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Fadraciclib (CYC-065) is a novel, 2nd generation, ATP-competitive and orally bioavailable inhibitor of cyclin dependent kinases 2, 5 and 9 (CDK2/5/9, IC50s of 5 and 26 nM for CDK2/9) with potential antineoplastic and chemoprotective activities. CYC065 selectively binds to and inhibits the activity of CDK2, 5 and 9, which leads to inhibition of CDK2, 5 and 9-dependent cellular pathways, downregulation of genes involved in the pro-survival pathway, prevention of the activation of DNA double-strand break repair pathways, and induction of both cell cycle arrest and apoptosis. This inhibits the proliferation of CDK2/5/9-overexpressing tumor cells. In addition, CYC065 protects hematopoietic stem and progenitor cells (HSPCs), prevents myelosuppression, and preserves the function of the bone marrow.
Fadraciclib (CYC065) is a novel cyclin-dependent kinase inhibitor that selectively targets CDK2 and CDK9, with IC50 values of 5 nM and 26 nM, respectively. Following the successful completion of Investigational New Drug (IND)-enabling studies, Fadraciclib received FDA clearance to proceed into first-in-human Phase 1 clinical trials. Objective: To establish the mechanistic rationale and define an optimal dosing schedule to support the clinical development of Fadraciclib in specific subsets of leukemias and lymphomas characterized by key molecular features that represent areas of high unmet medical need. Design: The activity of Fadraciclib was evaluated across three panels of cell lines derived from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and B-cell lymphoma. These panels encompassed a range of molecular profiles, including varying levels of Mcl-1 expression/dependence, MYC overexpression or amplification, and different MLL gene statuses. Comprehensive profiling was conducted to identify cellular sensitivity parameters that could serve as the foundation for patient selection biomarkers in the clinic. Additionally, the in vivo antitumor efficacy of Fadraciclib was assessed in two distinct AML xenograft models. Results: Consistent with its target inhibition profile, Fadraciclib treatment led to a rapid reduction in RNA polymerase II phosphorylation and the subsequent downregulation of key oncogenic target proteins, including Mcl-1 and MLL target genes, which in turn triggered a swift induction of apoptosis. Notably, even short-duration pulse exposures to the drug were sufficient to achieve ≥90% cell death in sensitive cell lines. Hematologic cancer cell lines harboring MLL rearrangements demonstrated enhanced sensitivity to Fadraciclib compared to wild-type counterparts; in wild-type AML cells, sensitivity was found to correlate with the expression levels of Bcl-2 family proteins. Furthermore, combining Fadraciclib with either Bcl-2 inhibitors or the standard chemotherapeutic agent cytarabine resulted in synergistic anti-leukemic effects. Conclusions: By leveraging its ability to induce rapid and robust downregulation of critical oncogenic transcripts, Fadraciclib shows significant therapeutic potential for a variety of leukemia and lymphoma indications with unmet clinical needs, including MLL-translocated leukemia, FLT3-ITD leukemia, and MYC-driven lymphoma. The compound has also demonstrated effective synergy when combined with standard cytotoxic agents like cytarabine, as well as with inhibitors targeting other key apoptotic regulators, such as Bcl-2/Bcl-xL inhibitors.| Targets |
Fadraciclib (CYC065) is a second-generation, orally available ATP-competitive inhibitor of cyclin-dependent kinase 2 (CDK2) and cyclin-dependent kinase 9 (CDK9). It targets the CDK2/cyclin E1 complex, which is critical for G1-S phase transition, and CDK9, which is involved in transcriptional regulation. The study does not provide specific IC₅₀ values for these targets [1].
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| ln Vitro |
Fadraciclib stops cells in the G1 phase of the cell cycle and reduces cell development, notably in uterine serous carcinoma (USC) overexpressing cyclin E1 (CCNE1). USC cell lines expressing high CCNE1 mRNA and protein levels were significantly more sensitive to in vitro Fadraciclib treatment compared to low CCNE1 expressing cell lines (IC50: mean ± sd = 124.1 ± in CCNE1 overexpressing USC cell lines vs. 415 cell lines 57.8 nM) and ±117.5 nM respectively in CCNE1 low-expression proteins; P=0.0003). Importantly, modest dosages of Fadraciclib (i.e., 100 nM) cause cell cycle G1 phase arrest only in CCNE1-overexpressing USC cell lines (i.e., USC-ARK-2, USC-ARK-7) [1].
