Cyclin-dependent kinase inhibitor fadraciclib (CYC065) depletes anti-apoptotic protein and synergizes with venetoclax in primary chronic lymphocytic leukemia cells.

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect.

Interrogation of novel CDK2/9 inhibitor fadraciclib (CYC065) as a potential therapeutic approach for AML.

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription.

Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer.

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer.

Implementing Patient-Derived Xenografts to Assess the Effectiveness of Cyclin-Dependent Kinase Inhibitors in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM.

Mitotic entry: Non-genetic heterogeneity exposes the requirement for Plk1.

The quest to develop novel antimitotic chemotherapy agents has led to the generation of several small molecule inhibitors targeting Plk1, a protein kinase required for multiple aspects of cell division. Previous studies have shown that upon exposure to Plk1 inhibitors, cells enter mitosis, delay briefly in prophase and then arrest in mitosis due to an inability to undergo centrosome separation. Here, we show that four different classes of Plk1 inhibitor block mitotic entry in several cancer cell lines and non-transformed RPE-1 cells.

Aurora kinase inhibitors: progress towards the clinic.

The Aurora kinases (serine/threonine kinases) were discovered in 1995 during studies of mutant alleles associated with abnormal spindle pole formation in Drosophila melanogaster. They soon became the focus of much attention because of their importance in human biology and association with cancer. Aurora kinases are essential for cell division and are primarily active during mitosis.

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A.

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2.

Discovery and characterization of 2-anilino-4- (thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents.

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e.

An integrated pharmacokinetic-pharmacodynamic model for an Aurora kinase inhibitor.

The spindle assembly checkpoint is a cell cycle surveillance mechanism that ensures the proper separation of chromosomes prior to cell division at mitosis. Aurora kinases play critical roles in mitotic progression and hence small-molecule inhibitors of Aurora kinases have been developed as a new class of potential anti-cancer drugs. In this paper we present for the first time an integrated pharmacokinetic-pharmacodynamic model of the functional effects of CYC116 (a known inhibitor of Aurora kinases A and B) on the spindle assembly checkpoint.

Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors.

Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds.

Design, synthesis, and evaluation of 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines as ring-constrained 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin-dependent kinase inhibitors.

Following the recent discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin dependent kinase (CDK) inhibitors, a program was initiated to evaluate related ring-constrained analogues, specifically, 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines for inhibition of CDKs. Here we report the rational design, synthesis, structure-activity relationships (SARs), and cellular mode-of-action profile of these second generation CDK inhibitors. Many of the analogues from this chemical series inhibit CDKs with very low nanomolar K(i) values.

Aurora kinases: structure, functions and their association with cancer.

Background: Aurora kinases are a recently discovered family of kinases (A, B & C) consisting of highly conserved serine\threonine protein kinases found to be involved in multiple mitotic events: regulation of spindle assembly checkpoint pathway, function of centrosomes and cytoskeleton, and cytokinesis. Aberrant expression of Aurora kinases may lead to cancer. For this reason the Aurora kinases are potential targets in the treatment of cancer.

Targeting glycogen synthase kinase-3 in insulin signalling.

The renewed interest in an enzyme first discovered over 25 years ago stems from the potential of inhibitors of this enzyme to treat conditions as diverse as diabetes, Alzheimer's disease, stroke and bipolar disorder, and even to enhance the repopulating capacity of transplanted haematopoietic stem cells. The emergence of the first few potent and specific glycogen synthase kinase-3 (GSK-3) inhibitors will end years of speculation on their potential and finally allow the impact of GSK-3 inhibitors to be evaluated clinically. The next few years are likely to be particularly exciting ones for fans of this old enzyme.

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding.

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e.

Structural and biochemical studies of human proliferating cell nuclear antigen complexes provide a rationale for cyclin association and inhibitor design.

The interactions between the tumor suppressor protein p21WAF1 and the cyclin-dependent kinase (CDK) complexes and with proliferating cell nuclear antigen (PCNA) regulate and coordinate the processes of cell-cycle progression and DNA replication. We present the x-ray crystal structure of PCNA complexed with a 16-mer peptide related to p21 that binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide previously undescribed structures of uncomplexed human PCNA and show that significant changes on ligand binding include rigidification of a number of flexible regions on the surface of PCNA.

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors: synthesis, SAR analysis, X-ray crystallography, and biological activity.

Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K(i)s against CDK2, are reported.

Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.

Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity.

The p53-Mdm2 pathway: targets for the development of new anticancer therapeutics.

The tumour suppressor p53 is at the centre of a network of regulatory pathways that guard over the continued integrity of the living cell and its progeny after exposure to different forms of stress, particularly those capable of inducing DNA damage. Tumour cells very frequently circumvent this control by disabling the function of p53, or other proteins in the p53 network, through mutation. Here we review the different therapeutic strategies that have been adopted to exploit common neoplastic aberrations in the p53 pathways.

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop.

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis.

Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.

The recognition of cyclin-dependent kinase (CDK)/cyclin complexes by various cell-cycle regulatory proteins, including certain tumour suppressors and transcription factors, occurs at least in part through a protein-protein interaction with a binding groove on the cyclin subunit. Since CDK function is generally deregulated in tumour cells, blocking of this recruitment site prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of checkpoint control in transformed cells. Here we discuss the finding that peptides derived from such cyclin-interacting proteins, and rendered permeable through conjugation to cellular delivery vectors, can apparently induce tumour cells to undergo apoptosis selectively.

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine).

CDK2 inhibitors have been proposed as effective anti-cancer therapeutics. We show here that CYC202 (R-roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC(50) = 0.10 microM) with an average cytotoxic IC(50) of 15.

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach.

Peptide inhibitors of CDK2-cyclin A that target the cyclin recruitment-site: structural variants of the C-terminal Phe.

A focused series of octapeptides based on the lead compound H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH(2) 1, in which the C-terminal phenylalanine residue was replaced by alpha and/or beta-modified variants, was synthesized using solid-phase chemistry. Both the L-threo-beta-hydroxy-phenylalanine (beta-phenylserine, Pse) and (2S)-phenylalaninol derivatives, as competitive binders at the cyclin-recruitment site, displayed potent inhibitory activity towards the CDK2-cyclin A complex. Unexpectedly, the D-threo-Pse derivatives also showed inhibitory activity.