US 2005.0014697A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0014697 A1 Stamler et al. (43) Pub. Date: Jan. 20, 2005 (54) COMPOSITIONS AND METHODS FOR on Feb. 18, 2004. Provisional application No. 60/550, MODULATING S-NITROSOGLUTATHONE 833, filed on Mar. 4, 2004. REDUCTASE Publication Classification (76) Inventors: Jonathan S. Stamler, Chapel Hill, NC (US); Limin Liu, Durham, NC (US) (51) Int. Cl." ........................ A61K 38/18; A61K 31/198 (52) U.S. Cl. .............................................. 514/18: 514/565 Correspondence Address: MINTZ, LEVIN COHN FERRIS GLOWSKY & POPEO (57) ABSTRACT 666 THIRDAVENUE Disclosed herein are methods and compositions for modu NEW YORK, NY 10017 (US) lating the levels and/or activity of S-nitroSoglutathione (21) Appl. No.: 10/861,304 reductase (GSNOR) in vivo or in vitro. Specifically dis closed are GSNOR deletion constructs, host cells and non (22) Filed: Jun. 4, 2004 human mammals comprising GSNOR deletions, and meth ods of screening employing GSNOR deletion mutants. Also Related U.S. Application Data Specifically disclosed are reagents and procedures for mea Suring, monitoring, or altering GSNOR levels or activity (as (60) Provisional application No. 60/476,055, filed on Jun. well as nitric oxide and S-nitroSothiol levels) in connection 4, 2003. Provisional application No. 60/545,965, filed with various medical conditions. Patent Application Publication Jan. 20, 2005 Sheet 1 of 27 US 2005/0014697 A1 A Wild-ild-type allelee Ab-SS) s Targetingtly vector- PGKn xs / B / PGK h N 2. 4 is 9 2. so Fse %2 5 E.a so g 2 4. is S is 40an s. 2 2 35 r 2.% wer they Heart Lung Spleen Thymus y: atweaning F.G. 1 Patent Application Publication Jan. 20, 2005 Sheet 2 of 27 US 2005/0014697 A1 A B C 80 92 10 360 a Se 2. 2 6 X 4 E 40 3 S O 9 E 5 20 g 9 2. is t WT KO WT KO SNO FeNO D Redblood Cell Pierra GSeoGye-ND Remi-Abuadr-SNO Largsgata Wasecarterestigato Wasodilation during ypgdstagfig demand No deficiency FIG 2 Patent Application Publication Jan. 20, 2005 Sheet 3 of 27 US 2005/0014697 A1 A 1 B 10 9 or 7 7 w ad o 6 2 2 e C a O 1 2 3 4. 5 6 O 1. 2 3 4. 5 6 Days after LPS Days after LPS C 1 D 1 9 s 2 7 7 3 3 S. O 1 2 3 4. S 6 Days after LPS Days after LPS E 2 2 t O 2 3 4 5 & 7 Days after CLP FIG. 3 Patent Application Publication Jan. 20, 2005 Sheet 4 of 27 US 2005/0014697 A1 A 40 ++ total B 1200 -- total 000 30 S -- low-mass 'o 2 s 800 asE 2 retS. 600 sE s 400 2 200 O O PBS LPS24 LPS48 PBS LPS24h LPS48h C 8 D 6 E 4. s g3 A 5 E3 22 re. 2 9. O D PBS LPS24h LPS48h LPS 24h LPS48h 0 FIG. 4 Patent Application Publication Jan. 20, 2005 Sheet 5 of 27 US 2005/0014697 A1 - PBS LPS24h LPS48h PBS LPS24h LPS48h PES is lish PBS LPS24h LPS48h ps ships G 140 H2 2 s 100 S. O a tha O s - - 2. t 2 3 4 5) 8) to PBS PS24h LPS48h SNO (pmol mg) F.G. 5 Patent Application Publication Jan. 20, 2005 Sheet 6 of 27 US 2005/0014697 A1 FIG 6 Patent Application Publication Jan. 20, 2005 Sheet 7 of 27 US 2005/0014697 A1 A 1200 B 40 C D 100 OO) 35 90 30 8 as 800 'o pa s E 25 s to 600 as 20 S. wn E 5 so O 3, 15 8 2. O 10 50 20 e 5 4. O 1 2 3 4 S 6 LPS 24h LPS 