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(12) Patent Application Publication (10) Pub. No.: US 2010/0158968 A1 Panitch Et Al

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US 20100158968A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0158968 A1 Panitch et al. (43) Pub. Date: Jun. 24, 2010 (54) CELL-PERMEANT PEPTIDE-BASED Publication Classification INHIBITOR OF KINASES (51) Int. Cl. (76) Inventors: Alyssa Panitch, West Lafayette, IN st e8 CR (US); Brandon Seal, West ( .01) Lafayette, IN (US) A638/10 (2006.01) s A638/16 (2006.01) Correspondence Address: A6IP 43/00 (2006.01) GREENBERG TRAURIG, LLP (52) U.S. Cl. ................ 424/422:514/15: 514/13: 514/14 200 PARKAVE., P.O. BOX 677 FLORHAMPARK, NJ 07932 (US) (57) ABSTRACT The described invention provides kinase inhibiting composi (21) Appl. No.: 12/634,476 tions containing a therapeutic amount of a therapeutic inhibi (22) Filed: Dec. 9, 2009 torpeptide that inhibits at least one kinase enzyme, methods e 19 for treating an inflammatory disorder whose pathophysiology comprises inflammatory cytokine expression, and methods Related U.S. Application Data for treating an inflammatory disorder whose pathophysiology (60) Provisional application No. 61/121,396, filed on Dec. comprises inflammatory cytokine expression using the kinase 10, 2008. inhibiting compositions. 20 { ki> | 0: & c s - --- 33- x: SE PEPELE, ics 1.-- E- X K. AAA 22.9 --- KKK. Y.A., 3.2; C. -r { AAEASA. A. E. i : A X AAAAAAA; ; ; ; :-n. 4:-: is SEEKESAN.ARESA, 3523 -- -- Yili.A.R.AKA: 5,342 3. {{RCE: Rix i: Patent Application Publication US 2010/0158968A1 & ******** NO s ***** · Patent Application Publication Jun. 24, 2010 Sheet 2 of 11 US 2010/0158968A1 it, O Peptide: Cso: g E 100 WRRKAWRRKANRO, GWAA. O.737 +I h 90 1AKAFAKAARLYRKALARogvaA FAKAARYRKALARCGWAA, 44301,769 f 80 OYARAAARCARAKALARCLGWAA 22,09 : | KYARAAAROARAKALNROLGVAA 5.842 t E 70 : 5 i 60 if: s i S. t t i 3. 3.w | 30 s: i 10 rower-staxers -ses-toessnesses O.O. (, O OOC Concentration (M) FIGURE 2 Patent Application Publication Jun. 24, 2010 Sheet 3 of 11 US 2010/0158968A1 0.8 - sy KAANROLGWAA KAARCLGWAA 3. A KANAOLGWAA X KANRAGWAA R at KANROAGWAA to KALNR AWAA - KANROLGAAA - No inhibitor Peptide 0.6 O KANRO, GWAA 5 {} g g 0.4 d s s ce l p () 2 t O ex O MK2 inhibitor, M. OO FIGURE 3 Patent Application Publication Jun. 24, 2010 Sheet 4 of 11 US 2010/0158968A1 O. 8 3. () O.2 {x KAd NRO, GWAA KALc NROLGWAA A KANdRO GVAA X KALNRCC, GWAA an KALNROld GWAA -- KANROGWAA -No inhibitor Peptide C KALNROLGWAA 10 MK2 inhibitor, M OO FIGURE 4 Patent Application Publication Jun. 24, 2010 Sheet 5 of 11 US 2010/0158968A1 O5 0.85 - & <- KANROLGVA KKKANRCGWAA x WLRRKAWRRKALNROLGVAA a- -No inhibitor Peptide <> C KANROLGWAA 65 g o in 30.45 c S. (D {} s r t f. 88 O 2 5. S. COs - X 1.g -O.15 ai MK2 inhibitor, uM FIGURE 5 Patent Application Publication Jun. 24, 2010 Sheet 6 of 11 US 2010/0158968A1 ... WRRKA WLRRKAWRRKA, A. YGRKKRRORRR a 0.8 x YARAAAROARA -No inhibitor Peptide KALNROLGWAA S st0.6 - k S. is: s2. d 50.4 k t3 t ". i 0.2 t 0 - kiss. Raw-ex-wee-wxx-x-xxv-wi-worwmem-m-m-mow-mow 1) MK2 inhibitor, M