How selective are clinical CDK4/6 inhibitors?

Pharmacological inhibition of cyclin-dependent kinase 4/6 (CDK4/6) has emerged as an efficient approach for treating breast cancer, and its clinical potential is expanding to other cancers. CDK4/6 inhibitors were originally believed to act by arresting proliferation in the G1 phase, but it is gradually becoming clear that the cellular response to these compounds is far more complex than this. Multiple context-dependent mechanisms of action are emerging, involving modulation of quiescence, senescence, autophagy, cellular metabolism, and enhanced tumor cell immunogenicity. These mechanisms may be driven by interactions with unexpected targets. We review cellular responses to the Food and Drug Administration-approved CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib, and summarize available knowledge of other drugs undergoing clinical trials, including data on their off-target landscapes. We emphasize the importance of comprehensively characterizing drugs' selectivity profiles to maximize their clinical efficacy and safety and to facilitate their repurposing to treat additional diseases based on their target spectrum.

Promising 2,6,9-Trisubstituted Purine Derivatives for Anticancer Compounds: Synthesis, 3D-QSAR, and Preliminary Biological Assays.

We designed, synthesized, and evaluated novel 2,6,9-trisubstituted purine derivatives for their prospective role as antitumor compounds. Using simple and efficient methodologies, 31 compounds were obtained. We tested these compounds in vitro to draw conclusions about their cell toxicity on seven cancer cells lines and one non-neoplastic cell line. Structural requirements for antitumor activity on two different cancer cell lines were analyzed with SAR and 3D-QSAR. The 3D-QSAR models showed that steric properties could better explain the cytotoxicity of compounds than electronic properties (70% and 30% of contribution, respectively). From this analysis, we concluded that an arylpiperazinyl system connected at position 6 of the purine ring is beneficial for cytotoxic activity, while the use of bulky systems at position C-2 of the purine is not favorable. Compound 7h was found to be an effective potential agent when compared with a currently marketed drug, cisplatin, in four out of the seven cancer cell lines tested. Compound 7h showed the highest potency, unprecedented selectivity, and complied with all the Lipinski rules. Finally, it was demonstrated that 7h induced apoptosis and caused cell cycle arrest at the S-phase on HL-60 cells. Our study suggests that substitution in the purine core by arylpiperidine moiety is essential to obtain derivatives with potential anticancer activity.

A Stepwise Approach for the Synthesis of Folic Acid Conjugates with Protein Kinase Inhibitors.

Herein, we report an alternative synthetic approach for selected 2,6,9-trisubstituted purine CDK inhibitor conjugates with folic acid as a drug-delivery system targeting folate receptors. In contrast to the previously reported approaches, the desired conjugates were constructed stepwise using solid-phase synthesis starting from immobilized primary amines. The ability of the prepared conjugates to release the free drug was verified using dithiothreitol (DTT) and glutathione (GSH) as liberating agents. Finally, binding to the folate receptor (FOLR1) overexpressed in a cancer cell line was measured by flow cytometry using a fluorescent imaging probe.

2,4-Diaryl-pyrimido[1,2-a]benzimidazole derivatives as novel anticancer agents endowed with potent anti-leukemia activity: Synthesis, biological evaluation and kinase profiling.

Acute myeloid leukemia (AML) stands as one of the most aggressive type of human cancer that can develop rapidly and thus requires immediate management. In the current study, the development of novel derivatives of pyrimido[1,2-a]benzimidazole (5a-p) as potential anti-AML agents is reported. The prepared compounds 5a-p were inspected for their in vitro anti-tumor activity at NCI-DTP and subsequently 5h was selected for full panel five-dose screening to assess its TGI, LC50 and GI50 values. Compound 5h showed effective anti-tumor activity at low micromolar concentration on all tested human cancer cell lines with GI50 range from 0.35 to 9.43 µM with superior sub-micromolar activity towards leukemia. Furthermore, pyrimido[1,2-a]benzimidazoles 5e-l were tested on a panel ofhuman acute leukemia cell lines, namely HL60, MOLM-13, MV4-11, CCRF-CEM and THP-1, where 5e-h reached single-digit micromolar GI50 values for all the tested cell lines. All prepared compounds were first tested for inhibitory action against the leukemia-associated mutant FLT3-ITD, as well as against ABL, CDK2, and GSK3 kinases, in order to identify the kinase target for the herein described pyrimido[1,2-a]benzimidazoles. However, the examined molecules disclosed non-significant activity against these kinases. Thereafter, a kinase profiling on a panel of 338 human kinases was then used to discover the potential target. Interestingly, pyrimido[1,2-a]benzimidazoles 5e and 5h significantly inhibited BMX kinase. Further investigation for the effect on cell cycle of HL60 and MV4-11 cells and caspase 3/7 activity was also performed. In addition, the changes in selected proteins (PARP-1, Mcl-1, pH3-Ser10) associated with cell death and viability were analyzed in HL60 and MV4-11 cells by immunoblotting.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Novel modified leucine and phenylalanine dipeptides modulate viability and attachment of cancer cells.

Here, we describe the synthesis and biological characterization of 32 novel phenylalanine and leucine dipeptides modified on both the N and C termini by salicylic acid and aromatic or alicyclic amines, respectively. All compounds displayed antiproliferative activity in the tested cancer cell lines and eight of the compounds exhibited single digit micromolar GI50 values. Treated cells rapidly detached from surface of tissue culture dishes and we found that focal adhesion kinase (FAK), p130CAS and paxillin, which are important regulators of cell adhesion, were dephosphorylated at Y397, Y410 and Y118, respectively. The most potent compound reduced proliferation in the HCT-116 cell line in a dose-dependent manner, as shown by a decrease in 5-bromo-2'-deoxyuridine incorporation into DNA. Furthermore, this compound increased the levels of several apoptotic markers, including activated caspases, and increased site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.

How Selective Are Pharmacological Inhibitors of Cell-Cycle-Regulating Cyclin-Dependent Kinases?

Cyclin-dependent kinases (CDKs) are an important and emerging class of drug targets for which many small-molecule inhibitors have been developed. However, there is often insufficient data available on the selectivity of CDK inhibitors (CDKi) to attribute the effects on the presumed target CDK to these inhibitors. Here, we highlight discrepancies between the kinase selectivity of CDKi and the phenotype exhibited; we evaluated 31 CDKi (claimed to target CDK1-4) for activity toward CDKs 1, 2, 4, 5, 7, 9 and for effects on the cell cycle. Our results suggest that most CDKi should be reclassified as pan-selective and should not be used as a tool. In addition, some compounds did not even inhibit CDKs as their primary cellular targets; for example, NU6140 showed potent inhibition of Aurora kinases. We also established an online database of commercially available CDKi for critical evaluation of their utility as molecular probes. Our results should help researchers select the most relevant chemical tools for their specific applications.