Novel CDK19-Targeted Radiotracers: A Potential Design Strategy to Improve the Pharmacokinetics and Tumor Uptake.

Cyclin-dependent kinase 19 (CDK19) is overexpressed in prostate cancer, making it an attractive target for both imaging and therapy. Since little is known about the optimized approach for radioligands of nuclear proteins, linker optimization strategies were used to improve pharmacokinetics and tumor absorption, including the adjustment of the length, flexibility/rigidity, and hydrophilicity/lipophilicity of linkers. Molecular docking was conducted for virtual screening and followed by IC50 determination. Both BALB/c mice and P-16 xenografts were used for tissue distribution and PET/CT imaging. The ligand 68Ga-10c demonstrated high absorption in tumor 5 min after injection and sustains long-term imaging within 3 h. Furthermore, 68Ga-10c exhibited slow clearance within the tumor and was predominantly metabolized in both the liver and kidneys, showing the potential to alleviate metabolic pressure and enhance tissue safety. Therefore, the linker optimization strategy is well suited for CDK19 and provides a reference for the radioactive ligands of other nuclear targets.

Design and Synthesis of Novel Macrocyclic Derivatives as Potent and Selective Cyclin-Dependent Kinase 7 Inhibitors.

The duality of function (cell cycle regulation and gene transcription) of cyclin-dependent kinase 7 (CDK7) makes it an attractive oncology target and the discovery of CDK7 inhibitors has been a long-term pursuit by academia and pharmaceutical companies. However, achieving selective leading compounds is still difficult owing to the similarities among the ATP binding pocket. Herein, we detail the design and synthesis of a series of macrocyclic derivatives with pyrazolo[1,5-a]-1,3,5-triazine core structure as potent and selective CDK7 inhibitors. The diverse manners of macrocyclization led to distinguished selectivity profiles of the CDK family. Molecular dynamics (MD) simulation explained the binding difference between 15- and 16-membered macrocyclic compounds. Further optimization generated compound 37 exhibiting good CDK7 inhibitory activity and high selectivity over other CDKs. This work clearly demonstrated macrocyclization is a versatile method to finely tune the selectivity profile of small molecules and MD simulation can be a valuable tool in prioritizing designs of the macrocycle.

The reversible inhibitor SR-4835 binds Cdk12/cyclin K in a noncanonical G-loop conformation.

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Alvocidib inhibits IRF4 expression via super-enhancer suppression and adult T-cell leukemia/lymphoma cell growth.

Adult T-cell leukemia/lymphoma (ATL) is an intractable hematological malignancy with extremely poor prognosis. Recent studies have revealed that super-enhancers (SE) play important roles in controlling tumor-specific gene expression and are potential therapeutic targets for neoplastic diseases including ATL. Cyclin-dependent protein kinase (CDK) 9 is a component of a complex comprising transcription factors (TFs) that bind the SE region. Alvocidib is a CDK9 inhibitor that exerts antitumor activity by inhibiting RNA polymerase (Pol) II phosphorylation and suppressing SE-mediated, tumor-specific gene expression. The present study demonstrated that alvocidib inhibited the proliferation of ATL cell lines and tumor cells from patients with ATL. RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) disclosed that SE regulated IRF4 in the ATL cell lines. Previous studies showed that IRF4 suppression inhibited ATL cell proliferation. Hence, IRF4 is a putative alvocidib target in ATL therapy. The present study revealed that SE-mediated IRF4 downregulation is a possible mechanism by which alvocidib inhibits ATL proliferation. Alvocidib also suppressed ATL in a mouse xenograft model. Hence, the present work demonstrated that alvocidib has therapeutic efficacy against ATL and partially elucidated its mode of action. It also showed that alvocidib is promising for the clinical treatment of ATL and perhaps other malignancies and neoplasms as well.

Discovery of a Highly Potent and Selective Dual PROTAC Degrader of CDK12 and CDK13.