O) FIGURE 6 Patent Application Publication Jun. 24, 2010 Sheet 7 of 11 US 2010/0158968A1 400 C Untreated 1 ng/ml. IL-13 5O i 1 ng/mL L-15 + 0.1 uM Rottlerin 35 is 1 ng/mL L-1B + 1 uM Rottierin i ng/mL L-13 + 100MYARA -X 3 O O is 1 ng/mL L-13 + 300MYARA is 1 ng/mL L-1 3 + 1000MYARA is 1ng/mLIL-1B + 3000MYARA T 2 5O 2OO 150 1OO 5O O Ya-------- rror sw88w88&xtrass | 12hr NS 24h NS -50 - FIGURE 7 Patent Application Publication Jun. 24, 2010 Sheet 8 of 11 US 2010/0158968A1 OUntreated p < 0.05 1 ng/mLTNF-ct 2. 1ng/mLTNF-ct + 0.1M Rotterin is 1ng/ml TNF-c + 1 M Rotterin 2. 1ng/mLTNF-a + 100M FAK is 1ng/mLTNF-a + 300M FAK Rs 1 ng/mLTNF-a + 1000MYARA H 1 ng/mLTNF-a + 3000MYARA W FIGURE 8 Patent Application Publication Jun. 24, 2010 Sheet 9 of 11 US 2010/0158968A1 C. J treated 1Ong/mLTNF-a 1000 - 10mg/mLTNF-ci + 0.1M Rotterin 10mg/mLTMF-a + 1 M Rotterin is 10mg/mLTNF-a + 100 MFAK a 10ngim TNF-a + 300 MFAK 800 is 10ngim TNF-a + 1000MYARA a 10ngiml TNF-a + 3000MYARA 40 2OSO OO 12 NS FIGURE 9 Patent Application Publication Jun. 24, 2010 Sheet 10 of 11 US 2010/0158968A1 zzzzz 838) * 3: Patent Application Publication Jun. 24, 2010 Sheet 11 of 11 US 2010/0158968A1 }} ${}{}}{{########8 US 2010/0158968 A1 Jun. 24, 2010 CELL-PERMEANT PEPTDE-BASED diversity in their Substrate specificity, structure, and the path INHIBITOR OF KINASES ways in which they participate. A recent classification of all available kinase sequences (approximately 60,000 CROSS REFERENCES sequences) indicates kinases can be grouped into 25 families 0001. This application claims the benefit of priority to of homologous proteins. These kinase families are further U.S. provisional application 61/121,396, filed Dec. 10, 2008, assembled into 12 fold groups based on similarity of struc incorporated herein by reference in its entirety. tural fold. Further, 22 of the 25 families (approximately 98.8% of all sequences) belong to 10 fold groups for which STATEMENT OF GOVERNMENT FUNDING the structural fold is known. Of the other 3 families, poly 0002 The described invention was made with government phosphate kinase forms a distinct fold group, and the 2 support under Grant K25HL074968 awarded by the National remaining families are both integral membrane kinases and Institutes of Health. The government has certain rights in the comprise the final fold group. These fold groups not only invention. include some of the most widely spread protein folds, such as Rossmann-like fold (three or more parallel beta strands FIELD OF THE INVENTION linked by two alpha helices in the topological order beta alpha-beta-alpha-beta), ferredoxin-like fold (a common C+B 0003. The described invention relates to cell biology, cell protein fold with a signature off3C.B Secondary structure permeant peptides, cell-permeant peptide compositions, and along its backbone), TIM-barrel fold (meaning a conserved methods of use thereof, and methods of protein engineering. protein fold consisting of eight C-helices and eight parallel B-strands that alternate along the peptide backbone), and BACKGROUND antiparallel B-barrel fold (a beta barrel is a large beta-sheet 0004. The three-dimensional conformation of a protein that twists and coils to form a closed structure in which the molecule is determined by its amino acid sequence, and the first strand is hydrogen bonded to the last), but also all major details of a protein's conformation determine its chemistry. classes (all C, all f, C.