48h LPS48h Days after LPS FIG. 7 Patent Application Publication Jan. 20, 2005 Sheet 8 of 27 US 2005/0014697 A1 5 to H H at at & 3s s ck O C 3. S bf) b) w b e b vu h O (oosiu? Huo) dugoseqiad) in O Patent Application Publication Jan. 20, 2005 Sheet 9 of 27 US 2005/0014697 A1 a S5 S 33> 2. a 3O s- O2 : : < > O O N4 t > O Od s CD L M A A. O NZ V A A. C O C O O C O C C O lf C b) O lf) N N y yu Patent Application Publication Jan. 20, 2005 Sheet 10 of 27 US 2005/0014697 A1 9. Ek s Sa 9 O O CD I O O N4 O S. S. 92 S C2 e e g e. Oe Ce Oi sCo seO OOO Oe Cot Oe O (I u?irl) el-TI ITV Patent Application Publication Jan. 20, 2005 Sheet 11 of 27 US 2005/0014697 A1 cysNO (M) O 500 50 5 a GRK2 - - - - - - - - - - - - - ISO - - - - - - - - - - - - B-AR cysNO (uM) GRK2 32P-rhodopsin FIG. 11B 5000 s 20000 L CPM 9.4000 a. 3000 s 10000 2000 3.t 5 1000 8 E O saxx, s O S s s . c) s e a ge Yala o 2-- o 39 FIG. 12A FIG. 12B 7 as s S s 80 92 x s 2 - SO s 34 g 40 33s a g 2 X cap g 20 2 1 s O : O PBS ISO GSNO ISO+GSNO PBS ISO GSNO SO-GSNO PBS ISO GSNO GSNO+SO F.G. 13A F.G. 13B FIG. 13C Patent Application Publication Jan. 20, 2005 Sheet 13 of 27 US 2005/0014697 A1 LOCUS NM 000671 2496 bp mRNA linear DEFINITION Homo sapi ens alcohol dehydrogenase 5 (class III), chi polypeptide (ADH5), mRNA. ACCESSION NM 000671 COMPLETENESS: full length. FEATURES Location/Qualifiers Solice . 2496 A chromosome="4" 1. 2496 /gene="ADH5" Anote= "synonyms: FDH, ADHX, ADH-3" misc feature 1. /gene="ADH5" Wnote= "alternative start site; active site" variation complement (4) /replace="T" /replace="C" /db Xref="dbSNP : 1154400" misc feature 83 /gene="ADH5" /note= "alternative start site; active site" Inisc feature 85 /gene="ADH5" /note= "alternative start site; active site" CDS 63 . .287 /gene="ADH5" /EC number="l. 1. l. 1" /EC number="1.2.1.1" /note= "Alcohol dehydrogenase (class III), chi polypeptide; glutathione-dependent formaldehyde dehydrogenase; go function : formaldehyde dehydrogenase (glutathione) activity (goid 0.004327) evidence TAS) (pinid 84.60164); go function: fatty acid binding Igoid OOO5504) (evidence TAS) pmid 84.60164); go function: electron transporter activity (goid 0005489) evidence TAS) pmid 846O164); go function: alcohol dehydrogenase activity, zinc-dependent (goid OOO4024) evidence IEA) ; go function : zinc ion binding (goid OOO827O) evidence IEA) ; go function: oxidoreductase activity goid OO16491) evidence IEA); go function: alcohol dehydrogenase activity, metal ion-independent goid 0004023) evidence IEA) ; go function: alcohol dehydrogenase activity, iron-dependent goid 0004 025) evidence IEA) ; go process: ethanol oxidation (goid 0006069) (evidence TAS) (pmid 84.60164); go process: alcohol metabolism (goid 0006066 (evidence NR) FIG. 15A Patent Application Publication Jan. 20, 2005 Sheet 14 of 27 US 2005/0014697 A1 /codon start=1 /product = "class III alcohol dehydrogenase 5 chi subunit" /protein id="NP OOO662. 