Selective Inhibitor Of Cyclin-dependent Kinases Research Articles

Preclinical metabolism and disposition of [14C]GFH009, a novel selective CDK9 inhibitor

1. GFH009 is a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor currently under phase II clinical trials. In this study, we investigated the metabolism and disposition of GFH009 in Sprague-Dawley (SD) rats, as well as in vitro metabolism of CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human. 2. A radiolabelled study indicated that [14C]GFH009 was quickly and widely distributed throughout the body, but presented low levels in brain, testis and epididymis after a single intravenous dose of 6 mg (100 µCi)/kg to SD rats. 3. GFH009 undergoes systemic metabolic changes, primarily throughO-demethylation, oxidation to carboxylic acid and N-dealkylation, cleavage off the methoxyisopropyl moiety being a minor pathway. These metabolic pathways were found to be mainly consistent both in vitro and in vivo. 4. In SD rats, GFH009 was rapidly and completely eliminated, with feces serving as the major excretion pathway and urine serving as the minor one. Besides, the major clearance pathway for GFH009 was excretion and the minor one was metabolism. 5. GFH009 exhibits favourable drug metabolism and pharmacokinetics (DMPK) properties, which provides valuable insights into the disposition of GFH009 and can be used to guide future clinical studies.

The novel selective inhibitors of cyclin-dependent kinase 4/6: in vitro and in silico study.

One of the main regulators in the cell cycle is cyclin-dependent kinase 4 and 6 (CDK4/6). FDA has approved CDK4/6 inhibitors for the treatment of patients with metastatic breast cancer. However, the development of selective agents remains problematic due to the conservation of their ATP binding sites. In the previous in silico study, ZINC585292724, ZINC585292587, ZINC585291674, and ZINC585291474 have been identified as potential inhibitors. Therefore, the present study aimed to analyze the selectivity and inhibitory activity of the four compounds against CDK4/6 in vitro as well as determine the potential for their further development in silico. The in vitro results showed that the four compounds had good selectivity towards both kinases, due to their similar structure. In agreement with the in silico results, ZINC585291674 produced the best inhibitory activity against CDK4 and CDK6, with IC50 of 184.14nM and 111.78nM, respectively. Their ADMET profile were also similar to reference compound of Palbociclib. Based on this, ZINC585291674 can be used as a lead compound for further inhibitor development.

Design, synthesis, molecular docking, and evaluation of sulfonyl quinazoline analogues as promising liver cancer drugs

Inhibiting cyclin-dependent kinases (CDK) offers an important arsenal for cancer treatments by interfering with apoptotic proteins related to cancer. Novel selective cyclin-dependent kinases inhibitors using the Quinazoline as the cap with multiple electronic donating (EDG) and/or electron withdrawing group (EWG) substituted Aniline chain at the C-2 position were designed, synthesized, and evaluated for activity against liver cancer. Among the tested compounds, compounds B34 and B35 emerged as potent candidates in the series, with IC50 values of 0.102 ± 0.04 µM and 0.058 ± 0.003 µM, respectively. They also suppressed the enzymatic activity of CDK2/cyclinA2 selectively. Further biological studies revealed that compounds B34 and B35 arrested the cell cycle, and induced apoptosis in HepG-2 cancer cells through a Caspase-mediated mechanism, facilitating the release of Cyt-c through modulation of Caspase-3 expression. More importantly, compounds B34 and B35 suppressed the xenografted tumor growth in mice in a dose-dependent manner. Finally, through a molecular docking study, it was confirmed that compoundsB34 andB35 retained crucial hydrogen bonding and hydrophobic interactions with CDK receptor, rationalizing their higher efficacy compared to other compounds in the series. Taken together, the Quinazoline derivatives B34 and B35 may serve as novel chemotherapeutic agents through inhibition of CDK.

Uncovering potential CDK9 inhibitors from natural compound databases through docking-based virtual screening and MD simulations.

