In vitro high throughput screening, what next? Lessons from the screening for aurora kinase inhibitors.

Based on in vitro assays, we performed a High Throughput Screening (HTS) to identify kinase inhibitors among 10,000 small chemical compounds. In this didactic paper, we describe step-by-step the approach to validate the hits as well as the major pitfalls encountered in the development of active molecules. We propose a decision tree that could be adapted to most in vitro HTS.

Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase.

Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

Hydrosoluble benzo[e]pyridoindolones as potent inhibitors of aurora kinases.

Aurora kinases play an essential role in mitotic progression and are potentially druggable targets in cancer therapy. We identified benzo[e]pyridoindoles (BePI) as powerful aurora kinase inhibitors. Their efficiency was demonstrated both in enzymatic inhibition studies and in cell culture assays. New BePI molecules were synthesized, and a structure-activity relationship study was conducted with the aim of improving the activity and solubility of the lead compound. Tetracyclic BePI derivatives are characterized by a particular curved shape, and the presence of an oxo group on the pyridine ring was found to be required for aurora kinase B inhibition. New hydrosoluble benzo[e]pyridoindolones were subsequently designed, and their efficacy was tested by a combination of cell-cycle analysis and time-lapse experiments in live cells. The most active BePI derivative, 13 b, inhibited the cell cycle, drove cells to polyploidy, and eventually induced apoptosis. It exhibited high antiproliferative activity in HeLa cells with an IC(50) value of 63 nM. Relative to compounds tested in clinical trials, this antiproliferative potency places 13 b among the top 10 aurora kinase inhibitors. Our results justify further in vivo evaluation in preclinical animal models of cancer.