Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, no functional assays are currently implemented clinically, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. Here we demonstrate that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defines the drug sensitivity or resistance of glioblastoma multiforme (GBM) and B-cell acute lymphocytic leukemia (B-ALL) cells. MAR reveals heterogeneity in drug sensitivity not only between different tumors but also within individual tumors and tumor-derived cell lines. MAR measurement predicts drug response using samples as small as 25 μL of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance within heterogeneous tumors.

Rationale p53 pathway alterations are key molecular events in glioblastoma (GBM). MDM2 inhibitors increase expression and stability of p53 and are presumed to be most efficacious in patients with TP53 wild-type and MDM2-amplified cancers. However, this biomarker hypothesis has not been tested in patients or patient-derived models for GBM. Methods We performed a preclinical evaluation of RG7112 MDM2 inhibitor, across a panel of 36 patient-derived GBM cell lines (PDCLs), each genetically characterized according to their P53 pathway status. We then performed a pharmacokinetic (PK) profiling of RG7112 distribution in mice and evaluated the therapeutic activity of RG7112 in orthotopic and subcutaneous GBM models. Results MDM2-amplified PDCLs were 44 times more sensitive than TP53 mutated lines that showed complete resistance at therapeutically attainable concentrations (avg. IC50 of 0.52 μM vs 21.9 μM). MDM4 amplified PDCLs were highly sensitive but showed intermediate response (avg. IC50 of 1.2 μM), whereas response was heterogeneous in TP53 wild-type PDCLs with normal MDM2/4 levels (avg. IC50 of 7.7 μM). In MDM2-amplified lines, RG7112 restored p53 activity inducing robust p21 expression and apoptosis. PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival. Conclusion These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2 amplified models suggests that additional markers of response to MDM2 inhibitors must be identified.

17 alpha-Hydroxylase/C17-20-lyase (P450 17, CYP 17) and 5 alpha-reductase are the key enzymes in androgen biosynthesis and targets for the treatment of prostate cancer and benign prostatic hyperplasia. In the search of inhibitors for both enzymes, 23 pregnenolone- or progesterone-based steroids were synthesized bearing an oxime group connected directly or via a spacer to the steroidal D-ring. Tested for inhibition of human and rat P450 17, some pregnenolone (9, 11, 14) and a series of progesterone compounds (17-20) turned out to be highly active inhibitors of the human enzyme. The most active compound was Z-21-hydroxyiminopregna-5, 17(20)-dien-3 beta-ol (9) showing K(i) values of 44 and 3.4 nM for the human and rat enzymes, respectively, and a type II UV-difference spectrum indicating a coordinate bond between the oxime group and the heme iron. In contrast to the pregnenolones which showed no inhibition of 5 alpha-reductase isozymes 1 and 2, the progesterones 16, 17, 20, 21, and 23 showed marked inhibition, especially toward the type 2 enzyme. Compounds 17 and 20 were identified as potent dual inhibitors of both P450 17 and 5 alpha-reductase. Tested for selectivity, the most potent P450 17 inhibitors 9, 10, and 14 showed no or only marginal inhibition of P450 arom, P450 scc, and P450 TxA(2). Selected compounds were tested for inhibition of the target enzymes using whole-cell assays. Compounds 9-11 strongly inhibited P450 17 being coexpressed with NADPH-P450 reductase in E. coli cells, and 16, 20, and 23 markedly inhibited 5 alpha-reductase expressed in HEK 293 cells. Tested for in vivo activity, 9 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats after 2 and 6 h by 57% and 44%.

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Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada). This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione), a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 µM.

A new series of furan embelin derivatives was synthesized and characterized as ATPcompetitive CK2 inhibitors. The new compounds were efficiently synthesized using a multicomponent approach from embelin (1), aldehydes and isonitriles through a Knoevenagel condensation/ Michael addition/ heterocyclization. Several compounds with inhibitory activities in the low micromolar or even submicromolar were identified.The most active derivative was compound 4l (2-(tert-butylamino)-3-(furan-3-yl)-5hydroxy-6-undecylbenzofuran-4,7-dione) with an IC 50 value of 0.63 µM. It turned out to be an ATP competitive CK2 inhibitor with a K i value determined to be 0.48 µM.Docking studies allowed the identification of key ligand-CK2 interactions, which could help to further optimize this family of compounds as CK2 inhibitors.

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