The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.nuclear phosphatases | enzyme regulation | enzyme specificity | X-ray crystal structure | nuclear magnetic resonance P rotein phosphatase 1 (PP1; ∼38.5 kDa), a single-domain protein, is the most widely expressed and abundant serine/threonine phosphatase (1). By dephosphorylating a variety of protein substrates, PP1 regulates diverse biological processes, including protein synthesis, muscle contraction, carbohydrate metabolism, neuronal signaling and, of specific interest for this work, cell-cycle progression. Although the intrinsic substrate specificity of PP1 is very low, by interacting with regulatory proteins (∼200 biochemically confirmed PP1 interactors), PP1 achieves high specificity (2-5). The majority of PP1 regulators and some substrates bind PP1 via a primary PP1-binding motif, the RVxF motif, which binds to a hydrophobic groove on PP1 ∼20 Å distal from its catalytic center (6). Outside of the RVxF motif, PP1 regulatory proteins mostly lack any apparent sequence similarity. Thus, additional interaction sites, such as the SILK (7), the MyPhoNE (8), and the recently identified ΦΦ motif (9) can only be identified by structural analysis, a major challenge for a comprehensive understanding of PP1 regulation. Only when the primary sequences of PP1 regulators are correlated with specific PP1 binding modes and activities will the PP1 interactome, and the biological processes it regulates, become a viable drug target for the multitude of PP1-associated diseases, such as multiple cancers.One of the key PP1 regulatory targeting proteins in the nucleus, in addition to nuclear inhibitor of PP1 (NIPP1) and Repoman, is the...

We present a novel class of dual modulators of gamma-secretase and peroxisome proliferator-activated receptor gamma (PPARgamma) based on the structure of 2-(bis(phenethoxy)pyrimidine-2-ylthio)hexanoic acid 8 (IC(50)(Abeta42) = 22.8 microM, EC(50)(PPARgamma) = 8.3 microM). The modulation of both targets with approved drugs (i.e., amyloid-beta 42 (Abeta42)-lowering NSAIDs for gamma-secretase and glitazones for PPARgamma) has demonstrated beneficial effects in in vitro and in vivo models of Alzheimer's disease (AD). However, although NSAIDs and PPARgamma agonists share similar structural features, no druglike compounds with dual activities as gamma-secretase modulators (GSMs) and PPARgamma agonists have been designed so far. On the basis of our initial lead structure 8, we present the structure-activity relationships (SARs) of broad structural variations. A significant improvement was reached by the introduction of p-trifluoromethyl substituents at the phenyl residues yielding compound 16 (IC(50)(Abeta42) = 6.0 microM, EC(50)(PPARgamma) = 11.0 microM) and the replacement of the two phenyl residues of 8 by cyclohexyl yielding compound 22 (IC(50)(Abeta42) = 5.1 microM, EC(50)(PPARgamma) = 6.6 microM).

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Various inflammatory diseases are associated with the excessive formation of leukotrienes (LTs) and prostaglandins (PGs). Herein, we present a novel class of dual inhibitors of 5-lipoxygenase (5-LO) and microsomal prostaglandin E(2) synthase-1 (mPGES-1), key enzymes in the formation of LTs and PGE(2), respectively. On the basis of the structure of 2-[(4,6-diphenethoxypyrimidin-2-yl)thio]hexanoic acid (1), we performed a detailed SAR analysis, and mechanistic studies were carried out to elucidate the mode of 5-LO inhibition. Interestingly, the pyrimidine ring including the thioether of 1 could be replaced by a simple benzyl or a benzylidene moiety yielding a novel series of bioactive 2-benzylidene- and 2-benzylhexanoic acids exemplified by 2-(2,3-diphenethoxybenzylidene)hexanoic acid, 29 (IC(50) 5-LO = 0.8 μM; mPGES-1 = 1.1 μM). Importantly, none of the novel bioactive derivatives strongly inhibited cyclooxygenase activities. Together, we provide novel promising lead compounds for the treatment of inflammatory diseases valuable for further investigations in vivo.