Compositions and methods for treating aberrant nitrosylation

Abstract

A method of treating a disease or disorder characterized by aberrant or dysregulated nitrosylation in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates SNO-CoA-associated Nitrosyltransferase (SCAN) mediated S-nitrosylation. In some embodiments, it includes an agent that modulates SNO-CoA- associated Nitrosyltransferase (SCAN)Zbiliverdin IXB reductase B (SCAN7BLVRB) mediated S-nitrosylation and its use in methods of treating diseases and disorders characterized by aberrant or dysregulated nitrosylation in a subject in need thereof.

Description

COMPOSITIONS AND METHODS FOR TREATING ABERRANT NITROSYLATION RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No.63 / 605,838, filed December 4, 2023, the subject matter of which is incorporated herein by reference in its entirety. GOVERNMENT FUNDING

[0002] This invention was made with government support under DK119506 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 3, 2024, is named CWR-032403WO ORD.st.26 and is 7,260 bytes in size. BACKGROUND

[0004] S-nitrosylation, the posttranslational modification (PTM) of proteins by NO to form S-nitrosothiols (SNOs), shares features with phosphorylation. Physiologically, S- nitrosylation provides ubiquitous control over cellular function, while in excess, it plays causal roles in disease, including Alzheimer’s, cancer, muscular dystrophy, heart failure and type 2 diabetes. But whereas aberrant phosphorylation of proteins is enzymatically-driven, convention holds that hypernitrosylation is mediated chemically by high amounts of NO and of low molecular weight (LMW) SNOs acting as NO-donors to indiscriminately modify proteins.

[0005] LMW SNOs, formed endogenously by oxidative coupling of NO to the thiols of molecules including cysteine, glutathione and CoA, stabilize NO bioactivity and expand NO’s repertoire. At low levels, each SNO is identified with distinct cellular functions through S-nitrosylation of specific protein substrates, and cellular SNO-protein levels are regulated by denitrosylases dedicated to individual LMW-SNOs. This is well exemplified in the newly discovered metabolic functions of SNO-CoA, whose levels are strictly regulated by the cognate enzyme SNO-CoA reductase (SCoR). Deletion of SCoRs leads to elevated SNO- CoA, thereby increasing S-nitrosylation of specific proteins. However, how SNO-CoAselectively modifies specific substrates is unknown. An additional major quandary in the field is the fact that the amount of SNO-CoA needed to S-nitrosylate substrates in vitro is orders of magnitude higher than is ever present in vivo, hinting that S-nitrosylation by SNO- CoA might utilize enzymatic mechanisms. SUMMARY

[0006] Embodiments described herein relate to an agent that modulates SNO-CoA- associated Nitrosyltransferase (SCAN) / biliverdin IXβ reductase B (SCAN / BLVRB) mediated S-nitrosylation and its use in methods of treating diseases and disorders characterized by aberrant or dysregulated nitrosylation in a subject in need thereof. In some embodiments, the methods include administering to the subject a therapeutically effective amount of an agent that modulates SCAN / BLVRB mediated S-nitrosylation.

[0007] In some embodiments, the disease or disorder treated is characterized by hypernitrosylation and the agent is an inhibitor of SCAN / BLVRB mediated S-nitrosylation. The inhibitor of SCAN / BLVRB mediated S-nitrosylation can inhibit SCAN / BLVRB S- nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression. The inhibitor of SCAN / BLVRB mediated S-nitrosylation can be provided in a pharmaceutical composition with at least one pharmaceutically acceptable carrier.