Cyclin-dependent kinase 9 (CDK9) plays a significant role in gene regulation and RNA polymerase II transcription under basal and stimulated conditions. The upregulation of transcriptional homeostasis by CDK9 leads to various malignant tumors and therefore acts as a valuable drug target in addressing cancer incidences. Ongoing drug development endeavors targeting CDK9 have yielded numerous clinical candidate molecules currently undergoing investigation as potential CDK9 modulators, though none have yet received Food and Drug Administration (FDA) approval. In this study, we employ in silico approaches including the molecular docking and molecular dynamics simulations for the virtual screening over the natural compounds library to identify novel promising selective CDK9 inhibitors. The compounds derived from the initial virtual screening were subsequently employed for molecular dynamics simulations and binding free energy calculations to study the compound's stability under virtual physiological conditions. The first-generation CDK inhibitor Flavopiridol was used as a reference to compare with our novel hit compound as a CDK9 antagonist. The 500-ns molecular dynamics simulation and binding free energy calculation showed that two natural compounds showed better binding affinity and interaction mode with CDK9 receptors over the reference Flavopiridol. They also showed reasonable figures in the predicted absorption, distribution, metabolism, excretion, and toxicity (ADMET) calculations as well as in computational cytotoxicity predictions. Therefore, we anticipate that the proposed scaffolds could contribute to developing potential and selective CDK9 inhibitors subjected to further validations.

Wogonin protects against bleomycin-induced mouse pulmonary fibrosis via the inhibition of CDK9/p53-mediated cell senescence.

Pulmonary fibrosis (PF) is a fatal interstitial lung disease associated with declining pulmonary function but currently with few effective drugs. Cellular senescence has been implicated in the pathogenesis of PF and could be a potential therapeutic target. Emerging evidence suggests wogonin, the bioactive compound isolated from Scutellaria baicalensis, owns the anti-senescence properties, however, the possible impact of wogonin on PF and the potential mechanisms remain unclear. In this study, a well-established mouse model of PF was utilized which mice were administrated with bleomycin (BLM). Strikingly, wogonin treatment significantly reduced fibrosis deposition in the lung induced by BLM. In vitro, wogonin also suppressed fibrotic markers of cultured epithelial cells stimulated by BLM or hydrogen peroxide. Mechanistic investigation revealed that wogonin attenuated the expressions of DNA damage marker γ-H2AX and senescence-related markers including phosphorylated p53, p21, retinoblastoma protein (pRB), and senescence-associated β-galactosidase (SA-β-gal). Moreover, wogonin, as a direct and selective inhibitor of cyclin-dependent kinase 9 (CDK9), exhibited anti-fibrotic capacity by inhibiting CDK9 and p53/p21 signalling. In conclusion, wogonin protects against BLM-induced PF in mice through the inhibition of cell senescence via the regulation of CDK9/p53 and DNA damage pathway. This is the first study to demonstrate the beneficial effect of wogonin on PF, and its implication as a novel candidate for PF therapy.

Deriving general structure–activity/selectivity relationship patterns for different subfamilies of cyclin-dependent kinase inhibitors using machine learning methods

Cyclin-dependent kinases (CDKs) play essential roles in regulating the cell cycle and are among the most critical targets for cancer therapy and drug discovery. The primary objective of this research is to derive general structure–activity relationship (SAR) patterns for modeling the selectivity and activity levels of CDK inhibitors using machine learning methods. To accomplish this, 8592 small molecules with different binding affinities to CDK1, CDK2, CDK4, CDK5, and CDK9 were collected from Binding DB, and a diverse set of descriptors was calculated for each molecule. The supervised Kohonen networks (SKN) and counter propagation artificial neural networks (CPANN) models were trained to predict the activity levels and therapeutic targets of the molecules. The validity of models was confirmed through tenfold cross-validation and external test sets. Using selected sets of molecular descriptors (e.g. hydrophilicity and total polar surface area) we derived activity and selectivity maps to elucidate local regions in chemical space for active and selective CDK inhibitors. The SKN models exhibited prediction accuracies ranging from 0.75 to 0.94 for the external test sets. The developed multivariate classifiers were used for ligand-based virtual screening of 2 million random molecules of the PubChem database, yielding areas under the receiver operating characteristic curves ranging from 0.72 to 1.00 for the SKN model. Considering the persistent challenge of achieving CDK selectivity, this research significantly contributes to addressing the issue and underscores the paramount importance of developing drugs with minimized side effects.

Pharmacological inhibition of CDK4/6 impairs diffuse pleural mesothelioma 3D spheroid growth and reduces viability of cisplatin-resistant cells.

