Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212's enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life.Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models.Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B , and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors.

Presenilins are integral membrane protein involved in the production of amyloid ␤-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter ␥-secretase cleavage of the ␤-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A␤, the 4-kDa ␤-peptide. Here, we show that radiolabeled ␥-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A␤ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N-and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of ␥-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective ␥-secretase inhibitors block A␤ formation by binding to presenilin-1 and -2.␤-Amyloid precursor protein (␤APP) 1 is a transmembrane protein that undergoes processing to A␤ by proteolytic activities known as ␤-and ␥-secretases (for review, see Refs. 1-3). The ␤-secretase cleavage occurs in the extracellular domain by a recently identified aspartyl protease variously termed BACE, memapsin, and Asp2 (4 -9), whereas the heterogeneous ␥-secretase cleavage occurs in the transmembrane domain (2, 10). Dominant mutations in either of the two human presenilin (PS-1 and PS-2) genes lead to familial Alzheimer's disease (AD). PS-1 and -2 are polytopic membrane proteins (for review, see Refs. 11-13). Presenilins are proteolytic processed. In vivo, only small amounts of the holoprotein can be detected, primarily in the nuclear envelope, whereas 30-kDa N-terminal and 20-kDa C-terminal fragments of presenilin are observed in all mammalian tissues and cell lines analyzed so far. Coimmunoprecipitation experiments revealed that presenilin fragments are assembled into a high molecular weight complex together with other proteins (for review see 11-13). The proposed mechanism through which the presenilin mutations cause AD is an alteration in the predominant ␥-secretase cleavage site which increases the amount of the longer, more amyloidogenic A␤ 1-42(43) fragments produced (11-13). A null mutation of the mouse PS-1 selectively reduces ␥-secretase activity (14), and site-directed mutagenesis of PS-1 and PS-2 at two conserved aspartyl residues, which resemble the catalytic center of aspartyl proteases, also reduces ␥-secretase activity (15, 16). These observations indicate that PS-1 and PS-2 either stimulate the activity of ␥-secretase by trafficking to appropriate cellular compartments, serve as cofactors of the ␥-secretase, or are ␥-secretase themselves.Here, we report that a series of potent and selective ␥-secretase inhibitors bind to mam...

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in AML patients’ cells. Our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)

methylidene]-

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.

␥-Secretase is a multimeric complex consisted of presenilins (PSs) and three other proteins. PSs appear to be key contributors for the enzymatic center, the potential target of a number of recently developed ␥-secretase inhibitors. Using radiolabeled and unlabeled inhibitors as ligands, this study was aimed to determine the in situ distribution of ␥-secretase in the brain. Characterization using PS-1 knock-out mouse embryos revealed 50 and 80% reductions of ␥-secretase inhibitor binding density in the heterozygous (PS-1 ϩ/Ϫ ) and homozygous (PS-1 Ϫ/Ϫ ) embryos, respectively, relative to the wild type (PS-1). The pharmacological profile from competition binding assays suggests that the ligands may target at the N-and C-terminal fragments of PS essential for ␥-secretase activity. In the adult rat brain, the binding sites existed mostly in the forebrain, the cerebellum, and discrete brainstem areas and were particularly abundant in areas rich in neuronal terminals, e.g., olfactory glomeruli, CA3-hilus area, cerebellar molecular layer, and pars reticulata of the substantia nigra. In the developing rat brain, diffuse and elevated expression of binding sites occurred at the early postnatal stage relative to the adult. The possible association of binding sites with neuronal terminals in the adult brain was further investigated after olfactory deafferentation. A significant decrease with subsequent recovery of binding sites was noted in the olfactory glomeruli after chemical damage of the olfactory epithelium. The findings in this study support a physiological role of PS or ␥-secretase complex in neuronal and synaptic development and plasticity.

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