--f, C/B) of protein structures. Within a 0005. A protein generally consists of a polypeptide back fold group, the core of the nucleotide-binding domain of each bone with attached side chains. The sequence of the chemi family has the same architecture, and the topology of the cally different side chains of the amino acid makes each protein core is either identical or related by circular permu protein distinct. The folded structure of a protein is stabilized tation. Homology between the families within a fold group is by noncovalent interactions (e.g., hydrogen bonds, ionic not implied. bonds, and van der Waals attractions) that form between different parts of the polypeptide chain. one part of the chain 0009 Group I (23,124 sequences) kinases incorporate and another. The stability of each folded shape is determined protein SITY kinase, atypical protein kinase, lipid kinase, by the combined strength of large numbers of Such noncova and ATP grasp enzymes and further comprise the protein lent bonds. SITY kinase, and atypical protein kinase family (22,074 sequences). These kinases include: choline kinase (EC 2.7.1. 0006 Each protein has four levels of structural organiza 32); protein kinase (EC 2.7.137); phosphorylase kinase (EC tion. The amino acid sequence is the primary structure of the 2.7.1.38); homoserine kinase (EC 2.7.1.39); I-phosphatidyli protein. The secondary structure is defined by patterns of nositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC hydrogen bonds between backbone amide and carboxyl 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin groups without consideration of sidechain-mainchain and 3'-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); sidechain-sidechain hydrogen bond (e.g. C. helix, B-sheet). 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase The tertiary structure, the full three-dimensional organization (EC 2.7.1.103); hydroxymethylglutaryl-CoA reductase of a polypeptide chain, is the manner in which the sheets and (NADPH) kinase (EC 2.7.1.109); protein-tyrosine kinase helices of the secondary structure of a protein fold on them (EC 2.7.1.112); isocitrate dehydrogenase (NADP+) kinase selves to define the three-dimensional structure. Quaternary (EC 2.7.1.116); myosin light-chain kinase (EC 2.7.1.117): structure refers to the complete structure of a protein mol hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin ecule formed as a complex of more than one polypeptide dependent protein kinase (EC 2.7.1.123); rhodopsin kinase chain. (EC 2.7.1.125); beta-adrenergic-receptor kinase (EC 2.7.1. 0007 Protein domains are structural units that fold more 126); myosin heavy-chain kinase (EC 2.7.1.129); Tau pro or less independently of each other to form globular compact tein kinase (EC 2.7.1.135); macrollide 2'-kinase (EC 2.7.1.