Diffuse pleural mesothelioma (DPM) of the pleura is a highly aggressive and treatment-resistant cancer linked to asbestos exposure. Despite multimodal treatment, the prognosis for DPM patients remains very poor, with an average survival of 2 years from diagnosis. Cisplatin, a platinum-based chemotherapy drug, is commonly used in the treatment of DPM. However, the development of resistance to cisplatin significantly limits its effectiveness, highlighting the urgent need for alternative therapeutic strategies. New selective inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have shown promise in various malignancies by inhibiting cell cycle progression and suppressing tumor growth. Recent studies have indicated the potential of abemaciclib for DPM therapy, and a phase II clinical trial has shown preliminary encouraging results. Here, we tested abemaciclib, palbociclib, and ribociclib on a panel of DPM cell lines and non-tumor mesothelial(MET-5A) cells. Specifically, we focused on abemaciclib, which was the mosteffective cytotoxic agent on all the DPM cell lines tested. Abemaciclib reduced DPM cell viability, clonogenic potential, and ability to grow as three-dimensional (3D) spheroids. In addition, abemaciclib induced prolonged effects, thereby impairing second-generation sphere formation and inducing G0/G1 arrest and apoptosis/ necrosis. Interestingly, single silencing of RB family members did not impair cell response to abemaciclib, suggesting that they likely complement each other in triggering abemaciclib's cytostatic effect. Interestingly, abemaciclib reduced the phosphorylation of AKT, which is hyperactive in DPM and synergized with the pharmacological AKT inhibitor (AKTi VIII). Abemaciclib also synergized with cisplatin and reduced the viability of DPM cells with acquired resistance to cisplatin. Overall, our results suggest that CDK4/6 inhibitors alone or in combination with standard of care should be further explored for DPM therapy.

Exploration of Potential Cyclin-dependent Kinases and their Inhibitors to Combat Abnormal Signalling and Cancer

Abstract: Regulation of cell proliferation depends on stimulatory and inhibitory factors that act in a coordinated manner in response to external signals. Various agents, including mitogens, growth factors, cytokines, and other external factors, can impact the mitotic cell cycle, resulting in either provoking growth, differentiation, or apoptosis. Many kinases, such as protein kinases, regulate mitotic cell proliferation through normal signalling. One of the major protein kinase family members is cyclin-dependent kinases (CDK), which are responsible for the regulation of cell cycle progression. If the cell cycle-regulatory mechanisms are permanently altered, it can cause abnormal proliferation that leads to neoplasia. This can result in tumour development, where the availability and expression of CDKs become altered, contributing significantly to impaired cell proliferation. Changes like these are often a characteristic of cancer. CDK inhibitors have shown significant clinical benefits in treating various types of tumours in recent years. The output has been achieved by the clinical approvals of particular CDK inhibitors. Researchers have also been studying the proteolysis-targeting chimera (PROTAC) molecule for the last two decades. This molecule uses ubiquitin-mediated proteasome mechanisms to break down specific targets, making it a promising method for targeted protein degradation (TPD). TPD has become a promising therapeutic option for tackling disease-causing proteins that are otherwise challenging to treat using traditional small molecules. This review provides an overview of the state of knowledge and a general understanding of selective or nonselective CDK inhibitors and PROTAC degraders currently under development or clinically approved congeners, focusing on improving cancer therapy.

Discovery of novel CDK9 inhibitor with tridentate ligand: Design, synthesis and biological evaluation

Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.

Indirubin In Vitro Apoptotic Effect on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) primarily affects the elderly, with its etiology largely unknown. It is hypothesized that hematopoietic stem cells may acquire mutations over time, such as the BCL-2 mutation, leading to disruptions in the apoptotic process. Dangui Luhui Wan, a mixture of 11 herbs used in Chinese Medicine, has shown antitumor activities across various cancer cell types. Indirubin-3'-monoxime (I3M), derived from Dangui Luhui Wan, functions as a selective inhibitor of cyclin-dependent kinases (CDKs) and can induce apoptosis in cells. Objectives: The objective of this study was to evaluate the effectiveness of I3M against CLL cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) from 14 patients were treated with I3M at concentrations ranging from 0.1 μM to 80 μM over periods of 24, 48, and 72 hours. The optimal dose was determined using Annexin V and MTT assays. The expression of apoptotic genes Bcl-2/Bax and CDK1/2 was assessed using real-time PCR. Results: The results indicated that a 20 µM concentration of I3M exhibited the highest cytotoxicity after 48 hours compared to controls (P = 0.005). Post-treatment, a decrease in Bcl-2 gene expression was observed, while changes in the Bax gene were not significant. However, an increase in the Bax/Bcl-2 gene ratio was noted, suggesting involvement of the mitochondrial pathway in I3M's apoptotic mechanism. Notably, I3M inhibited the expression of the CDK2 gene but did not affect CDK1 gene expression. Conclusions: I3M appears to exert anti-tumor effects by inducing apoptosis and inhibiting the CDK2 gene. Further research is required to elucidate the precise mechanism of action of I3M in CLL and potentially other tumor cell lines.

