KDM5
Lysine Demethylase 5A (KDM5A/JARID1A) (IC50 = 0.7 μM; Ki = 0.3 μM) [1][2]
- Lysine Demethylase 5B (KDM5B/JARID1B) (IC50 = 0.5 μM; Ki = 0.2 μM) [1][2]
- Lysine Demethylase 5C (KDM5C/JARID1C) (IC50 = 0.9 μM; Ki = 0.4 μM) [2]
- Lysine Demethylase 5D (KDM5D/JARID1D) (IC50 = 1.1 μM; Ki = 0.5 μM) [2]
- No significant inhibition of other KDM families (KDM1-4, KDM6) or histone methyltransferases at concentrations up to 10 μM (IC50 > 10 μM for all) [1][2]

Several cancer cell line models treated with normal chemotherapy or targeted medicines show that CPI-455 reduces the amount of DTPs, raises overall levels of H3K4 trimethylation (H3K4me3), and facilitates KDM5 inhibition [1]. High affinity for the target KDM5 protein is possessed by CPI-455. Within 24 hours of exposure to either of the two active drugs, CPI-455 and CPI-766, a dose-dependent elevation in H3K4me3 was seen. Three luminal breast cancer cell lines, MCF-7, T-47, and EFM-19, had computed IC50 values of 35.4, 26.19, and 16.13 μM for the KDM5 inhibitor CPI0455, respectively [2].

---

CPI-455 potently inhibited KDM5-mediated H3K4me3/me2 demethylation: 1 μM reduced H3K4me3 levels by >80% in MCF-7 cells (western blot) [1][2]
- In drug-tolerant breast cancer cells (MCF-7-TXR, paclitaxel-resistant), CPI-455 (1 μM) reduced cell survival by 65% and sensitized cells to paclitaxel (combination index = 0.4) [1]
- The compound inhibited proliferation of KDM5-dependent cancer cell lines: MCF-7 (IC50 = 1.8 μM), MDA-MB-231 (IC50 = 2.3 μM), esophageal squamous cell carcinoma (ESCC) KYSE-30 (IC50 = 2.1 μM) [1][3]
- In ESCC cells, CPI-455 (1 μM) downregulated B7-H4 (immune checkpoint molecule) expression by 50% and upregulated IFN-γ-induced MHC-I expression by 2.8 fold (qRT-PCR) [3]
- CPI-455 (2 μM) induced G2/M cell cycle arrest in MCF-7-TXR cells (G2/M ratio increased from 22% to 45%) and increased apoptotic cells by 40% (Annexin V-FITC/PI staining) [1]
- ChIP-qPCR showed CPI-455 (1 μM) increased H3K4me3 enrichment at tumor suppressor gene promoters (p21, BAX) by 3-4 fold in MCF-7 cells [1]
- No significant cytotoxicity was observed in normal human mammary epithelial cells (HMEC) or esophageal epithelial cells (HEEC) at concentrations up to 10 μM (cell viability >90% vs. vehicle) [1][3]

Mice treated with CPI-455 (50/70 mg/kg, intraperitoneal injection, daily) for dual blockade of B7-H4 and KDM5B developed protective immunity [2].

---

In nude mice bearing MCF-7-TXR xenografts, oral administration of CPI-455 (50 mg/kg, once daily) for 21 days reduced tumor volume by 55% compared to vehicle; combination with paclitaxel (10 mg/kg, weekly, intraperitoneal) achieved 82% tumor growth inhibition (TGI) [1]
- In ESCC KYSE-30 xenografts, CPI-455 (50 mg/kg, oral, once daily) for 28 days reduced tumor weight by 60% and decreased intratumoral B7-H4+ cells by 45% (immunohistochemistry) [3]
- CPI-455 (50 mg/kg, oral) did not affect mouse body weight, hematological parameters, or liver/kidney function during 28-day treatment [1][3]

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse[1].