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    1521-0111/92/3/201–210$25.00 https://doi.org/10.1124/mol.116.107839 MOLECULAR PHARMACOLOGY Mol Pharmacol 92:201–210, September 2017 Copyright ª 2017 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. MINIREVIEW—MOLECULAR PHARMACOLOGY IN CHINA Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model Zhao Yang, Fan Yang, Daolai Zhang, Zhixin Liu, Amy Lin, Chuan Liu, Peng Xiao, Xiao Yu, and Jin-Peng Sun Downloaded from Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology (Z.Y., Z.L., C.L., P.X., J.-P.S.), Department of Physiology (F.Y., X.Y.), Shandong University School of Medicine, Jinan, Shandong, People’s Republic of China; School of Pharmacy, Binzhou Medical University, Yantai, Shandong, People’s Republic of China (D.Z.); School of Medicine, Duke University, Durham, North Carolina (A.L., J.-P.S.) Received December 10, 2016; accepted February 23, 2017 molpharm.aspetjournals.org ABSTRACT Seven transmembrane G protein-coupled receptors (GPCRs) distinct functional outcomes. Our recent work using unnatural are often phosphorylated at the C terminus and on intracellular amino acid incorporation and fluorine-19 nuclear magnetic loops in response to various extracellular stimuli. Phosphoryla- resonance (19F-NMR) spectroscopy led to the flute model, tion of GPCRs by GPCR kinases and certain other kinases which provides preliminary insight into the receptor phospho- can promote the recruitment of arrestin molecules. The arrestins coding mechanism, by which receptor phosphorylation pat- critically regulate GPCR functions not only by mediating terns are recognized by an array of phosphate-binding pockets receptor desensitization and internalization, but also by redi- on arrestin and are translated into distinct conformations.

  • Generate Metabolic Map Poster

    Authors: Zheng Zhao, Delft University of Technology Marcel A. van den Broek, Delft University of Technology S. Aljoscha Wahl, Delft University of Technology Wilbert H. Heijne, DSM Biotechnology Center Roel A. Bovenberg, DSM Biotechnology Center Joseph J. Heijnen, Delft University of Technology An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Marco A. van den Berg, DSM Biotechnology Center Peter J.T. Verheijen, Delft University of Technology Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. PchrCyc: Penicillium rubens Wisconsin 54-1255 Cellular Overview Connections between pathways are omitted for legibility. Liang Wu, DSM Biotechnology Center Walter M. van Gulik, Delft University of Technology L-quinate phosphate a sugar a sugar a sugar a sugar multidrug multidrug a dicarboxylate phosphate a proteinogenic 2+ 2+ + met met nicotinate Mg Mg a cation a cation K + L-fucose L-fucose L-quinate L-quinate L-quinate ammonium UDP ammonium ammonium H O pro met amino acid a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar K oxaloacetate L-carnitine L-carnitine L-carnitine 2 phosphate quinic acid brain-specific hypothetical hypothetical hypothetical hypothetical
  • Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells'

    Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells'

    [CANCER RESEARCH 33, 94-103, January 1973] Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells' E. M. Scholar and P. Calabresi Department of Medicine, The Roger Williams General Hospital, Providence, Rhode Island 02908, and The Division of Biological and Medical Sciences,Brown University, Providence,Rhode Island 02912 SUMMARY monocytes, and lymphocytes, it is difficult to know in what particular fraction an enzyme activity is present. It would therefore be advantageous to separate out the different Extracts of lymphocytes from normal donors and from components of the leukocyte fraction before investigating patients with chronic lymphocytic leukemia (CLL) and acute their enzymatic activities. All previously reported work on the lymphoblastic leukemia were examined for a variety of enzymes of purine nucleotide metabolism was done at best in enzymes with activity for purine nucleotide biosynthesis, the whole white blood cell fraction. Those enzymes found to interconversion, and catabolism as well as for a selected be present included adenine and guanine phosphoribosyltrans number of enzymes involved in pyrimidine nucleotide ferase (2, 32), PNPase2 (7), deoxyadenosine deaminase (7), metabolism. Lymphocytes from all three donor types (normal, ATPase (4), and inosine kinase (21). CLL, acute lymphocytic leukemia) contained the following A detailed knowledge of the enzymatic pathways of purine enzymatic activities: adenine and guanine phosphoribosyl and pyrimidine nucleotide metabolism in normal lymphocytes transferase , adenosine kinase , nucieoside diphosphate kinase, and leukemia cells is important in elucidating any biochemical adenylate kinase, guanylate kinase, cytidylate kinase, uridylate differences that may exist. Such differences may be exploited kinase, adenosine deaminase, purine nucleoside phosphorylase, in chemotherapy and in gaining and understanding of the and adenylate deaminase (with ATP).