6.03; F, 7.11; N, 10.48; O, 8.98
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| CAS # |
2222941-37-7
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| Related CAS # |
2222941-37-7
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| PubChem CID |
134457551
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| Appearance |
White to off-white solid
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
|
| Heavy Atom Count |
39
|
| Complexity |
860
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
CC1=C(C(=NO1)C)C2=CC3=C(C=C2)N(C(=N3)[C@@H]4CCCC(=O)N4C5=CC(=C(C=C5)F)F)C6CCC(CC6)OC
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| InChi Key |
SKDNDJWEBPQKCS-CLHVYKLBSA-N
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| InChi Code |
InChI=1S/C30H32F2N4O3/c1-17-29(18(2)39-34-17)19-7-14-26-25(15-19)33-30(36(26)20-8-11-22(38-3)12-9-20)27-5-4-6-28(37)35(27)21-10-13-23(31)24(32)16-21/h7,10,13-16,20,22,27H,4-6,8-9,11-12H2,1-3H3/t20-,22-,27-/m0/s1
|
| Chemical Name |
(S)-1-(3,4-difluorophenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one
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| Synonyms |
CBP-IN-1CBP IN-1Inobrodib CBP-IN 1CCS-1477 CCS 1477 CCS1477
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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 (~187.06 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5.25 mg/mL (9.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 52.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume 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: ≥ 5.25 mg/mL (9.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 52.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.68 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.5 mg/mL (4.68 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8706 mL | 9.3528 mL | 18.7056 mL | |
| 5 mM | 0.3741 mL | 1.8706 mL | 3.7411 mL | |
| 10 mM | 0.1871 mL | 0.9353 mL | 1.8706 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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