In primary uterine serous carcinoma (USC) cell lines, Fadraciclib demonstrates potent antiproliferative activity. CCNE1-amplified USC cell lines (USC-ARK-1, USC-ARK-2, USC-ARK-7) are significantly more sensitive to Fadraciclib compared with non-amplified lines, with mean IC₅₀ values of 124.1 ± 57.8 nM versus 415 ± 117.5 nM, respectively (P = 0.0003) [1]. Cell cycle analysis reveals that treatment with 100 nM Fadraciclib for 48 hours induces G1 phase arrest specifically in CCNE1-amplified USC cell lines (USC-ARK-2, USC-ARK-7), but not in non-amplified lines [1]. Apoptosis induction is observed at higher concentrations: treatment of USC-ARK-7 cells with 500 nM and 1000 nM Fadraciclib for 6 hours increases the percentage of Annexin V/PI-positive cells by 1.78 ± 0.3-fold and 2.25 ± 0.7-fold, respectively [1]. Knockdown of CCNE1 using siRNA in USC-ARK-2 cells results in a 9.29-fold increase in the IC₅₀ of Fadraciclib compared with control cells (P = 0.021), confirming target specificity. CCNE1 knockdown alone also inhibits cell growth by 49.5 ± 14.4% (P = 0.027), indicating dependency of USC cells on CCNE1 for proliferation [1]. Combination studies with the PIK3CA inhibitor Taselisib show synergistic effects. In USC-ARK-1 (CCNE1-amplified, PIK3CA-mutated) and USC-ARK-2 (CCNE1-amplified, PIK3CA-wild-type) cells, the combination of Fadraciclib and Taselisib significantly inhibits cell growth more effectively than either single agent. Combination index (CI) values calculated by the Chou-Talalay method indicate synergism (CI < 1) across multiple effect levels (Fa = 0.5 to 0.95) in both cell lines [1]. Western blot analysis shows that treatment with Fadraciclib (200 nM, 6 h) inhibits Rb phosphorylation, and the combination with Taselisib (10 nM) potently inhibits both pRb and pS6 (downstream effector of PI3K pathway) in USC-ARK-1 and USC-ARK-2 cells. Increased expression of CCNE1 is observed after Fadraciclib treatment [1]. |
| ln Vivo |
USC-ARK-2-derived xenografts were treated with Fadraciclib (22.5 mg/kg) daily for three weeks in order to assess the therapeutic potential of the drug as a stand-alone treatment. Twice a week, the growth of the tumor and the weight of the mice were noted. When compared to mice treated with a vehicle, daily dose of Fadraciclib significantly slowed the growth of the tumor (beginning on day 9 of therapy, P=0.012). Throughout the whole course of the medication, no discernible weight reduction was noted [1].
In USC-ARK-2-derived xenografts in SCID mice, oral administration of Fadraciclib (22.5 mg/kg daily for 3 weeks) significantly inhibits tumor growth compared with vehicle control (P = 0.012 from day 9). No significant weight loss is observed during treatment [1]. In USC-ARK-1-derived xenografts (CCNE1-amplified, PIK3CA-mutated), the combination of Fadraciclib (22.5 mg/kg daily for 3 weeks) and Taselisib (10 mg/kg, 5 days/week for 3 weeks) demonstrates significantly greater tumor growth inhibition than either single agent. At day 21, mean tumor volumes are 1.04 ± 0.5 cm³ (CYC065 alone), 0.72 ± 0.29 cm³ (Taselisib alone), and 0.37 ± 0.11 cm³ (combination; P < 0.03). A slight but non-significant weight decrease is noted in the combination group after day 19 [1]. |
| Cell Assay |
Cell viability assay: USC primary cell lines are plated in six-well plates and treated with scalar concentrations of Fadraciclib (ranging from 100 to 500 nM) for 72 hours. Cells are harvested, washed, stained with propidium iodide (5 μg/mL), and counted by flow cytometry. The percentage of viable cells is normalized to vehicle-treated controls, and IC₅₀ values are calculated using nonlinear regression in GraphPad Prism [1].
Cell cycle analysis: Cells are treated with 100 nM Fadraciclib for 48 hours, permeabilized with ice-cold 70% ethanol, fixed at 4°C for 30 minutes, treated with RNase (100 μg/mL), stained with propidium iodide (50 μg/mL), and analyzed by flow cytometry using FACSCalibur and FlowJo software [1]. Apoptosis assay: Cells are treated with Fadraciclib (500 or 1000 nM) for 6 hours, then stained with Annexin V/PI and analyzed by flow cytometry [1]. siRNA knockdown: USC-ARK-2 cells are transfected with CCNE1-specific siRNA (10 nM) or MOCK siRNA using Lipofectamine RNAiMAX. After 24 hours, cells are treated with scalar doses of Fadraciclib for 72 hours, then harvested for viability assays or RNA extraction [1]. Western blotting: Cells are treated with Fadraciclib (200 nM), Taselisib (10 nM), or their combination for 6 hours. Lysates are prepared in RIPA buffer with protease inhibitors, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against HER2/neu, pHER2/neu, Rb, pRb, CCNE1, S6, pS6, and GAPDH. Detection uses HRP-conjugated secondary antibodies and chemiluminescence [1]. |
| Animal Protocol |
For single-agent efficacy studies, 5-7 week old SCID mice are subcutaneously injected with USC-ARK-2 cells. One week after implantation, when tumors reach 0.125-0.150 cm³, mice are randomized into two groups (minimum n=5 per group). The treatment group receives Fadraciclib (22.5 mg/kg) daily by oral gavage for 3 weeks; the control group receives vehicle. Tumor size and body weight are recorded twice weekly. Tumor volume is calculated as V = length × (width)²/2 [1].
