A Dual-Readout F2 Assay That Combines Fluorescence Resonance Energy Transfer and Fluorescence Polarization for Monitoring Bimolecular Interactions

Forster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed -Dual-Readout F2 assay- with F2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90469/1/adt-2E2010-2E0292.pd

Mutated Ptpn11 alters leukemic stem cell frequency and reduces the sensitivity of acute myeloid leukemia cells to Mcl1 inhibition

PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 data set. We examined the role of mutated Shp2 in leukemias harboring MLL translocations, which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9-mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. Although the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K, suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small-molecule-mediated Mcl1 inhibition, suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2

Regulation of Wnt Signaling Target Gene Expression by the Histone Methyltransferase DOT1L

The histone methyltransferase DOT1L, solely responsible for histone H3 lysine 79 (H3K79) methylation, is associated with gene activation. Human leukemias carrying MLL gene rearrangements aberrantly recruit DOT1L to leukemogenic genes leading to increased H3K79 methylation and their transcriptional activation. Recent studies suggest that Wnt-targeted genes also depend on H3K79 methylation. Employing a chemical biology approach, the requirement for H3K79 methylation was investigated in Wnt pathway-inducible HEK293 cells and human colon adenocarcinoma-derived cell lines by inhibiting DOT1L with EPZ004777, a selective and potent S-adenosylmethionine competitive inhibitor. Our findings indicate that H3K79 methylation is not essential for the canonical Wnt signaling pathway, in particular for maintenance or activation of Wnt pathway target gene expression. Furthermore, H3K79 methylation is not elevated in human colon carcinoma samples in comparison with normal colon tissue. Therefore, our findings indicate that inhibition of DOT1L histone methyltransferase activity is likely not a viable therapeutic strategy in colon cancer

Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2

Bcl-2 is an antiapoptotic protein that is up-regulated in several tumor types, and its expression levels have strong correlation, to development of resistance to therapy and poor prognosis. We have shown recently that Bcl-2 also functions as a proangiogenic signaling molecule that activates a nuclear factor-kappa B-mediated pathway resulting in up-regulation of the angiogenic chemokines CXCL1 and CXCL8 by neovascular endothelial cells. Here, we evaluate the antiangiogenic effect of the novel small-molecule inhibitor of Bcl-2 (TW37) developed using a structure-based design strategy. We observed that TW37 has an IC50 of 1.8 mu mol/L for endothelial cells but showed no cytotoxic effects for fibroblasts at concentrations up to 50 mu mol/L. The mechanism of TW37-induced endothelial cell death was apoptosis, in a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3. The effect of TW37 on endothelial cell apoptosis was not prevented by coexposure to the growth factor milieu secreted by tumor cells. Inhibition of the angiogenic potential of endothelial cells (i.e., migration and capillary sprouting assays) and expression of the angiogenic chemokines CXCL1 and CXCL8 were accomplished at sub-apoptotic TW37 concentrations (0.005-0.05 mu mol/L). Notably, administration of TW37 i.v. resulted in a decrease in the density of functional human microvessels in the severe combined immunodeficient mouse model of human angiogenesis. In conclusion, we describe functionally separate proapoptotic and antiangiogenic mechanisms for a small-molecule inhibitor of Bcl-2 and show the potential for Bcl-2 inhibition as a target for antiangiogenic therapy

Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database

The X-linked inhibitor of apoptosis (XIAP) is a promising new molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells to chemotherapeutic agents and radiation therapy. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Through computational structure-based screening of an in-house traditional herbal medicine three-dimensional structure database of 8221 individual natural products, followed by biochemical testing of selected candidate compounds, we discovered embelin from the Japanese Ardisia herb as a small-molecular weight inhibitor that binds to the XIAP BIR3 domain. We showed that embelin binds to the XIAP BIR3 protein with an affinity similar to that of the natural Smac peptide using a fluorescence polarization-based binding assay. Our NMR analysis further conclusively confirmed that embelin interacts with several crucial residues in the XIAP BIR3 domain with which Smac and caspsase-9 bind. Embelin inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP. In stably XIAP-transfected Jurkat cells, embelin effectively overcomes the protective effect of XIAP to apoptosis and enhances the etoposide-induced apoptosis and has a minimal effect in Jurkat cells transfected with vector control. Taken together, our results showed that embelin is a fairly potent, nonpeptidic, cell-permeable, small-molecule inhibitor of XIAP and represents a promising lead compound for designing an entirely new class of anticancer agents that target the BIR3 domain of XIAP