[0008] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity can include a SCAN / BLVRB inhibitor selected from phloxine B, erythrosin B, NSC130813, NSC12516, PH001924, Lumichrome, PH006888, ZINC4366439 / NSC 12516, xanthene, proflavine, alizarin red S, NSC ID 371876, NSC ID 179187, NSC ID 53396, NSC ID 10936, NSC ID 169534, NSC ID 117269, NSC ID 143491, NSC ID 305821, NSC ID 130813, ZINC ID ZINC0977089, ZINC ID ZINC27528243, ZINC ID ZINC09330686, ZINC ID ZINC71767103, ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC ID ZINC21093196, ZINC ID ZINC71767097, asunaprevir (BMS-650032), micafungin, tamibarotene, TSU-68 (SU6668, Orantinib), sulfasalazine, febuxostat, crenolanib (CP- 868596), olsalazine, PTC124 (ataluren), deferasirox, flunixin in combination with meglumin, azelastine, benzbromarone, triclabendazole, nifedipine, nisoldipine, zafirlukast, pyrantel in combination with pamoate, candesartan cilexetil, and azilsartan medoxomil, prodrugs thereof, metabolites thereof, and / or pharmaceutically acceptable salts thereof, or any combination thereof.

[0009] In other embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity or expression can include a small interfering RNA (siRNA) molecule or an antisense oligonucleotide specific to a region in the mRNA of BLVRB gene. In certain embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity or expression is a microRNA (miRNA), preferably, miR-127-5p.

[0010] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation or S- nitrosylation of SCAN / BLVRB can include an NOS inhibitor, such as a nNOS inhibitor, iNOS inhibitor, and / or eNOS inhibitor. In some embodiments, the NOS inhibitor can be selected from L-NMMA (Nγ-Monomethyl-L-arginine acetate), L-NIO dihydrochloride, ZZL7 (N-Acetyl-L-alanyl-L-valine methyl ester), Diphenyleneiodonium Chloride, Nω-Nitro-L- arginine Methyl Ester, Hydrochloride, (L-NAME), a caveolin-1(Cav-1) peptide, or combinations thereof. In certain embodiments, the NOS inhibitor includes L-NMMA.

[0011] In some embodiments, the disease or disorder treated is associated with insulin resistance and the inhibitor of SCAN / BLVRB mediated S-nitrosylation is administered at an amount effective to inhibit hyper S-nitrosylation of INSRβ / IRS1.

[0012] In some embodiments, the disease or disorder characterized by hyper-S- nitrosylation can include at least one of diabetes, heart failure, atherosclerosis, atherosclerosis-induced ischemic stroke, ventricular arrhythmia in individuals with Duchenne muscular dystrophy, sickle cell anemia, neurodegenerative diseases and disorders, cancers, infections, inflammatory disorders, and shock states.

[0013] In some embodiments, the disease or disorder treated is diabetes and the inhibitor of SCAN / BLVRB mediated S-nitrosylation can be administered at an amount effective to inhibit hyper S-nitrosylation of INSRβ / IRS1.

[0014] In some embodiments, the heart failure treated is selected from post-myocardial infarction heart failure and heart failure with preserved ejection fraction (HFpEF).

[0015] In some embodiments, the neurodegenerative disease treated is selected from amyloid lateral sclerosis (ALS), Alzheimer's, Parkinson’s, or Huntington’s disease.

[0016] In some embodiments, the cancer can be selected from hepatocellular carcinoma (HCC), cholangiocarcinoma, prostate cancer, or acute lymphoblastic leukemia.

[0017] In some embodiments, where the disease or disorder is cancer, the method can further include co-administering one or more additional cancer therapeutic agents, such as a chemotherapeutic, with the inhibitor of SCAN / BLVRB mediated S-nitrosylation.

[0018] In other embodiments, the disease or disorder can be characterized by hyponitrosylation and the agent is a promoter of SCAN / BLVRB mediated S-nitrosylation. The promoter of SCAN / BLVRB mediated S-nitrosylation can promote SCAN / BLVRB S- nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

[0019] In some embodiments, the promoter of SCAN / BLVRB mediated S-nitrosylation is an expression vector or DNA construct that promotes expression or expresses SCAN / BLVRB in a cell of the subject.

[0020] Other embodiments, described herein relate to a method of modulating S- nitrosylation of INSRβ / IRS1 in a subject in need thereof. The method includes administering to the subject an amount of an agent that modulates SCAN / BLVRB mediated S-nitrosylation.