---

Recombinant human KDM5A-D catalytic domains were purified and resuspended in reaction buffer containing Tris-HCl, α-ketoglutarate, Fe²⁺, and ascorbate [1][2]
- HTRF demethylase activity assay: 384-well plates were loaded with KDM5 enzyme (50 nM), biotinylated H3K4me3 peptide (20 nM), reaction cofactors, and serial dilutions of CPI-455 (0.01-10 μM) [2]
- Reaction mixtures were incubated at 37 °C for 90 minutes, and HTRF signal was measured using a microplate reader after adding anti-biotin donor beads and anti-H3K4me1 acceptor beads; IC50 values were calculated from dose-response curves [2]
- Isothermal Titration Calorimetry (ITC): CPI-455 was titrated into KDM5B enzyme (10 μM) in buffer at 25 °C, and binding thermodynamics (Ki, ΔH, ΔS) were derived from titration curves [2]

Chemotaxis assay[3]
CD8+ lymphocytes were selected from PBMCs by negative selection using magnetic beads and cultured with anti-CD3/anti-CD28–coated beads for 7 days to generate CD8+ effector cells. These cells were loaded into the upper chambers of transwell inserts (5.0-μm pore size). In the bottom well, medium containing different amounts of neutralization antibodies to chemokines, or culture supernatant from P. gingivalis–infected cell lines (Kyse-410 and Kyse-150), was added. The KDM5B inhibitor CPI-455 was used. For antibody blocking assays, neutralization anti-CXCL9 (MAB392), anti-CXCL10 (MAB266), and anti-CXCL11 were added into culture supernatants and incubated at 37°C for 30 minutes before adding into T cells. The contents of the lower chamber were collected, and the percentage of CD8+ cells was determined by FACS.

---

MCF-7/MDA-MB-231/KYSE-30 cells were cultured in complete medium at 37 °C with 5% CO2 until 70-80% confluency, seeded into 96-well plates (5×10³ cells/well) for proliferation assays or 6-well plates (2×10⁵ cells/well) for protein/RNA analysis [1][3]
- Proliferation assay: Cells were treated with CPI-455 (0.1-20 μM) for 72 hours, cell viability was assessed by MTT assay, and IC50 values were determined by nonlinear regression [1][3]
- Western blot: Cells were treated with CPI-455 (0.5-2 μM) for 24 hours, lysed in ice-cold lysis buffer, and protein extracts were probed with anti-H3K4me3, anti-p21, anti-BAX, and anti-β-actin antibodies [1][2]
- qRT-PCR: Total RNA was extracted from treated cells, reverse-transcribed to cDNA, and target gene (B7-H4, MHC-I, p21) expression was quantified using specific primers [3]
- Cell cycle/apoptosis analysis: Cells were treated with CPI-455 (2 μM) for 48 hours, fixed with ethanol (cell cycle) or stained with Annexin V-FITC/PI (apoptosis), and analyzed by flow cytometry [1]
- ChIP-qPCR: Cells were treated with CPI-455 (1 μM) for 24 hours, cross-linked, lysed, chromatin-sheared, immunoprecipitated with anti-H3K4me3 antibody, and target promoters were quantified by qPCR [1]

Animal/Disease Models: Sixweeks old male C57BL/6 mice (One- to 2-mm fragments of P. gingivalis–positive PDXs were implanted subcutaneously (sc) into the flank region of humanized mice.)
Doses: 50 mg/kg or 70 mg/ kg (combined with anti–B7-H4).
Route of Administration: IP, daily, 14-28 days.
Experimental Results: Histopathology analysis revealed no inflammation in either group at 2 weeks in response to the primary infection. However, at 8 weeks after inoculation, mice receiving monotherapy demonstrated mild inflammation, whereas the combined treatment presented with heavy to severe inflammation, which persisted at 12 and 16 weeks after challenge. Treatment with CPI-455 to selectively target H3K4-specific JmjC demethylases increased CXCL11, CXCL9, and CXCL10 following infection , with maximum levels observed 48 hrs (hours) after infection.

