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      Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing

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          There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).


          ► We report a highly selective nanomolar inhibitor KH-CB19 for the kinases CLK1/4 and DYRK1 ► KH-CB19 cocrystal structures revealed an ATP competitive but not ATP mimetic binding mode ► KH-CB19 formed halogen bonds with the kinase hinge region ► KH-CB19 led to dephosphorylation of SR proteins and effected splicing of TF isoforms in cells

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          Most cited references 53

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          Coot: model-building tools for molecular graphics.

          CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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            A short history of SHELX.

            An account is given of the development of the SHELX system of computer programs from SHELX-76 to the present day. In addition to identifying useful innovations that have come into general use through their implementation in SHELX, a critical analysis is presented of the less-successful features, missed opportunities and desirable improvements for future releases of the software. An attempt is made to understand how a program originally designed for photographic intensity data, punched cards and computers over 10000 times slower than an average modern personal computer has managed to survive for so long. SHELXL is the most widely used program for small-molecule refinement and SHELXS and SHELXD are often employed for structure solution despite the availability of objectively superior programs. SHELXL also finds a niche for the refinement of macromolecules against high-resolution or twinned data; SHELXPRO acts as an interface for macromolecular applications. SHELXC, SHELXD and SHELXE are proving useful for the experimental phasing of macromolecules, especially because they are fast and robust and so are often employed in pipelines for high-throughput phasing. This paper could serve as a general literature citation when one or more of the open-source SHELX programs (and the Bruker AXS version SHELXTL) are employed in the course of a crystal-structure determination.
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              Refinement of macromolecular structures by the maximum-likelihood method.

              This paper reviews the mathematical basis of maximum likelihood. The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estimating sigma(A) using 'free' reflections is described and its effects analysed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial alpha-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement.

                Author and article information

                Chem Biol
                Chem. Biol
                Chemistry & Biology
                28 January 2011
                28 January 2011
                : 18
                : 1
                : 67-76
                [1 ]University of Oxford, Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Old Road Campus Research Building, Oxford OX3 7DQ, UK
                [2 ]Ludwig-Maximilians Universität, Department of Pharmacy-Center for Drug Research, Butenandtstrasse 5-13, 81377 Munich, Germany
                [3 ]Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Centrum für Herz-und Kreislaufmedizin, Berlin, Germany
                [4 ]NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Denmark
                [5 ]Novartis Pharma AG, Klybeckstrasse 141, CH-4002 Basel, Switzerland
                [6 ]University of Oxford, Department of Clinical Pharmacology, Old Road Campus Research Building, Oxford OX3 7DQ, UK
                Author notes
                []Corresponding author franz.bracher@
                [∗∗ ]Corresponding author stefan.knapp@

                These authors contributed equally to this work

                © 2011 Elsevier Ltd.

                This document may be redistributed and reused, subject to certain conditions.




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