[0021] In some embodiments, the INSRβ / IRS1 is hypernitrosylated and the agent is an inhibitor of SCAN / BLVRB mediated S-nitrosylation that is administered at amount effective to inhibit hypernitrosylation. The inhibitor of SCAN / BLVRB mediated S-nitrosylation can inhibit SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

[0022] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity is a SCAN / BLVRB inhibitor selected from phloxine B, erythrosin B, NSC130813, NSC12516, PH001924, Lumichrome, PH006888, ZINC4366439 / NSC 12516, xanthene, proflavine, alizarin red S, NSC ID 371876, NSC ID 179187, NSC ID 53396, NSC ID 10936, NSC ID 169534, NSC ID 117269, NSC ID 143491, NSC ID 305821, NSC ID 130813, ZINC ID ZINC0977089, ZINC ID ZINC27528243, ZINC ID ZINC09330686, ZINC ID ZINC71767103, ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC ID ZINC21093196, ZINC ID ZINC71767097, asunaprevir (BMS-650032), micafungin, tamibarotene, TSU-68 (SU6668, Orantinib), sulfasalazine, febuxostat, crenolanib (CP- 868596), olsalazine, PTC124 (ataluren), deferasirox, flunixin in combination with meglumin, azelastine, benzbromarone, triclabendazole, nifedipine, nisoldipine, zafirlukast, pyrantel in combination with pamoate, candesartan cilexetil, and azilsartan medoxomil, prodrugs thereof, metabolites thereof, and / or pharmaceutically acceptable salts thereof, or any combination thereof.

[0023] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity or expression is a small interfering RNA (siRNA) molecule or an antisense oligonucleotide specific to a region in the mRNA of BLVRB gene.

[0024] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation activity or expression is a microRNA (miRNA), preferably, miR-127-5p.

[0025] In other embodiments, an inhibitor of SCAN / BLVRB S-nitrosylation or S- nitrosylation of SCAN / BLVRB can include a nitric oxide synthase (NOS) inhibitor, such as a nNOS inhibitor, iNOS inhibitor, and eNOS inhibitor.

[0026] In some embodiments, the NOS inhibitor for use in a method or compositions described herein can be selected from ʟ-NMMA (Nγ-Monomethyl-L-arginine acetate), ʟ- NAME (Nω-Nitro-L-arginine Methyl Ester, Hydrochloride), ronopterin (VAS203), 2- Iminobiotin, MTR104, ʟ-NG-benzylarginine, ʟ-NG-aminoarginine, pimagedine (aminoguanidine), OsteoDex, iminoethylornithine, or combinations thereof.

[0027] In certain embodiments, the NOS inhibitor includes ʟ-NMMA.

[0028] In some embodiments, the subject in need of S-nitrosylation of INSRβ / IRS1 modulation has an elevated or increased body mass index. For example, the subject can be obese. In some embodiments, the subject has type 2 diabetes.

[0029] In some embodiments, the INSRβ / IRS1 is hyponitrosylated and the agent is a promoter of SCAN / BLVRB mediated S-nitrosylation that is administered at an amount effective to inhibit hyponitrosylation or promote SCAN / BLVRB mediated S-nitrosylation.

[0030] In some embodiments, the promoter of SCAN / BLVRB mediated S-nitrosylation can promote SCAN S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB mediated S- nitrosylation, or SCAN / BLVRB mediated S-nitrosylation expression.