Selective degradation of the cyclin-dependent kinases 12 and 13 (CDK12/13) presents a novel therapeutic opportunity for triple-negative breast cancer (TNBC), but there is still a lack of dual CDK12/13 degraders. Here, we report the discovery of the first series of highly potent and selective dual CDK12/13 degraders by employing the proteolysis-targeting chimera (PROTAC) technology. The optimal compound 7f effectively degraded CDK12 and CDK13 with DC50 values of 2.2 and 2.1 nM, respectively, in MDA-MB-231 breast cancer cells. Global proteomic profiling demonstrated the target selectivity of 7f. In vitro, 7f suppressed expression of core DNA damage response (DDR) genes in a time- and dose-dependent manner. Further, 7f markedly inhibited proliferation of multiple TNBC cell lines including MFM223, with an IC50 value of 47 nM. Importantly, 7f displayed a significantly improved antiproliferative activity compared to the structurally similar inhibitor 4, suggesting the potential advantage of a CDK12/13 degrader for TNBC targeted therapy.

Cell Cycle-Related Clinical Applications.

The cell cycle is a highly regulated and orchestrated mechanism of life that ensures successive division of a cell and precise replication of cellular contents. Cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors are three of the most critical cell cycle regulatory proteins that enable the smooth progression of cells through the different phases of cell cycle before and after division. The alteration of cell cycle-related proteins causes aberration in the normal cell cycle process, which is one of the pivotal causes of cancer and other diseases. Targeting cell cycle components has proven to be a valuable therapeutic strategy and leads to the development of novel anticancer therapeutic. The purpose of this book chapter is to summarize the literature and discuss the clinical significance of cell cycle-related proteins in cancers and other diseases, with a focus on identifying potential targets as therapeutic interventions for cancer patients.

Cyclin-dependent kinases in breast cancer: expression pattern and therapeutic implications.

Presently, breast cancer (BC) is one of the most common malignancies diagnosed and the leading cause of tumor-related deaths among women worldwide. Cell cycle dysregulation is one of the hallmarks of cancer, resulting in uncontrolled cell proliferation. Cyclin-dependent kinases (CDKs) are central to the cell cycle control system, and deregulation of these kinases leads to the development of malignancies, including breast cancer. CDKs and cyclins have been reported as crucial components involved in tumor cell proliferation and metastasis. Given the aggressive nature, tumor heterogeneity, and chemoresistance, there is an urgent need to explore novel targets and therapeutics to manage breast cancer effectively. Inhibitors targeting CDKs modulate the cell cycle, thus throwing light upon their therapeutic aspect where the progression of tumor cells could be inhibited. This article gives a comprehensive account of CDKs in breast cancer progression and metastasis and recent developments in the modulation of CDKs in treating malignancies. We have also explored the expression pattern and prognostic significance of CDKs in breast cancer patients. The article will also shed light on the Implications of CDK inhibition and TGF-ß signaling in breast cancer.

CDK Inhibition Reverses Acquired 5-Fluorouracil Resistance in Hepatocellular Carcinoma Cells.

Background: 5-Fluorouracil (5-FU) has been widely applied in treating cancers. However, its usage is largely limited in hepatocellular carcinoma (HCC), due to acquired resistance. Here, we aim to identify target proteins and investigate their roles in 5-FU sensitivity of HCC cells. Methods: Mass spectrometry (MS) proteomics was performed on 5-FU-resistant cell line (BEL7402/5-FU) and its parental cell line (BEL7402) with 5-FU treatment. In order to identify potential targets, we compared the proteomics between two cell line groups and used bioinformatics tools to select hub proteins from all differentially expressed proteins. Results: We finally focused on a group of cell cycle-related kinases (CDKs). By CCK8 assay, we confirmed that the CDK inhibitor significantly decreased the IC50 of 5-FU-resistant cells. Conclusions: Our study verified that CDK inhibition can reverse 5-FU resistance of HCC cells.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics.

Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and Senexin B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship (SAR). An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.

Discovery and Optimization of Highly Selective Inhibitors of CDK5.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including GFB-12811, that are highly selective over the other tested kinases. In cellular assays, our compounds demonstrate CDK5 target engagement while avoiding anti-proliferative effects associated with inhibiting other CDKs. In addition, we show that the compounds in this series exhibit promising in vivo PK profiles, enabling their use as tool compounds for interrogating the role of CDK5 in ADPKD and other diseases.

p53 directs leader cell behavior, migration, and clearance during epithelial repair.