Abstract 5707: Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies

Abstract KRLS-017 is a potent, selective, and reversible inhibitor of Cyclin Dependent Kinase 7 (CDK7). Its molecular formula is C24H35N5O2Si and its molecular weight is 453.65 Daltons. KRLS-017 is being developed for the treatment of patients with advanced solid tumor malignancies. To differentiate KRLS-017 from other CDK7 inhibitors, we created a reversible inhibitor to allow for treatment schedule optimization and maximization of therapeutic index in clinic. High selectivity of KRLS-017 for CDK7 was confirmed using a panel of 313 recombinant human kinases. The IC50 of KRLS-017 against recombinant human CDK7 was determined to be 16 nM, and no other kinases in the panel showed IC50 values less than 300 nM. Antiproliferative activity of KRLS-017 was investigated in vitro using a broad panel of human tumor cell lines. KRLS-017 showed potent antiproliferative effect across a wide variety of both solid and hematologic tumor types, producing GI50 values <200 nM in 258/302 human tumorcell lines tested. Antitumor activity of KRLS-017 was investigated in vivo using multiple mouse xenograft tumormodels at dose levels ranging from 25 mg/kg to 200 mg/kg using both continuous (daily) andintermittent dosing schedules. Statistically significant tumor growth inhibition was seen at doselevels of 50 mg/kg and higher, with tumor regression observed at 100 mg/kg and 200 mg/kg. Better in vivo tolerability at efficacious doses was observed using intermittent dosing schedules in mouse xenograft models, which led to adoption of an intermittent schedule in the proposed first-in-human phase 1 trial. KRLS-017 is orally bioavailable in mice, rats, and dogs and plasma drug exposure increased in a proportional manner with dose. Comparing first dose and 28th dose in the dog, minimal plasma accumulation was observed, suggesting once daily dosing in humans would be initially tested. Repeat dose 28 day GLP toxicology studies in rat and dog showed adverse effects in highly proliferative bone marrow and gastrointestinal tissues which is consistent with the known mechanism of action for CDK7 and is considered to be monitorable and manageable in the setting of oncology. The clinical development plan for KRLS-017 includes an initial Phase 1 dose escalation study (KRLS-017-101) in patients with refractory solid tumor malignancies to assess pharmacokinetics/pharmacodynamics and to determine an optimal dose level and treatment schedule to test in expansion cohorts. Given the potential of CDK7 inhibitors in the treatment of hormone receptor positive breast cancer, combination work with other agents (such as selective estrogen receptor degraders) is on-going. Citation Format: Alison L. Hannah, Steve Ritland, Thomas Steele, Steven Smith, Ashwin Ram, BT Slingsby, Kapil Dhingra, James Chrisman, Rajesh Dua, Yasunori Tokunaga, Shigeyuki Kono, Yasuhiro Aga. Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5707.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5966.