[0031] In some embodiments, the promoter of SCAN / BLVRB mediated S-nitrosylation mediated S-nitrosylation is an expression vector or DNA construct that promotes expression or expresses SCAN / BLVRB mediated S-nitrosylation in a cell of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figs.1(A-T) illustrate identification and characterization of a SNO-CoA- assisted nitrosyltransferase (SCAN) Purification of SNO-CoA-binding proteins from bovine liver using SNO-CoA-resin. SNO-thiopropyl- and CoA-resin were used as controls. Eight protein bands identified (including BLVRB) on silver-stained SDS-PAGE gel are indicated by gene name and arrows; n = 3. Interaction of SNO-CoA resin with endogenous BLVRB in HEK cell lysate (upper) or with recombinant BLVRB (lower), detected by western blot. Other resins (amylose resin, thiopropyl Sepharose 6B [Thio], SNO-thiopropyl Sepharose 6B[SNO-thio], glutathione [GSH]-agarose, glutathione-SNO [GSNO]-Sepharose 4B, CoA- agarose, acetyl-CoA-agarose and palmitoyl-CoA-agarose) were used as controls; n = 2. Proteins found in both the BLVRB-dependent nitrosoproteome and the BLVRB cytosolic interactome. Endogenous S-nitrosylation of HO2(SNO-HO2) in untargeted HEK cells (HEK- WT) and BLVRB-knockout HEK cells (HEK-BLVRB- / -), each expressing eNOS. Results are from two independent cell lines. In vitro S-nitrosylation of HO2 by BLVRB in the presence of increasing amounts of SNO-CoA. GST (glutathione S-transferase) is used as control. (F and G) Quantification of SNO-HO2 (from D, n = 4) and (from E, n = 3). SNO-HO2level is normalized to expression of HO2 (input). (H) Enzymatic activity of BLVRB to S-nitrosylate HO2; n = 3. (I and J) Competition for SCAN binding to SNO-CoA resin by a fixed dose (50 mM) of SNO-cysteamine, NADH, or NADPH (I) and by varying amounts of NADPH (J); n = 2. Reduced binding of mutant SCAN (QTG / NAA) to SNO-CoA resin; n = 2. Endogenous SNO-HO2 in HEK-WT cells (WT), HEK-SCAN- / -cells, wild-type SCAN-re-expressing HEK cells (WT-SCAN), and mutant SCAN-re-expressing HEK cells (QTG-NAA), respectively, each expressing eNOS. Quantification of SNO-HO2 levels in (L); n = 4. S-nitrosylation of HO2 by recombinant WT-SCAN and SCAN-QTG / NAA protein in vitro with increasing SNO-CoA. Quantification of SNO-HO2 in (N); n = 5. Identification of SNO sites within SCAN. Amount of SNO-SCAN in HEK cell lines overexpressing wild-type SCAN or three mutated SCAN forms (C109R, C188R, or C109 / 188R); n = 3. Amount of SNO-HO2in HEK-WT versus four HEK-SCAN- / -cell lines overexpressing empty vector: wild-type SCAN (SCAN-WT), SCAN-R35G, SCAN-QTG / NAA, and SCAN-C109 / 188R; n = 3. S- nitrosylation of HO2 by recombinant WT-SCAN and SCAN-C109 / 188R protein in vitro with increasing SNO-CoA. Quantification of SNO-HO2 in (R); n R 3. Working model of ‘‘ping- pong’’ mechanism utilized by SCAN. The samples of WT-SCAN in (O) and (S) are the same. All results are presented as mean ± SD. Two-tailed Student’s t test was used to detect significance in (F). One-way ANOVA with Tukey post hoc was used to detect significance in (G), (M), (O), (P) and (S). *p < 0.05; **p < 0.01; and ****p < 0.0001. See also Figs.7 and 8 and Tables 1 and 2.