Epithelial cells migrate across wounds to repair injured tissue. Leader cells at the front of migrating sheets often drive this process. However, it is unclear how leaders emerge from an apparently homogeneous epithelial cell population. We characterized leaders emerging from epithelial monolayers in cell culture and found that they activated the stress sensor p53, which was sufficient to initiate leader cell behavior. p53 activated the cell cycle inhibitor p21WAF1/CIP1, which in turn induced leader behavior through inhibition of cyclin-dependent kinase activity. p53 also induced crowding hypersensitivity in leader cells such that, upon epithelial closure, they were eliminated by cell competition. Thus, mechanically induced p53 directs emergence of a transient population of leader cells that drive migration and ensures their clearance upon epithelial repair.

Transcriptional cyclin-dependent kinases: Potential drug targets in cancer therapy.

In the wake of the development of the concept of cell cycle and its limiting points, cyclin-dependent kinases (CDKs) are considered to play a central role in regulating cell cycle progression. Recent studies have strongly demonstrated that CDKs also has multiple functions, especially in response to extracellular and intracellular signals by interfering with transcriptional events. Consequently, how to inhibit their function has been a hot research topic. It is worth noting that the key role of CDKs in regulating transcription has been explored in recent years, but its related pharmacological targets are less developed, and most inhibitors have not entered the clinical stage. Accordingly, this perspective focus on the biological functions of transcription related CDKs and their complexes, some key upstream and downstream signals, and inhibitors for cancer treatment in recent years. In addition, some corresponding combined treatment strategies will provide a more novel perspective for future cancer remedy.

Small-molecule degraders of cyclin-dependent kinase protein: a review.

Proteolysis-targeting chimeras are a new modality of chemical tools and potential therapeutics involving the induction of protein degradation. Cyclin-dependent kinase (CDK) protein, which is involved in cycles and transcription cycles, participates in regulation of the cell cycle, transcription and splicing. Proteolysis-targeting chimeras targeting CDKs show several advantages over traditional CDK small-molecule inhibitors in potency, selectivity and drug resistance. In addition, the discovery of molecule glues promotes the development of CDK degraders. Herein, the authors describe the existing CDK degraders and focus on the discussion of the structural characteristics and design of these degraders.

Resistance to endocrine therapy in HR + and/or HER2 + breast cancer: the most promising predictive biomarkers.

Breast cancer is the most common cancer in women. It is a heterogeneous disease, encompassing different biological subtypes that differ in histological features, outcomes, clinical behaviour and different molecular subtypes. Therapy has progressed substantially over the past years with a reduction both for locoregional and systemic therapy. Endocrine therapies have considerably reduced cancer recurrence and mortality. Despite the major diagnostic and therapeutic innovations, resistance to therapy has become a main challenge, especially in metastatic breast cancer, and became a major factor limiting the use of endocrine therapeutic agents in ER positive breast cancers. Approximately 50% of patients with ER positive metastatic disease achieve a complete or partial response with endocrine therapy. However, in the remaining patients, the benefit is limited due to resistance, intrinsic or acquired, resulting in disease progression and poor outcome.Tumour heterogeneity as well as acquired genetic changes and therapeutics pressure have been involved in the endocrine therapy resistance. Nowadays, targeted sequencing of genes involved in cancer has provided insights about genomic tumour evolution throughout treatment and resistance driver mutations. Several studies have described multiple alterations in receptor tyrosine kinases, signalling pathways such as Phosphoinositide-3-kinase-protein kinase B/Akt/mTOR (PI3K/Akt/mTOR) and Mitogen-activated protein kinase (MAPK), cell cycle machinery and their implications in endocrine treatment failure.One of the current concern in cancer is personalized therapy. The focus has been the discovery of new potentially predictive biomarkers capable to identify reliably the most appropriate therapy regimen and which patients will experience disease relapse. The major concern is also to avoid overtreatment/undertreatment and development of resistance.This review focuses on the most promising predictive biomarkers of resistance in estrogen receptor-positive breast cancer and the emerging role of circulating free-DNA as a powerful tool for longitudinal monitoring of tumour molecular profile throughout treatment.