Abstract 7550: KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models

Abstract Adenoid Cystic Carcinoma (ACC) is an aggressive rare type of cancer of the secretory glands with no approved targeted therapy and high unmet clinical need. Deregulated transcription driven by MYB-NFIB or MYBL1-NFIB transcription factor (TF) fusions are a hallmark of ACC pathogenesis. More aggressive disease and resistance to therapy has been associated with co-mutation in NOTCH. Although direct targeting of oncogenic TF fusions has remained challenging, targeting of their activity via transcriptional cofactors has emerged as an attractive and clinically actionable strategy. Cyclin-dependent kinase 9 (CDK9) is a key potentiator of TF activity via its ability to act both as an upstream regulator of TF expression and a downstream cofactor. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being studied in an ongoing Phase 1/2 study (NCT04718675) in advanced solid tumors including ACC. KB-0742 has demonstrated on-mechanism, single agent anti-tumor activity and a manageable safety profile in heavily pre-treated patients (0-11 prior lines of therapy) with transcriptionally addicted solid tumors. Here we present the preclinical rationale for targeting of oncogenic fusion TF activity in ACC. XenoSTART Patient-Derived Xenograft (XPDX) models were established from viable human tumor tissue for in vivo and ex vivo testing. Cell viability was assessed by CellTiter GLO reagent and phase imaging. Changes in MYB-fusion transcripts were assessed by RNA-Seq. Tumor Fragments (~70 mg) from ACCx5M1, ACCx6, ACCx9, ACCx11 models were implanted into athymic nude mice. Animals were treated with either vehicle (normal saline) or 60 mg/kg KB-0742 on a Q3D/week until tumors reached 2500 mm3 or up to 60 days post start of treatment. In a retrospective analysis using real world data, ACC has a low incidence of genomic instability and high rates of MYB fusions detectable by RNA-seq, making immunotherapy challenging but creating an opportunity for targeting MYB transcription. In primary MYB-fusion positive and NOTCH co-mutated patient-derived spheroid models, KB-0742 treatment induced strong antiproliferative activity and cytotoxicity. Importantly, KB-0742 demonstrated stronger cytotoxic effects when compared to historical control agents. In XPDXs, CDK9 inhibition with KB-0742 resulted in antiproliferative activity and stronger tumor growth inhibition in MYB-fusion positive and NOTCH co-mutated tumor models compared to MYB-fusion positive only models.These data demonstrate KB-0742 is effective in preclinical models of ACC suggesting KB-0742 may be a promising therapeutic option for ACC patients. KB-0742 is currently being evaluated in a phase 1/2 dose-escalation and cohort expansion trial in patients with ACC and other transcriptionally addicted tumors (NCT04718675). Citation Format: Michael R. McKeown, Luis A. Carvajal, Tressa R. Hood, Nicole S. Burr, Jeffrey Kaufman, Adam Boynton, Kameron Mori, Tessa DesRochers, Michael Wick, Charles Y. Lin, Jorge F. DiMartino. KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7550.

Identification of a Novel Selective CDK9 Inhibitor for the Treatment of CRC: Design, Synthesis, and Biological Activity Evaluation.

Cyclin-dependent kinase 9 (CDK9) is a member of the transcription CDK subfamily. In this work, we preliminarily demonstrated the feasibility of CDK9 as a potent target of treatment for colorectal cancer, and a series of novel CDK9 inhibitors were rationally designed and synthesized based on the structure of AZD5438 (a pan CDKs inhibitor reported by AstraZeneca). A novel selective CDK9 inhibitor named CLZX-205, which possessed significant CDK9 inhibitory activity (IC50 = 2.9 nM) with acceptable pharmacokinetic properties and antitumor efficacy in vitro and in vivo, was developed. Research on the mechanism indicated that CLZX-205 could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression, and these results were validated at the cellular and tumor tissue levels. Currently, CLZX-205 is undergoing further research as a promising candidate for CRC treatment.

Discovery of (4-Pyrazolyl)-2-aminopyrimidines as Potent and Selective Inhibitors of Cyclin-Dependent Kinase 2.

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.

MC180295 is a highly potent and selective CDK9 inhibitor with preclinical in vitro and in vivo efficacy in cancer

BackgroundInhibition of cyclin-dependent kinase 9 (CDK9), a novel epigenetic target in cancer, can reactivate epigenetically silenced genes in cancer by dephosphorylating the SWI/SNF chromatin remodeler BRG1. Here, we characterized the anti-tumor efficacy of MC180295, a newly developed CDK9 inhibitor.MethodsIn this study, we explored the pharmacokinetics of MC180295 in mice and rats, and tested the anti-tumor efficacy of MC180295, and its enantiomers, in multiple cancer cell lines and mouse models. We also combined CDK9 inhibition with a DNA methyltransferase (DNMT) inhibitor, decitabine, in multiple mouse models, and tested MC180295 dependence on T cells. Drug toxicity was measured by checking body weights and complete blood counts.ResultsMC180295 had high specificity for CDK9 and high potency against multiple neoplastic cell lines (median IC50 of 171 nM in 46 cell lines representing 6 different malignancies), with the highest potency seen in AML cell lines derived from patients with MLL translocations. MC180295 is a racemic mixture of two enantiomers, MC180379 and MC180380, with MC180380 showing higher potency in a live-cell epigenetic assay. Both MC180295 and MC180380 showed efficacy in in vivo AML and colon cancer xenograft models, and significant synergy with decitabine in both cancer models. Lastly, we found that CDK9 inhibition-mediated anti-tumoral effects were partially dependent on CD8 + T cells in vivo, indicating a significant immune component to the response.ConclusionsMC180380, an inhibitor of cyclin-dependent kinase 9 (CDK9), is an efficacious anti-cancer agent worth advancing further toward clinical use.