For combination studies, USC-ARK-1-derived xenografts are established similarly. Mice are divided into four groups: vehicle (0.5% methylcellulose-0.2% Tween-80), Fadraciclib alone (22.5 mg/kg daily for 3 weeks), Taselisib alone (10 mg/kg daily, 5 days/week for 3 weeks), and combination of both drugs. Treatments begin one week after implantation when tumors reach 0.125-0.150 cm³. Tumor volume and body weight are monitored twice weekly [1]. |
| ADME/Pharmacokinetics |
Fadraciclib is described as an oral compound, but this study [1] did not provide specific pharmacokinetic parameters (absorption, distribution, metabolism, excretion, half-life, bioavailability).
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| Toxicity/Toxicokinetics |
In in vivo studies of both monotherapy and combination therapy, treatment with Fadraciclib at a daily dose of 22.5 mg/kg for up to 3 weeks was well tolerated, with no significant weight loss observed compared to the placebo control group. When used in combination with Taselisib, a slight but insignificant weight loss was observed after day 19, but no other toxicities were reported [1].
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| References | |
| Additional Infomation |
CYC-065 is currently undergoing clinical trial NCT03739554 (a study of CYC065 CDK inhibitors in combination with venetoc for the treatment of relapsed/refractory chronic lymphocytic leukemia). Fadracib is an orally bioavailable cyclin-dependent kinase 2, 5, and 9 (CDK2/5/9) inhibitor with potential antitumor and chemoprotective activity. After oral administration, fadracib selectively binds to and inhibits the activity of CDK2, 5, and 9, thereby inhibiting CDK2, 5, and 9-dependent cellular pathways, downregulating genes involved in pro-survival pathways, preventing activation of DNA double-strand break repair pathways, and inducing cell cycle arrest and apoptosis. This can inhibit the proliferation of CDK2/5/9 overexpressing tumor cells. Furthermore, CYC065 can protect hematopoietic stem cells and progenitor cells (HSPCs), prevent myelosuppression, and maintain bone marrow function. CDKs are serine/threonine kinases involved in cell cycle regulation and may be overexpressed in certain cancer cell types; they play a crucial role in tumor cell proliferation, transcriptional regulation, and DNA damage repair. Fadraciclib (CYC065) is a second-generation oral ATP-competitive CDK2 and CDK9 inhibitor developed by Cyclacel Ltd. It is designed to target cancers with CCNE1 amplification, which occurs in approximately 48% of uterine serous carcinomas. This compound blocks G1-S phase transition by inhibiting CDK2 and induces apoptosis at higher concentrations by inhibiting CDK9 and downregulating Mcl-1. Preclinical studies have shown that Fadraciclib has significant antitumor activity against CCNE1-amplified USC cell lines and xenograft tumors. In addition, the combination of Fadraciclib and Taselisib targeting CCNE1 and PIK3CA showed synergistic antitumor effects in vitro and in vivo, suggesting that it may be a promising treatment strategy for relapsed, chemotherapy-resistant USC patients with CCNE1 amplification and PIK3CA mutations [1].
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| Molecular Formula |
C21H31N7O
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|---|---|
| Molecular Weight |
397.51714348793
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| Exact Mass |
397.25900864
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| Elemental Analysis |
C, 63.45; H, 7.86; N, 24.66; O, 4.02
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| CAS # |
1070790-89-4
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| Related CAS # |
1070790-89-4;1315571-38-0 (HCl);1315571-33-5 (tartrate);1315571-34-6 (citrate); 1315571-36-8 (besylate); 1315571-40-4 (mesylate);
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| PubChem CID |
24983461
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| Appearance |
White to off-white solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
29
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| Complexity |
507
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O[C@H](C)[C@H](CC)NC1=NC(=C2C(=N1)N(C=N2)C(C)C)NCC1C=NC(C)=CC=1C
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| InChi Key |
DLPIYBKBHMZCJI-WBVHZDCISA-N
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| InChi Code |
InChI=1S/C21H31N7O/c1-7-17(15(6)29)25-21-26-19(18-20(27-21)28(11-24-18)12(2)3)23-10-16-9-22-14(5)8-13(16)4/h8-9,11-12,15,17,29H,7,10H2,1-6H3,(H2,23,25,26,27)/t15-,17+/m1/s1
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| Chemical Name |
(2R,3S)-3-((6-(((4,6-dimethylpyridin-3-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)amino)pentan-2-ol
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| Synonyms |
CYC065; CYC-065; fadraciclib; 1070790-89-4; YET2XNU791; CYC 065
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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 (~251.56 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.29 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 25.0 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: ≥ 2.5 mg/mL (6.29 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 to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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 (6.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5156 mL | 12.5780 mL | 25.1560 mL | |
| 5 mM | 0.5031 mL | 2.5156 mL | 5.0312 mL | |
| 10 mM | 0.2516 mL | 1.2578 mL | 2.5156 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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