Discovery of SY-5609: A Selective, Noncovalent Inhibitor of CDK7.

CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

Noncovalent CDK12/13 dual inhibitors-based PROTACs degrade CDK12-Cyclin K complex and induce synthetic lethality with PARP inhibitor.

Cyclin-dependent kinase 12 (CDK12) plays a crucial role in DNA-damage response gene transcription and has recently been validated as a promising target in cancer therapy. However, existing CDK12 inhibitors potently inhibit its closest isoform CDK13, which could cause potential toxicity. Therefore, the development of CDK12 inhibitors with isoform-selectivity against CDK13 continues to be a challenge. By taking advantage of the emerging PROteolysis-TArgeting Chimeras (PROTACs) approach, we have synthesized a potent PROTAC degrader PP-C8 based on the noncovalent dual inhibitors of CDK12/13 and demonstrated its specificity for CDK12 over CDK13. Notably, PP-C8 induces profound degradation of cyclin K simultaneously and downregulates the mRNA level of DNA-damage response genes. Global proteomics profiling revealed PP-C8 is highly selective toward CDK12-cyclin K complex. Importantly, PP-C8 demonstrates profound synergistic antiproliferative effects with PARP inhibitor in triple-negative breast cancer (TNBC). The potent and selective CDK12 PROTAC degrader developed in this study could potentially be used to treat CDK12-dependent cancers as combination therapy.

Cyclin-dependent kinase (CDK) inhibitors in solid tumors: a review of clinical trials.

Cyclin-dependent kinases (CDKs) play a key regulating role in the cell cycle, which is almost universally altered in cancer, leading to sustained proliferation. Early pan-CDK inhibitors showed poor results in clinical trials for solid malignancies, as the lack of selectivity produced significant toxicity. The production of more selective inhibitors led to significant developments in cancer therapy, as CDK4/6 inhibitors in combination with endocrine therapy changed the landscape of the treatment of hormone-receptor positive (HR +) metastatic breast cancer. Recently, Trilaciclib demonstrated benefits regarding hematological toxicity compared to placebo when administered in combination with chemotherapy in small cell lung cancer. Newer agents, such as SY-5609, a selective CDK7 inhibitor, have also shown promising results in early clinical trials. In this paper, we review the data from clinical trials of CDK inhibitors in solid tumors, either as a monotherapy or in combination with other agents, with an emphasis on novel agents and potential new indications for this drug class.

Naturally Sourced CDK Inhibitors and Current Trends in Structure-Based Synthetic Anticancer Drug Design by Crystallography.

Cyclin-Dependent Kinases (CDKs) are the chief regulators in cell proliferation; the kinase activities are largely regulated by their interactions with CDK Inhibitors (CKIs) and Cyclins. The association of different CDKs with CDKIs and Cyclins at the cell-cycle checkpoints of different stages of mitotic cell cycle function act more likely as the molecular switches that regulate different transcriptional events required for progression through the cell cycle. A fine balance in response to extracellular and intracellular signals is highly maintained in the orchestrated function of CDKs along with Cyclins and CDKIs for normal cell proliferation. This fine-tuning in mitotic cell cycle progression sometimes gets lost due to dysregulation of CDKs. The aberrant functioning of the CDKIs is therefore studied for its contributions as a vital hallmark of cancers. It has attracted our focus to maneuver cancer therapy. Hence, several synthetic CDKIs and their crystallography-based drug design have been explained to understand their mode of action with CDKs. Since most of the synthetic drugs function by inhibiting the CDK4/6 kinases by competitively binding to their ATP binding cleft, these synthetic drugs are reported to attack the normal, healthy growing cells adjacent to the cancer cells leading to the decrease in the life span of the cancer patients. The quest for traditional natural medicines may have a great impact on the treatment of cancer. Therefore, in the present studies, a search for naturally sourced CDK inhibitors has been briefly focused. Additionally, some synthetic crystallography-based drug design has been explained to elucidate different avenues to develop better anticancer chemotherapeutics, converting natural scaffolds into inhibitors of the CDK mediated abnormal signal transduction with lesser side effects.