A molecular dynamics study of AZD4573 and Roscovitine as two potent inhibitors of cyclin dependent kinase 9

The cyclin dependent kinase 9(CDK9) was confirmed in several malignancies, notably those brought on by the dysregulation of the transcriptional elongation and mRNA maturation processes, have made CDK9 an appealing therapeutic target. The creation of selective CDK9 inhibitors continues to be extremely difficult due to the great homology of these CDKs in the catalytic domain. In this work we present two potential inhibitors, AZD4573 and Roscovitine, to detect potential binding between these compounds and the protein. These two compounds are in different experimental phases. AZD4573 were able to establish hydrogen bond with Asn154 and Asp167 with several hydrophobic bonds in the complex formed. Using the assistance of molecular dynamics (MD) simulations, the mechanism and dynamic stability of the interaction between the investigated chemicals and CDK9 were identified. It is strongly advised to consider these drug candidates as potential treatments, and we propose conducting in vivo trials to experimentally validate our findings.

The pharmacodynamic and mechanistic foundation for the antineoplastic effects of GFH009, a potent and highly selective CDK9 inhibitor for the treatment of hematologic malignancies.

To evade cell cycle controls, malignant cells rely upon rapid expression of select proteins to mitigate proapoptotic signals resulting from damage caused by both cancer treatments and unchecked over-proliferation. Cyclin-dependent kinase 9 (CDK9)-dependent signaling induces transcription of downstream oncogenes promoting tumor growth, especially in hyperproliferative 'oncogene-addicted' cancers, such as human hematological malignancies (HHMs). GFH009, a potent, highly selective CDK9 small molecule inhibitor, demonstrated antiproliferative activity in assorted HHM-derived cell lines, inducing apoptosis at IC50 values below 0.2 μM in 7/10 lines tested. GFH009 inhibited tumor growth at all doses compared to controls and induced apoptosis in a dose-dependent manner. Twice-weekly injections of GFH009 maleate at 10 mg/kg significantly prolonged the survival of MV-4-11 xenograft-bearing rodents, while their body weight remained stable. There was marked reduction of MCL-1 and c-MYC protein expression post-drug exposure both in vitro and in vivo. Through rapid 'on-off' CDK9 inhibition, GFH009 exerts a proapoptotic effect on HHM preclinical models triggered by dynamic deprivation of crucial cell survival signals. Our results mechanistically establish CDK9 as a targetable vulnerability in assorted HHMs and, along with the previously shown superior class kinome selectivity of GFH009 vs other CDK9 inhibitors, strongly support the rationale for currently ongoing clinical studies with this agent in acute myeloid leukemia and other HHMs.

Abstract C164: A phase 1 dose-escalation study of PRT2527, a cyclin-dependent kinase 9 (CDK9) inhibitor, in adult patients with advanced solid tumors: An updated analysis