[0033] Figs.2(A-L) illustrate SCAN mediates S-nitrosylation of INSRb / IRS1 and insulin resistance on high-fat diet (A) expression of SCAN in the indicated organs from wild- type mice (SCAN+ / +) and SCAN-knockout mice (SCAN- / -), vs. p97 as loading control; n = 2. (B) Expression of SCAN and iNOS in hindlimb skeletal muscle (lateral gastrocnemius) fromWT mice fed chow vs. high-fat diet (HFD) for 16 weeks (upper) and from 12-week-old WT vs. mutant obese mice (ob / ob; lower). Data are shown for two independent mice, and actin is loading control; n = 3 mice. (C and D) Blood glucose (C) and plasma insulin levels (D) in chow-fed vs.16-week-HFD-fed SCAN+ / +and SCAN- / -male mice; n = 15–27 overnight- fasted mice in and n = 7–105-h-fasted mice in (D), per group. (E) Insulin tolerance test. Blood glucose in 5-h-fasted 16-week-HFD-fed male SCAN+ / +and SCAN- / -mice, immediately before and at the indicated time points after injection of human insulin (1 U / kg body weight, intraperitoneal [i.p.]); n = 22–27 mice per group. (F) Glucose tolerance test. Blood glucose levels in overnight-fasted 16-week-HFD-fed male SCAN+ / +and SCAN- / -mice, immediately before and at the indicated time points after injection of glucose (2 g / kg body weight, i.p.); n = 16–23 mice per group. (G) Glucose uptake in ex vivo soleus muscles from HFD-fed SCAN+ / +and SCAN- / -mice, measured with the non-metabolizable glucose analog 2-deoxyglucose, in the absence or presence of insulin (12 nM); n = 10 (5 female and 5 male mice). (H) S-nitrosylation of INSRb (INSR) and IRS1 in skeletal muscle (lateral gastrocnemius) of 5-h-fasted chow-fed or HFD-fed SCAN+ / +and SCAN- / -mice. (I) Quantification of (H); n = 4 mice per group. (J) SCAN increases SNO-CoA-induced in vitro S-nitrosylation of IRS1 (purified from IRS1-overexpressing HEK cells) compared to GST control (lower). (K) Quantification of (J); n = 3. (L) Effect of SCAN on insulin signaling activity markers. Phosphorylation of INSR (pTyr1162), IRS1 (pTyr608), AKT (pSer473), and AS160 (pThr642) in 5-h-fasted SCAN+ / +and SCAN- / -mice, 30 min and 60 min after insulin administration (1 U / kg body weight, i.p.); ‘‘-’’ indicates no insulin administration. Data are from two independent mice at each time point, and spliced blots for IRS1 / p-IRS1 are indicated. All results are presented as mean ± SD. One-way ANOVA with Tukey’s post hoc test was used to detect significance in (C), (D), (G), (I), and (K). ITT and GTT in (E) and (F) were analyzed by two-way (time 3 treatment) repeated measures analysis of variance followed by Sidak’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. See also Figs.9 and 10.

[0034] Figs.3(A-J) illustrate insulin induces S-nitrosylation of INSRb / IRS1 to inhibit signal transduction (A) SNO-INSRb and SNO-IRS1 in overnight-starved wild-type L6 cells (L6-WT) and SCAN-knockout L6 cells (L6-SCAN- / -) at 1, 30, 60, 120, and 240 min after removal of insulin following 10-min insulin treatment (100 nM). (B) Quantification of SNO- IRS1 in (A), compared to total IRS1; n = 3. (C) Quantification of SNO-INSRb in (A),compared to total INSRb; n = 3. (D) SNO-INSRb and SNO-IRS1 in skeletal muscle (lateral gastrocnemius) from 5-h-fasted SCAN+ / +and SCAN- / -mice, 60 min after insulin administration (1 U / kg body weight, i.p.) or after no insulin administration (-). (E) Quantification of (D); n = 3 mice per group. (F) Phosphorylation of INSRb (pTyr1162), IRS1 (pTyr608), AKT (pSer473), and AS160 (pThr642) in overnight-starved L6-WT and L6- SCAN- / -cells at 1, 30, 60, 120, and 240 min after removal of insulin, following 10-min insulin treatment (100 nM); n = 4, quantitation shown in Figs.11I–11L. (G and H) Normal (G) and severe (H) insulin tolerance test. Blood glucose level in 5-h-fasted chow-fed SCAN+ / +and SCAN- / -male mice, immediately before and at the indicated time points after injection of human insulin (1 U / kg body weight in G; 2.5 U / kg body weight in H, i.p.); n = 10 mice in (G) and (H). (I) SNO-INSRb in skeletal muscle from two representative mice during severe insulin challenge as in (H). (J) Quantification of (I) (n = 4 mice per condition). All results are presented as mean ± SD. ANOVA with Tukey’s post hoc test was used to detect significance in (E) and (J). (B), (C), (G), and (H) were analyzed by two- way (time 3 treatment) repeated measures analysis of variance followed by Sidak’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. See also Fig.11.