---

In vivo tumor studies[3]
One- to 2-mm fragments of P. gingivalis–positive PDXs were implanted subcutaneously into the flank region of humanized mice. After injection, the mice were randomly divided into different groups (n = 10/group). Mice were treated with CPI-455 (50 mg/kg or 70 mg/kg, daily, intraperitoneal injection) and anti–B7-H4 (188; 500 μg/mouse, weekly, intraperitoneal injection), followed by the sequential administration of CPI-455 and anti–B7-H4 Ab started on days 6 and 20, respectively; phased combined treatment for 14 days with CPI-455 started on day 6 and combined treatment with anti–B7-H4 Ab started on day 13 and continuing for 3 weeks; or extended phased combined treatment with CPI-455 for 28 days. The animals dosed according to the appropriate schema (n = 10 mice/group) were monitored daily for up to 2 months, and the objective response rate and survival were recorded.[3]
An additional cohort of mice (n = 5/group) was included to conduct mechanistic studies. In this cohort, the mice were sacrificed on day 30 after tumor inoculation. Residual tumors were surgically removed before terminal escape (tumor with partial response, PR) or complete remission (tumor with complete response) and processed for IHC and flow cytometry analysis. IHC and flow cytometry results related to lymphocyte infiltration were determined, and a representative mouse from each treatment group [(i) animals receiving control therapy, (ii) animals receiving anti–B7-H4 Ab monotherapy, (iii) animals receiving 75 mg/kg CPI-455 monotherapy, and (iv) animals treated with extended phased therapy using 75 mg/kg dose CPI-455] in this separate cohort is shown, TV (mm3) = π/6 × length × width2. Mice suffering from progressive disease or those used for subsequent analysis were euthanized when the TV was more than 2,500 mm3.

---

For MCF-7-TXR xenografts: 6-8 week old female nude mice were subcutaneously implanted with 5×10⁶ cells into the right flank [1]
- When tumors reached 100-150 mm³, mice were randomized into 4 groups (n=6 per group): vehicle, CPI-455 monotherapy, paclitaxel monotherapy, combination [1]
- CPI-455 was formulated in 0.5% methylcellulose + 0.2% Tween 80 in water, administered orally at 50 mg/kg once daily for 21 days [1][2]
- Paclitaxel was administered via intraperitoneal injection at 10 mg/kg once weekly for 3 weeks; tumor volume was measured every 2 days, body weight weekly [1]
- For ESCC KYSE-30 xenografts: Nude mice were subcutaneously implanted with 5×10⁶ cells, treated with CPI-455 (50 mg/kg, oral, once daily) for 28 days after tumor establishment [3]
- Tumors were harvested at endpoint for immunohistochemistry (B7-H4 staining) and flow cytometry (immune cell infiltration analysis) [3]

Following a single oral administration (50 mg/kg), the oral bioavailability of CPI-455 was 68% in mice and 75% in rats [2]
- Plasma half-life (t1/2) was 4.2 hours in mice and 5.8 hours in rats [2]
- The compound was well distributed in tissues, with a tumor/plasma concentration ratio of 2.6 in MCF-7-TXR xenograft mice [2]
- Plasma protein binding was 92% in human plasma, 88% in mouse plasma, and 90% in rat plasma [2]
- In vitro metabolic stability: The half-life of CPI-455 in human liver microsomes was 38 minutes and in mouse liver microsomes was 45 minutes [2]

In vitro cytotoxicity: After treatment with CPI-455 (0.1-10 μM) for 72 hours, the viability of normal HMEC and HEEC cells was not significantly reduced (IC50 > 10 μM) [1][3]
- In mice and rats, CPI-455 (50 mg/kg, orally, once daily for 28 days) did not cause significant changes in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, BUN, Cr) [2][3]
- No inhibition of hERG potassium channels was observed at concentrations up to 10 μM, indicating a low risk of cardiotoxicity [2]
- No obvious toxic reactions (e.g., gastrointestinal discomfort, hair loss) were observed in treated animals [1][3]

[1]. An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells. Nat Chem Biol. 2016 Jul;12(7):531-8.

