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Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

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.

Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is present only in proliferating cells, has become a novel and attractive anticancer target with potential for reduced side effects compared to currently available therapies. We report herein the discovery of the first known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D130V). In a murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP inhibitor versus tumors that are resistant to the other known KSP inhibitors.

The (Z)-allene-ene-yne 10, a precursor to a reactive alpha,3-dehydrotoluene biradical, was covalently linked to a minor groove binding element, and the resultant conjugate (9) was shown to bind and cleave DNA with sequence selectivity.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Kinesin spindle protein (KSP), a mitotic kinesin responsible for bipolar spindle establishment and maintenance, is currently the target of intense research for the development of novel anticancer therapeutics. Several inhibitors of KSP have progressed into clinical trials and many others are in preclinical development. A majority of these inhibitors are ATP-uncompetitive and bind in an allosteric loop L5 binding pocket, but recently, inhibitors with an alternative mechanism of action (ATP-competitive) have also been identified and characterized. In this review, an update of the clinical trial results with ATP-uncompetitive KSP inhibitors is provided and recent progress in the identification of additional KSP inhibitors is discussed.

The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.

Phosphoinositide 3-kinase (PI3K) is a critical regulator of cell growth and transformation and its signaling pathway is one of the most commonly mutated pathways in human cancer. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of the PI3K/AKT pathway. GSK1059615, our first PI3K clinical compound, progressed to a dose escalation study in patients with refractory malignancies. Following the discovery of GSK1059615, we sought to identify a second inhibitor with improved potency, selectivity, and pharmacokinetics. Key to our approach to achieving the desired levels of PI3K activity was to pursue structure-based design utilizing crystallography of the more amenable PI3K as a surrogate protein. Following a chemistry lead optimization effort, the pyridylsulfonamide GSK2126458 was identified as a highly potent, orally bioavailable, pan-PI3K and mTOR inhibitor (PI3K app Ki = 19 pM; mTORC1 app Ki = 180 pM; mTORC2 app Ki = 300 pM). Consistent with potent PI3K and mTORC2 enzyme inhibition, GSK2126458 decreased cellular levels of phosphorylated AKT (BT474 pAKT IC50 = 180 pM) and inhibited cell proliferation in a large panel of cancer cell lines (e.g. BT474 growth IC50 = 2 nM). GSK2126458 showed good exposure in four pre-clinical animal species and exhibited in vivo activity in both pharmacodynamic and tumor growth efficacy models. GSK2126458 is being evaluated currently in human clinical trials for the treatment of cancer. The discovery, design, and optimization of GSK2126458 and related analogs will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C62.