Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach

Cyclin‐dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508–8522) have found a ligand BS194 that has a high affinity with CDK2 (IC₅₀ = 3 nm ) but shows low affinity with CDK1 (IC₅₀ = 30 nm ). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free‐energy calculation to analyze the interactions. A rational three‐dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non‐polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1. Copyright © 2012 John Wiley & Sons, Ltd.     

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2? breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Ų), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpK_a = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse K_p,uu = 0.20–0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.     

Discovery of novel CDK inhibitors via scaffold hopping from CAN508

Cyclin-dependent kinases (CDKs) are promising drug targets for various human diseases, especially for cancers. Scaffold hopping strategy was applied on CAN508, a known selective CDK9 inhibitor, and a series of pyrazolo[3,4-b]pyridine compounds were synthesized and evaluated in vitro as CDK2 and CDK9 inhibitors. Most compounds exhibited moderate to potent inhibitory activities against both CDK2/cyclin A and CDK9/cyclin T1 systems. Among them, compound 2e showed IC₅₀ values of 0.36?μM for CDK2 and 1.8?μM for CDK9, respectively. Notably, the scaffold alteration seems to cause a shift in the selectivity profile of the inhibitors. In contrast to CAN508, compound 2k demonstrated remarkable selectivity toward CDK2 (265-fold over CDK9). Docking studies on compound 2k provided hints for further design of more potent and selective CDK2/CDK9 inhibitors.     

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.Subject terms: Senescence, Tumour biomarkers     

Design,synthesis and biological evaluation of novel 1H-1,2,4-triazole,benzothiazole and indazole-based derivatives as potent FGFR1 inhibitors viafragment-based virtual screening

Abstract

Fibroblast growth-factor receptor (FGFR) is a potential target for cancer therapy. We designed three novel series of FGFR1 inhibitors bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via fragment-based virtual screening. All the newly synthesised compounds were evaluated in vitro for their inhibitory activities against FGFR1. Compound 9d bearing an indazole scaffold was first identified as a hit compound, with excellent kinase inhibitory activity (IC₅₀ = 15.0?nM) and modest anti-proliferative activity (IC₅₀ = 785.8?nM). Through two rounds of optimisation, the indazole derivative 9?u stood out as the most potent FGFR1 inhibitors with the best enzyme inhibitory activity (IC₅₀ = 3.3?nM) and cellular activity (IC₅₀ = 468.2?nM). Moreover, 9?u also exhibited good kinase selectivity. In addition, molecular docking study was performed to investigate the binding mode between target compounds and FGFR1.     

Resistance to CDK4/6 inhibition: Mechanisms and strategies to overcome a therapeutic problem in the treatment of hormone receptor-positive metastatic breast cancer

Selective CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, have been approved in combination with hormone therapy for the treatment of patients with HR+, HER2-negative advanced or metastatic breast cancer (mBC). Despite their promising activity, approximately 10 % of patients have de novo resistance, while the rest of them will develop acquired resistance after 24–28 months when used as first-line therapy and after a shorter period when used as second-line therapy. Various mechanisms of resistance to CDK4/6 inhibitors have been described, including cell cycle-related mechanisms, such as RB loss, p16 amplification, CDK6 or CDK4 amplification, and cyclin E-CDK2 amplification. Other bypass mechanisms involve the activation of FGFR or PI3K/AKT/mTOR pathways. Identifying the different mechanisms by which resistance to CDK4/6 inhibitors occurs may help to design new treatment strategies to improve patient outcomes. This review presents the currently available knowledge on the mechanisms of resistance to CDK4/6 inhibitors, explores possible treatment strategies that could overcome this therapeutic problem, and summarizes relevant recent clinical trials.     

Design and synthesis of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazol-7-yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine as a highly potent and selective cyclin-dependent kinases 4 and 6 inhibitors and the discovery of structure-activity relationships

Cyclin-dependent kinases 4/6 play an important role in regulation of cell cycle, and overexpress in a variety of cancers. Up to now, new CDK inhibitors still need to be developed due to its poor selectivity. Herein we report a novel series of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazole-7-yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine anologues as potent CDK 4/6 inhibitors based on LY2835219 (Abemaciclib). Compound 10d, which exhibits approximate potency on CDK4/6 (IC₅₀?=?7.4/0.9?nM), has both good pharmacokinetic characters and high selectivity on CDK1 compared with LY2835219. Overall, compound 10d could be a promising candidate and a good starting point as anticancer drugs.     

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.     

Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.     

Expression of cell cycle regulating factor mRNA in small cell lung cancer xenografts

We have investigated the expression of cyclins, cyclin dependent kinases (CDK), and CDK inhibitors (CKI) at the mRNA level in a panel of small-cell lung cancer (SCLC) cell linesin vitro andin vivo as xenografts in nude mice. The results showed that the cell lines expressed varying amounts of most cyclin and CDK’s but only a few of the cell lines expressed cyclin D₁ and/or D₂ and some lacked expression of CDK6. Most cell lines expressed mRNA for the CKI’s but two cell lines lacked expression of p15^INK4B and p16^INK4A. The mRNA expression differed for a few of the cell lines regarding cyclin D₂ and CDK6 whenin vitro andin vivo data were compared. Two of the cell lines that express the retinoblastoma (Rb) protein had no sign of a deregulated Rb pathway but further studies at the protein level are necessary to demonstrate whether these two cell lines should have a normal Rb pathway or whether they will join the majority of cell lines with deregulated Rb pathway.     

Potent pro-apoptotic combination therapy is highly effective in a broad range of cancers

Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities.Subject terms: Cancer models, Cell biology, Preclinical research     

Whether to target single or multiple CDKs for therapy? That is the question

Complexes consisting of cyclin‐dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK–cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16^INK4A or p27^Kip1, and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad ‐ spectrum unspecific, pan‐specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan‐specific and highly selective CDK inhibitors in therapy. J. Cell. Physiol. 226: 341–349, 2011. © 2010 Wiley‐Liss, Inc.     

Transient treatment with CDK inhibitors eliminates proliferative potential even when their abilities to evoke apoptosis and DNA damage are blocked

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell-cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.     

Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations

Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.     

Synthesis and biological evaluation of N9-cis-cyclobutylpurine derivatives for use as cyclin-dependent kinase (CDK) inhibitors

A novel 6-aminopurine scaffold bearing an N9-cis-cyclobutyl moiety was designed using structure-based molecular design based on two known CDK inhibitors, dinaciclib and Cmpd-27. A series of novel 6-aminopurine compounds was prepared for structure–activity relationship (SAR) studies of CDK2 and CDK5 inhibitors. Among the compounds synthesized, compound 8l displayed potent CDK2 and CDK5 inhibitory activities with low nanomolar ranges (IC₅₀ = 2.1 and 4.8 nM, respectively) and showed moderate cytotoxicity in HCT116 colon cancer and MCF7 breast cancer cell lines. Here, we report the synthesis and evaluation of novel 6-aminopurine derivatives and present molecular docking models of compound 81 with CDK2 and CDK5.     

Identifying Tumor Cell Growth Inhibitors by Combinatorial Chemistry and Zebrafish Assays

Cyclin-dependent kinases (CDKs) play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos. We report the successful identification of a novel lead compound that displays selective inhibitory effects on CDK2 activity, cancer cell proliferation, and tumor progression in vivo. Our approaches should have general applications in developing cell proliferation inhibitors using an efficient combinatorial chemical genetic method and integrated biological assays. The novel cell growth inhibitor we identified should have potential as a cancer therapeutic agent.