Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan. KeywordsClk1; Clk2; Clk3; Clk4; Dyrk1A; Dyrk1B; Pre-mRNA splicing; kinase inhibition; quinazoline The cdc2-like kinase (Clk) family contains four isoforms (Clk1-4) and is proposed to alter the function of the spliceosome by phosphorylating serine-arginine-rich (SR) proteins within the spliceosome assembly.1 The spliceosome regulates the processing, or splicing, of premRNAs, yielding mature protein-encoding mRNAs.2 , 3 Many human genes express more than one mRNA via alternative splicing, leading to protein diversity,4 however, misregulation of alternative splicing is involved in the pathogenesis of cancer and other diseases.5 , 6 Studies have revealed that Clk isoforms are associated with alternative splicing of PkcβII7, TF8, β-globin9 and E1A pre-mRNA.10 The Clks also regulate the alternative splicing of microtubule-associated protein tau and are implicated in frontotemporal dementia and Parkinson's disease through the phosphorylation of splicing factors (SF).11 Inhibitors of Clk isoforms may alter these events and could prove to be useful agents in disease phenotypes characterized by abnormal splicing. Hagiwara reported TG003 (1), a small molecule benzothiazole, as having low-nanomolar IC 50 values versus Clk1 and Clk4. The patent literature revealed structurally similar benzothiazole 1a from Sirtris Pharmaceuticals12 and a quinoline 3 from Chronogen, Inc13 reported to have activity versus Clk1 (Figure 1). Indole 2 was recently revealed as a potent (20 nM) ATP competitive Clk1 inhibitor with good selectivity over Clk3 via a unique binding mode.14 We have previously Send proofs to: Dr. Craig J. Thomas, NIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892-3370 USA, Phone: 301-217-4079; Fax: 301-217-5736, craigt@mail.nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The Dyrk family contains several additional isoforms (Dyrk1B, Dyrk2, Dyrk3, and Dyrk4). 20 The Dyrk1B homolog (also referred to as Mirk) was first isolated and described by Friedman and coworkers.30 , 31 Several reports suggest that Dyrk1B plays a significant role in cancer biology and muscle differentiation. Extensive studies on the Dyrk1B gene found that overexpression is associated with pancreatic and other cancers potentially due to its downstream effect ...

A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure activity-relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4 and Dyrk1A.

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC1280)) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.