[0035] Figs.4(A-K) illustrate insulin-stimulated S-nitrosylation of INSRb / IRS1 is coupled to NOS activity (A) Phosphorylation of eNOS (pS1177) in skeletal muscle from 5-h- fasted C57BL / 6 mice, 10 min after insulin administration (1 U / kg body weight, i.p.). (B) Quantification of (A); n = 5 mice. (C) Phosphorylation of nNOS (pS1412) in skeletal muscle from 5-h-fasted C57BL / 6 mice, 10 min after insulin administration (1 U / kg body weight, i.p.). (D) Quantification of (C); n = 4 mice. (E) Phosphorylation of eNOS (pS1177) in overnight serum-starved L6 cells at the indicated time after removal of insulin, following 10-min insulin treatment (100 nM). (F) Quantification of (E); n = 3. (G) Amounts of SNO- INSRb, SNO-IRS1, and SNO-SCAN in overnight-starved PBS-treated (control) and L- NMMA-treated (100 mM) L6 cells at 1, 30, 60, 120, and 240 min after removal of insulin following 10-min insulin treatment (100 nM). (H–J) Quantification of SNO-IRS1 (H), SNO- INSRb (I), and SNO-SCAN (J) from (G); wo (without); n = 3. (K) Phosphorylation of AKT (pSer473) and AS160 (pThr642) in PBS-treated (control) and L-NMMA-treated (100 mM) L6 cells at 1, 30, 60, 120, and 240 min after removal of insulin following 10-min insulin treatment (100 nM); n = 3, quantitation shown in Figs.11P and 11Q. Two-tailed Student’s t test was used to detect significance in (B) and (D). One-way ANOVA with Tukey’s post hoctest was used to detect significance in (F). (H)–(J) were analyzed by two-way (time 3 treatment) repeated measures analysis of variance followed by Sidak’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

[0036] Figs.5(A-J) illustrate S-nitrosylation of INSRb by SNO-CoA-dependent SCAN activity regulates insulin signaling (A) SNO-INSRb and SNO-IRS1 in L6-SCAN-WT, L6- SCAN-QTG / NAA, and L6-SCAN-C109 / 188R cell lines. (B) Quantification of SNO-INSRb and SNO-IRS1 in (A); n = 3. (C) Phosphorylation of IRS1 (pTyr608) and AKT (pSer473) in overnight serum-starved L6-SCAN-WT, L6-SCAN-QTG / NAA, and L6-SCAN-C109 / 188R cell lines, 30 min after a 10-min insulin treatment (100 nM). (D) Quantification of phosphorylation level of IRS1 and AKT in (C); n = 3. (E) Four peptides containing single candidate SNO sites (cysteine residues, red) within INSR identified by SNO-RAC-coupled mass spectroscopy. (F) Identification of primary SNO site within INSRb. SNO-INSRb with the indicated mutations of candidate SNO sites expressed in HEK cells. (G) Quantification of SNO-INSRb in (F); n = 3. (H) Phosphorylation of AKT (pSer473) and AS160 (pThr642) in overnight serum-starved INSR-WT and INSR-C1083A expressing L6 cells at 1, 30, 60, 120, and 240 min after removal of insulin, following a 10-min insulin treatment (100 nM). (I and J) Quantification of phosphorylation of AKT (pSer473) (I) and AS160 (pThr642) (J) in (H); n = 3. All results are presented as mean ± SD. Two-tailed Student’s t test was used to detect significance in (B) and (D). One-way ANOVA with Tukey’s post hoc test was used to detect significance in (G), (I), and (J). *p < 0.05 and **p < 0.01. See also Fig.12.

