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Dasgupta S.,University of Chicago | Suslov N.B.,University of Chicago | Suslov N.B.,Takeda California | Piccirilli J.A.,University of Chicago
Journal of the American Chemical Society | Year: 2017

The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage. © 2017 American Chemical Society.

Goldfarb D.S.,New York University | MacDonald P.A.,Global Medical Affairs | Gunawardhana L.,Clinical Science | Chefo S.,Takeda Global Research and Development Center Inc | McLean L.,Takeda California
Clinical Journal of the American Society of Nephrology | Year: 2013

Background and objectives Higher urinary uric acid excretion is a suspected risk factor for calcium oxalate stone formation. Febuxostat, a xanthine oxidoreductase inhibitor, is effective in lowering serum urate concentration and urinary uric acid excretion in healthy volunteers and people with gout. This work studied whether febuxostat, compared with allopurinol and placebo, would reduce 24-hour urinary uric acid excretion and prevent stone growth or new stone formation. Design, setting, participants, & measurements In this 6-month, double-blind, multicenter, randomized controlled trial, hyperuricosuric participants with a recent history of calcium stones and one or more radio-opaque calcium stone ≥3 mm (as seen by multidetector computed tomography) received daily febuxostat at 80 mg, allopurinol at 300 mg, or placebo. The primary end point was percent change from baseline to month 6 in 24-hour urinary uric acid. Secondary end points included percent change from baseline to month 6 in size of index stone and change from baseline in the mean number of stones and 24-hour creatinine clearance. Results Of 99 enrolled participants, 86 participants completed the study. Febuxostat led to significantly greater reduction in 24-hour urinary uric acid (-58.6%) than either allopurinol (-36.4%; P=0.003) or placebo (-12.7%; P<0.001). Percent change from baseline in the size of the largest calcium stone was not different with febuxostat compared with allopurinol or placebo. There was no change in stone size, stone number, or renal function. No new safety concerns were noted for either drug. Conclusions Febuxostat (80 mg) lowered 24-hour urinary uric acid significantly more than allopurinol (300 mg) in stone formers with higher urinary uric acid excretion after 6 months of treatment. There was no change in stone size or number over the 6-month period. © 2013 by the American Society of Nephrology.

Zhang Y.,Takeda California | Zeng L.,Takeda California | Pham C.,Takeda California | Xu R.,Takeda California
Journal of Chromatography A | Year: 2014

A new preparative two-dimensional liquid chromatography/mass spectrometry system (2D LC-LC/MS) has been designed and implemented to enhance capability and resolving power for the separation and purification of pharmaceutical samples. The system was constructed by modifications of a conventional preparative LC/MS instrument with the addition of a set of switching valves and a sample loop, as well as interfacing a custom software program with MassLynx. The system integrates two chromatographic separations from the first and second dimensions into a single automated run to perform the purification of a target compound from a complex mixture without intermediate steps of sample preparation. The chromatography in the first dimension, operated in the heart-cutting mode, separates the target compound from the impurities by mass-triggered fractionation based on its molecular weight. This purified fraction from the first dimension is stored in the sample loop, and then gets transferred to the second column by using at-column dilution. A control software program, coined Prep 2D LCMS, was designed to integrate with MassLynx to retrieve data acquisition status. All of the chromatographic hardware components used in this preparative 2D LC-LC/MS system are from the original open access preparative LC/MS system, which has high level of robustness and affords easy and user-friendly operation. The new system is very versatile and capable of collecting multiple fractions with different masses under various purification modes as configured in the methods, such as conventional one-dimensional (1D) purification and/or 2D purification. This new preparative 2D LC-LC/MS system is therefore the ideal tool for medicinal chemistry lab in drug discovery environment. © 2013 Elsevier B.V.

Huang H.,University of Chicago | Suslov N.B.,University of Chicago | Suslov N.B.,Takeda California | Li N.-S.,University of Chicago | And 5 more authors.
Nature Chemical Biology | Year: 2014

Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers. © 2014 Nature America, Inc. All rights reserved.

Zhu J.,Scripps Research Institute | Hixon M.S.,Takeda California | Globisch D.,Scripps Research Institute | Kaufmann G.F.,Scripps Research Institute | Janda K.D.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2013

In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s-1), Km,ATP (150 ± 30 μM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria. © 2013 American Chemical Society.

Wang H.,Takeda California | Klein M.G.,Takeda California | Zou H.,Takeda California | Lane W.,Takeda California | And 4 more authors.
Nature Structural and Molecular Biology | Year: 2015

Stearoyl-coenzyme A desaturase-1 (SCD1) has an important role in lipid metabolism, and SCD1 inhibitors are potential therapeutic agents for the treatment of metabolic diseases and cancers. Here we report the 3.25-Å crystal structure of human SCD1 in complex with its substrate, stearoyl-coenzyme A, which defines the new SCD1 dimetal catalytic center and reveals the determinants of substrate binding to provide insights into the catalytic mechanism of desaturation of the stearoyl moiety. The structure also provides a mechanism for localization of SCD1 in the endoplasmic reticulum: human SCD1 folds around a tight hydrophobic core formed from four long α-helices that presumably function as an anchor spanning the endoplasmic reticulum membrane. Furthermore, our results provide a framework for the rational design of pharmacological inhibitors targeting the SCD1 enzyme. © 2015 Nature America, Inc. All rights reserved.

Takeda California | Date: 2012-03-21

Compounds of the following formula are provided for use with glucokinase: wherein the variables are as defined herein. Also provided are pharmaceutical compositions, kits and articles of manufacture comprising such compounds; methods and intermediates useful for making the compounds; and methods of using said compounds.

Takeda California | Date: 2011-03-04

A process for the production of fluorinated compound represented by the formula (I): or salts thereof wherein R^(1 )and R^(2 )are the same or different and each is selected from the group consisting of a hydrogen atom, a carbonyl group, a sulfonyl group and a phosphoryl group.

Takeda California | Date: 2013-10-02

Compounds are provided for use with glucokinase that comprise the formula: wherein the variables are as defined herein. Also provided are pharmaceutical compositions, kits and articles of manufacture comprising such compounds; methods and intermediates useful for making the compounds; and methods of using said compounds.

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