2" /db xref="GI: 11496891" /db xref="GenerD: 128." /db Xref="Locus ID: 128." /db xref="MIM:103710" /translation= "MANEVIKCKAAVAWEAGKPLSIEEIEVAPPKAHEVRIKIIATAV CHTDAYTLSGADPEGCFPVILGHEGAGIVESVGEGVTKLKAGDTVIPLYIPOCGECKF CLNPKTNLCOKIRVTOGKGLMPDGTSRFTCKGKTILHYMGTSTFSEYTVVADISVAKI DPLAPLYKVCILGCGSTGYGAAVNTAKLEPGSWCAVFGLGGWGLAVIMGCKVAGASR IIGVDINKDKFARAKEFGATECINPQDSKPIOEVLIEMTDGGVDYSFECIGNVKVMR AALEACHKGWGVSVVVGVAASGEEIATRPFQLVTGRTWKGTAFGGWKSVESVPKLVSE YMSKKIKVDEFWTHNLSFDEINKAFELMHSGKSIRTVVK I misc feature 63. 1284 /gene="ADH5" /note="KOG0022; Region: Alcohol dehydrogenase, class III Secondary metabolites biosynthesis, transport and catabolism.)" /db xref="CDD: 1782.0" misc feature 559 .56 /gene="ADH5" /note= "important for formaldehyde dehydrogenase activity and activation by fatty acids; binding site" variation complement (359) /replace="T" /replace="C" /db xref="dbSNP: 94.45600." variation complement (475) /replace="T" /replace= "A" /db xref="dbSNP: 94.45599." variation 66. /gene="ADH5" /note= "WARNING: map location ambiguous" /replace="T" /replace="G" /db xref="dbSNP:1050636." variation 900 /gene="ADH5" /replace="T" /replace= "A" /db xref="dbSNP:1050637" variation complement (932) /replace="G" /replace= "A" /db xref="dbSNP:43927 09" variation 153 /gene="ADH5" /replace="T" /replace="C" /db Xref="dbSNP:2229168" FIG. 15B Patent Application Publication Jan. 20, 2005 Sheet 15 of 27 US 2005/0014697 A1 variation 158 /gene="ADH5" /note="WARNING: map location ambiguous" /replace="G" /replace="C" /db xref="dbSNP: 1803039." variation complement (1400) /note= "WARNING: map location ambiguous" /replace="T" /replace="C" /db xref="dbSNP:30883 06" variation 1478 /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP:12697" variation 504 /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP: 12106" polyA signal 527 . 1532 /gene="ADH5" /note= "alternative polyA signal." /evidence=experimental variation 1703 /gene="ADH5" /replace="G" /replace="A" /db xref="dbSNP:1803037" variation 779 /gene="ADH5" /replace="T" /replace="G" /db xref="dbSNP:13832" variation complement (1859) /replace="T" /replace="C" /db xref="dbSNP: 682 7292." variation 2OOO /gene="ADH5" /note="WARNING: map location ambiguous" /replace="T" /replace="C" /db xref="dbSNP:1061187." variation 217. /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP:1803.038" polyA signal 2376. .238 /gene="ADH5" /note= "alternative polyA signal" /evidence=experimental polyA site 2443 /gene="ADH5" /note= "alternative polyA site" /evidence=experimental FIG. 15C Patent Application Publication Jan. 20, 2005 Sheet 16 of 27 US 2005/0014697 A1 ORIGIN agC9gCaC9C accacagctic gag caccgCC Cttccggttg gagcCattgc aag.ccc.cccc 6. CacgCCCC gC ccccct cqct aggcgct.cgc cacgc.cCatg cctc.cgtogc tgcgc.ggCCC 121 accCCggatg totagoccCCC gCGCCdacca gaatc.cgtga acatggcgaa cgaggittatc 181 aagttgcaagg CtgcagttgC ttgggaggCt ggaaag.cctic totccataga ggagatagag 241 gtggcacccc caaaggct Ca tgaagttcga atcaagatca ttgccactgc ggtttgccac 301 accgatgcct at accCtgag tggagctgat Cctgagggitt gtttitccagt gatcttggga 361 Catgaaggtg Ctggaattgt ggaaagttgtt g9tgagggag ttactaagct galagg.cgggt 42 gacactgtca tCccact teta catcccacag tgttggaga at gcaaattittg totaaat CCt 48 aaala Ctala CC tttgccagaa gatalaga.gtc act Calaggga aaggattaat gccagatggit 54.