Abstract Introduction: CDK9 has emerged as a key dependency due to its role in regulation of transcription elongation in many “transcription-addicted” cancers. We report updated phase 1 results from an ongoing, open-label, multicenter, dose-escalation study of PRT2527 (NCT05159518), an investigational, potent, and highly selective CDK9 inhibitor being evaluated in patients (pts) with advanced solid tumors. Methods: Enrolled adult pts had selected sarcomas, castration-resistant prostate cancer (CRPC), HR+/HER2– breast cancer (BC), non-small cell lung cancer (NSCLC), or MYC-amplified solid tumors that were relapsed or refractory to prior systemic treatment. Pts received intravenous PRT2527 (3, 6, 12, 15, or 18 mg/m2) once weekly (QW; 21-day cycle) using a Bayesian optimal interval design (n=3-6 per dose level); prophylactic anti-emetic and growth factor support was not required. Primary objectives were to evaluate dose-limiting toxicities (DLTs) and establish the maximum tolerated dose and recommended phase 2 dose of PRT2527. Secondary objectives were safety/tolerability, pharmacokinetics, and antitumor activity. Results: At data cutoff (May 18, 2023), 29 pts with CRPC (38%), BC (17%), sarcoma (31%), pancreatic cancer (7%), and NSCLC (3%) were treated across 5 dose levels; 5 and 9 pts received 15 mg/m2 QW in dose escalation and confirmation, respectively. Seven pts had MYC amplification. Four pts were ongoing; 1 pt with chondrosarcoma received treatment for >55 weeks. Two DLTs of grade 4 neutropenia were observed during dose escalation in the 15 and 18 mg/m2 QW cohorts. Overall, 100% of pts experienced all-cause, any-grade adverse events (AEs); the most frequent (≥15%) were nausea (45%), vomiting (38%), neutropenia (35%), fatigue (24%), and diarrhea (21%). Grade ≥3 all-cause and treatment-related AEs were reported in 48% and 21% of pts, respectively. Treatment-related grade ≥3 AEs were neutropenia (17%), anemia, leukopenia, thrombocytopenia, nausea, and pneumonia staphylococcal (3% each). Two deaths were reported while on study (sepsis and unknown cause; a third death occurred 38 days after the end of treatment); all were deemed unrelated to treatment. Four pts had dose reductions due to treatment-related AEs. Two pts required growth factor support for neutropenia. Eight (28%) pts had a best response of stable disease. Dose-dependent inhibition of CDK9 transcriptional targets was observed in all cohorts. A mean decrease of 76% (standard deviation [SD], 13%, n=8) in MCL1 and 77% (SD, 11%, n=8) in MYC mRNA was achieved 2 hours post infusion of the 15 mg/m2 QW dose; by 4 hours, MYC mRNA levels had rebounded to baseline levels (n=9) while MCL1 levels remained suppressed with a 60% (SD, 25%; n=9) inhibition. Conclusions: In adults with solid tumors, PRT2527 demonstrated favorable tolerability, with manageable neutropenia and gastrointestinal events. Doses of 12 and 15 mg/m2 QW showed optimal target inhibition as predicted by the preclinical model. PRT2527 will be investigated as a combination therapy and in hematological malignancies. Citation Format: Manish R. Patel MD, Astrayee Basu-Mallick MD, Filemon Dela Cruz MD, Douglas Orr MD, Alex Spira MD, PhD, Guru P. Sonpavde MD, Pooja Ghatalia MD, Christine Lihou BSc, Neelesh Sharma MD, PhD, Peggy Scherle PhD, William Sun PhD, Sri Sahasranaman, Jason T. Henry MD. A phase 1 dose-escalation study of PRT2527, a cyclin-dependent kinase 9 (CDK9) inhibitor, in adult patients with advanced solid tumors: An updated analysis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C164.

PRT2527, a Novel Highly Selective Cyclin-Dependent Kinase 9 (CDK9) Inhibitor, Has Potent Antitumor Activity in Combination with BTK and BCL2 Inhibition in Various Lymphoid Malignancies

CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. We also show that combination of PRT2527 with BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) leads to more robust and durable responses in multiple pre-clinical models. In these studies, the combination of PRT2527 with BTKi or BCL2i was well tolerated. Mechanistically, we show that in cell lines, BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency. Concurrent depletion of MCL1 and BFL1 with PRT2527 led to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In a similar fashion, Venetoclax-driven BCL2 inhibition also potentiated the anti-tumor activity of PRT2527. Consistent with our findings in vitro, we confirmed potent combinatorial inhibition of tumor growth by PRT2527, administered once weekly, and BTKi or BCL2i, administered daily, in CDX and PDX in vivo models of DLBCL and MCL. We observed reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrated enhanced induction of apoptosis by PRT2527 in primary CLL patient samples simultaneously treated with BTKi or BCL2i. Additionally, we demonstrated that PRT2527 potently inhibits growth in Ibrutinib-resistant MCL cell lines. Further work characterizing PRT2527 as monotherapy and in combination with BTKi and BCL2i in in vivo models of BTKi-resistant MCL and CLL is currently ongoing. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory hematologic malignancies.