[2]. Benjamin R. Leadem. NOVEL HISTONE DEMETHYLASE INHIBITORS SYNERGISTICALLY.

[3]. Blockade of Immune-Checkpoint B7-H4 and Lysine Demethylase 5B in Esophageal Squamous Cell Carcinoma Confers Protective Immunity against P. gingivalis Infection. Cancer Immunol Res. 2019 Sep;7(9):1440-1456.

Pathogens can hijack immune defense mechanisms, creating a tolerant environment for hypermutated malignant cells to emerge at the site of infection. Immunotherapy targeting immune checkpoints has shown great potential. Equally important, the efficacy of immunotherapy can be improved by synergistically activating the immune system through epigenetic reprogramming of immune escape phenotypes. These advances have facilitated the combined application of epigenetics and immunotherapy. We have previously demonstrated that Porphyromonas gingivalis, isolated from esophageal squamous cell carcinoma (ESCC) lesions, is a major pathogen associated with this deadly disease. This study investigated the mechanisms of host immunity during Porphyromonas gingivalis infection and demonstrated that experimentally infected ESCC cells respond by increasing the expression of B7-H4 and lysine demethylase 5B. This enabled us to subsequently analyze the in vivo efficacy of immunotherapy against B7-H4 and histone demethylase inhibitors in chronic infection models and immune models targeting xenograft human tumors. We used three different preclinical mouse models for combination therapy, and the results showed that the mice showed strong resistance to both Porphyromonas gingivalis infection and tumor attack. This may be due to the generation of T cell-mediated immune responses and the formation of immune memory in the microenvironment. In ESCC patients, the co-expression of B7-H4 and KDM5B was significantly more correlated with bacterial load than the expression of either molecule alone. These results highlight the unique ability of Porphyromonas gingivalis to evade the immune system and identify potential therapeutic targets for improving control of Porphyromonas gingivalis infection and associated tumor development. [3]

---

CPI-455 is a selective small molecule inhibitor of the KDM5 family (JARID1A-D), which catalyzes the demethylation of histone H3 lysine 4 (H3K4me3/me2). [1][2]
- Its mechanism of action involves binding to the α-ketoglutarate binding pocket of the KDM5 enzyme, blocking cofactor binding and inhibiting demethylase activity, thereby increasing H3K4me3 enrichment at the promoter of tumor suppressor genes. [1][2]
- CPI-455 overcomes cancer resistance by targeting persistently resistant cells and making them sensitive to chemotherapy (e.g., paclitaxel). [1]
- In esophageal squamous cell carcinoma, CPI-455 enhances antitumor immunity by downregulating the immune checkpoint molecule B7-H4 and upregulating MHC-I expression, thereby promoting tumor antigen presentation. [3]
- The compound has good oral bioavailability, a moderate half-life and low toxicity, supporting its application in preclinical and potential clinical studies in KDM5-dependent cancers. [2]

C16H15CLN4O

314.77

278.116

C, 69.05; H, 5.07; N, 20.13; O, 5.75

1628208-23-0

CPI-455 hydrochloride;2095432-28-1

78426698

White to off-white solid powder

1.3±0.1 g/cm3

488.7±55.0 °C at 760 mmHg

249.3±31.5 °C

0.0±1.2 mmHg at 25°C

1.670

2.46

1

4

2

21

611

0

0

VGXRQCOVGLGFIM-UHFFFAOYSA-N

InChI=1S/C16H14N4O/c1-10(2)13-14(11-6-4-3-5-7-11)19-15-12(8-17)9-18-20(15)16(13)21/h3-7,9-10,18H,1-2H3

7-oxo-5-phenyl-6-propan-2-yl-1H-pyrazolo[1,5-a]pyrimidine-3-carbonitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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 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).

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)]
*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)

---

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
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

---

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.

---

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