Human cancers require fatty acid synthase (FASN)-dependent de novo long-chain fatty acid synthesis for proliferation. FASN is therefore an attractive drug target, but fast technologies for reliable label-free cellular compound profiling are lacking. Recently, MALDI-mass spectrometry (MALDI-MS) has emerged as an effective technology for discovery of recombinant protein target inhibitors. Here we present an automated, mechanistic MALDI-MS cell assay, which monitors accumulation of the FASN substrate, malonyl-coenzyme A (CoA), in whole cells with limited sample preparation. Profiling of inhibitors, including unpublished compounds, identified compound 1 as the most potent FASN inhibitor (1 nM in A549 cells) discovered to date. Moreover, cellular MALDI-MS assays enable parallel profiling of additional pathway metabolites. Surprisingly, several compounds triggered cytidine 5'-diphosphocholine (CDP-choline) but not malonyl-CoA accumulation indicating that they inhibit diacylglycerol generation but not FASN activity. Taken together, our study suggests that MALDI-MS cell assays may become important tools in drug profiling that provide additional mechanistic insights concerning compound action on metabolic pathways.

A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.

BACKGROUND:Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916.METHODS:59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response.RESULTS:20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test).CONCLUSIONS:High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.

Novel Aurora inhibitors were identified truncating clinical candidate GSK1070916. Many of these truncated compounds retained potent activity against Aurora B with good antiproliferative activity. Mechanistic studies suggested that these compounds, depending on the substitution pattern, may or may not exert their antiproliferative effects via inhibition of Aurora B. The SAR results from this investigation will be presented with an emphasis on the impact structural changes have on the cellular phenotype.

The Aurora kinases play critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. Selective inhibitors may provide a new therapy for the treatment of tumors with Aurora kinase amplification. Herein we describe our lead optimization efforts within a 7-azaindole-based series culminating in the identification of GSK1070916 (17k). Key to the advancement of the series was the introduction of a 2-aryl group containing a basic amine onto the azaindole leading to significantly improved cellular activity. Compound 17k is a potent and selective ATP-competitive inhibitor of Aurora B and C with K(i)* values of 0.38 +/- 0.29 and 1.5 +/- 0.4 nM, respectively, and is >250-fold selective over Aurora A. Biochemical characterization revealed that compound 17k has an extremely slow dissociation half-life from Aurora B (>480 min), distinguishing it from clinical compounds 1 and 2. In vitro treatment of A549 human lung cancer cells with compound 17k results in a potent antiproliferative effect (EC(50) = 7 nM). Intraperitoneal administration of 17k in mice bearing human tumor xenografts leads to inhibition of histone H3 phosphorylation at serine 10 in human colon cancer (Colo205) and tumor regression in human leukemia (HL-60). Compound 17k is being progressed to human clinical trials.

The phosphoinositide 3‐kinase (PI3K) pathway is among the most commonly activated pathways in human cancer. The biological role of PI3K in growth and survival of cancer cells and the prevalence of activating mutations in human cancers are well documented and a significant proportion of tumors would be predicted to benefit from inhibition of this pathway. Here we report on the characterization of the pan‐PI3K pyridylsulfonamide inhibitor GSK2126458: a very potent (PI3K app K i = 19 pM), reversible, ATP‐competitive inhibitor of wild‐type PI3K and the ‘hotspot’ activating mutants of p110 found in human cancer. GSK2126458 demonstrated good selectivity for the PI3K family of enzymes including the mTORC1 and mTORC2 complexes, when evaluated in a large panel of protein kinases. Consistent with potent PI3K enzyme inhibition, GSK2126458 decreased the cellular levels of phosphorylated AKT, p70 S6K , PRAS40 and ERK in a concentration and time dependent manner, the IC50 for pAKT in the HCC1954 breast carcinoma cell line was 2 nM. GSK2126458 induced the nuclear translocation of the FOXO3a transcription factor in a concentration dependent manner that mechanistically appeared bimodal. Growth inhibition was time and concentration dependent with a 3 day exposure resulting in a growth IC 50 (gIC 50 ) of 9 nM in HCC1954 cells with evidence of cell death. Cell death correlated with induction of caspase 3 and 7 activity suggesting apoptosis was the mechanism of cell death. GSK2126458 had a breadth of activity for potent cell growth inhibition and induction of cell death in a variety of human cancer cells. GSK2126458 demonstrated robust, dose dependent in vivo pharmacodynamic activity as measured by inhibition of phospho‐AKT in advanced BT474 breast cancer cell xenograft tumors. Inhibition of AKT phosphorylation was rapid and lasted for several hours after a single oral administration of GSK2126458. Transient increases in plasma insulin and blood glucose were observed. Evaluation of in vivo tumor growth effects of GSK2126458 in xenograft models demonstrated dose dependent tumor growth delay in several models of diverse tumor lineage and tumor regression in a breast cancer xenograft. The biological profile of GSK2126458 supports the clinical advancement of this compound and GSK2126458 has entered Phase I human clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C63.

The protein kinases, Aurora A, B, and C have critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. GSK1070916, is a novel ATP competitive inhibitor that is highly potent and selective for Aurora B/C kinases. Human tumor cells treated with GSK1070916 show dose-dependent inhibition of phosphorylation on serine 10 of Histone H3, a substrate specific for Aurora B kinase. Moreover, GSK1070916 inhibits the proliferation of tumor cells with EC(50) values of <10 nmol/L in over 100 cell lines spanning a broad range of tumor types. Although GSK1070916 has potent activity against proliferating cells, a dramatic shift in potency is observed in primary, nondividing, normal human vein endothelial cells, consistent with the proposed mechanism. We further determined that treated cells do not arrest in mitosis but instead fail to divide and become polyploid, ultimately leading to apoptosis. GSK1070916 shows dose-dependent inhibition of phosphorylation of an Aurora B-specific substrate in mice and consistent with its broad cellular activity, has antitumor effects in 10 human tumor xenograft models including breast, colon, lung, and two leukemia models. These results show that GSK1070916 is a potent Aurora B/C kinase inhibitor that has the potential for antitumor activity in a wide range of human cancers.