S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents.

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr(15) Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

Synthesis of Cis-fused pyran indolocarbazole derivatives that inhibit FLT3 kinase and the DNA damage kinase, checkpoint kinase 1.

Protein kinases are important enzymes in solid tumour and leukaemia pathologies. Their structures are well understood at the atomic level and their key role in the progression of certain cancers makes them valuable targets for anti-cancer therapy. Through medicinal chemical approaches, we developed an efficient preparative stereospecific synthesis of N12, N13 pyran-bridged indolocarbazoles that opens access to functional diversity within this previously challenging series. We focused upon the indolocarbazole class of chemical inhibitors, which includes S27888, an inhibitor we previously identified. We used biochemical and cell-based assays to identify small molecule inhibitors of Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase that is activated when cancer cells are treated with genotoxic agents. These compounds show a promising inhibitory profile on Chk1. Furthermore, these compounds are active against FLT3, which is a tyrosine kinase that is frequently activated in human leukaemias. These data suggest that this chemical class may provide a source of therapeutic compounds for a broad range of human cancers.

Characterization of novel checkpoint kinase 1 inhibitors by in vitro assays and in human cancer cells treated with topoisomerase inhibitors.

AIMS: We have developed biochemical and cell based assays to characterize small therapeutic molecules that inhibit the DNA damage checkpoint enzyme, Chk1 (Checkpoint kinase 1). MAIN METHODS: To prepare a screen of large chemical libraries, we purified the full-length and the catalytic domain versions of human Chk1. We characterized their properties and then selected full-length Chk1 as the variant most suitable for screening. We then identified and characterized structurally different Chk1 inhibitors in cell based-assays by measuring cytotoxicity and checkpoint bypass activity. KEY FINDINGS: We treated human cells with topoisomerase I inhibitors and demonstrated that at the time of Chk1 inhibitor addition, the cells have damaged DNA and activated Chk1. One Chk1 inhibitor, the indolocarbazole S27888, was active in the checkpoint bypass assay. SIGNIFICANCE: Knowing that the protein kinase inhibitory properties are different for each inhibitor, it seems that only a limited range of inhibitory activity is tolerated by cells. Chk1 has an essential role in determining how cancer cells respond to genotoxic treatments, therefore, inhibitors of this protein kinase are of great medical interest.

An unusual DNA binding compound, S23906, induces mitotic catastrophe in cultured human cells.

The biochemical pathways that lead cells to mitotic catastrophe are not well understood. To identify these pathways, we have taken an approach of treating cells with a novel genotoxic compound and characterizing whether cells enter mitotic catastrophe or not. S23906 is a novel acronycine derivative that forms adducts with the N2 residue of guanine in the minor groove of the DNA helix and destabilizes base pairing to cause helix opening. We observed, in HeLa and HT-29 cells, that S23906 induced gamma-H2AX and activated checkpoint kinase 1, as did bleomycin, camptothecin, and cisplatin, when tested under equi-toxic conditions. S23906 also induced cyclin E1 protein, although this activity was not required for cytotoxicity because knock down of cyclin E1 by RNA interference did not affect the number of dead cells after treatment. Cyclin B1 levels first decreased and then increased after treatment with S23906. Cyclin B1 was associated with Cdk1 kinase activity, which correlated with an increase in the number of mitotic cells. By 32 h after treatment, at least 20% of the cells entered mitotic catastrophe as determined by microscopy. Suppression of the DNA checkpoint response by co-treatment with caffeine increased the number of cells in mitosis. These results suggest that mitotic catastrophe is one of the cellular responses to S23906 and that mitotic catastrophe may be a common cellular response to many different types of DNA damage.

Expression of UCP3 in CHO cells does not cause uncoupling, but controls mitochondrial activity in the presence of glucose.

The proton-transport activity of UCP1 (uncoupling protein 1) triggers mitochondrial uncoupling and thermogenesis. The exact role of its close homologues, UCP2 and UCP3, is unclear. Mounting evidence associates them with the control of mitochondrial superoxide production. Using CHO (Chinese-hamster ovary) cells stably expressing UCP3 or UCP1, we found no evidence for respiration uncoupling. The explanation lies in the absence of an appropriate activator of UCP protonophoric function. Accordingly, the addition of retinoic acid uncouples the respiration of the UCP1-expressing clone, but not that of the UCP3-expressing ones. In a glucose-containing medium, the extent of the hyperpolarization of mitochondria by oligomycin was close to 22 mV in the five UCP3-expressing clones, contrasting with the variable values observed with the 15 controls. Our observations suggest that, when glycolysis and mitochondria generate ATP, and in the absence of appropriate activators of proton transport, UCPs do not transport protons (uncoupling), but rather other ions of physiological relevance that control mitochondrial activity. A model is proposed using the known passive transport of pyruvate by UCP1.