[0037] Figs.6(A-I) illustrate S-nitrosylation of INSRb is associated with BMI and SCAN expression in human adipose tissue and skeletal muscle. (A) Expression of SCAN in 14 human subcutaneous adipose samples (Hsad#) with indicated BMI, and quantification (lower panel). GAPDH is used as internal loading control. Expression of SCAN was first normalized with expression of internal control GAPDH, and then versus the average expression level of SCAN in the same group of samples. (B and C) Expression of SCAN in 14 human skeletal muscle samples (B, see Fig.12E) and 28 human adipose tissue samples (C, see Fig.12F), plotted against patient BMI. (D) SNO-INSRb in 14 human subcutaneous adipose samples from patients with the indicated different BMI, and quantification (lower). SNO-INSRb was first normalized with input of INSRb, and versus the average SNO-INSRb level in the same group of samples. (E and F) SNO-INSRb in 14 human skeletal muscle samples (E, see Fig.12G) and 26 human adipose tissue samples (F, see Fig.12H), plottedagainst patient BMI. (G) SNO-INSRb in 14 human skeletal muscle samples is plotted against SCAN expression level. (H) SNO-INSRb in 26 human adipose tissue samples is plotted against SCAN expression level. (I) S-nitrosylation-based inhibition of insulin signaling and insulin resistance. Under healthy conditions, insulin induces S-nitrosylation of INSRb / IRS1 via eNOS / nNOS-coupled SCAN / SNO-CoA activity, promoting termination of insulin signaling. In obesity, pro-inflammatory cytokines and free fatty acids (FFAs) induce sustained S-nitrosylation of INSRb / IRS1 through iNOS-coupled SCAN / SNO-CoA activity, leading to insulin resistance. Simple linear regression was performed to identify the relationships among BMI, SCAN expression level, and SNO-INSRb level (using blot data in A and D and in Figs.12E–12H). R-squared (r2) and slope significance p values show the goodness of fit of the regression model. See also Fig.12.

[0038] Figs.7(A-F) illustrate the characterization of BLVRB / SCAN, related to Fig.1 (A). Quantification of binding of BLVRB in HEK lysates to seven different types of resins, from Fig.1B upper; n = 2. (B) Quantification of recombinant BLVRB protein binding to six different types of resins, from Fig.1B lower; n = 2. (C)Recombinant BLVRB does not oxidize NADPH in the presence of SNO-CoA, indicating that NADPH-dependent SNO-CoA reductase activity is absent from BLVRB. SNO-CoA Reductase (SCoR) was used as positive control; n = 5. (D) Recombinant BLVRB does not oxidize NADH in the presence of SNO- CoA, indicating that NADH-dependent SNO-CoA reductase activity is absent from BLVRB; n = 5. (E) Immunostaining of SCAN and cytochrome c (mitochondrial marker) in HEK cells, with DAPI nuclear staining. Overnight serum-starved HEK cells were treated by insulin for 10 min. (F) Expression of SCAN in three cellular fractions. ACADVL is a mitochondrial protein; GAPDH is a cytosolic protein; Histone3 is a nuclear protein.

[0039] Figs.8(A-O) illustrate SCAN / SNO-CoA-dependent S-nitrosylation of HO2, related to Fig.1 (A) SNO-HO2 level in eNOS-overexpressing HEK cells in the absence or presence of NOS inhibitor (L-NMMA, 100 mM). Reaction without ascorbate (-Ascorbate) is used as a negative control; n = 2. (B) SNO-HO2 level in LPS / interferon-induced RAW264.7 cells in the absence or presence of iNOS inhibitor (1400W, 100 mM); n = 2. (C) SNO-HO2 in HEK cell lines overexpressing wild-type HO2 or four candidate SNO site HO2 mutants (C127R, C265R, C282R, or C265 / 282A); n = 3. (D) Interaction between BLVRB and HO2 in HEK cells. Upper gel: IP of endogenous proteins with anti-rabbit BLVRB antibody or non- immune IgG; IB with anti-mouse HO2 antibody; n = 2. Lower gel: interaction betweenexpressed BLVRB and HO2 after treatment with the NO donor DPTA (200 mM for 20 h). Flag-HO2 and Myc-SCAN were co-expressed in HEK cells. IP with anti-rabbit myc antibody or non-immune IgG; probe with anti-mouse Flag antibody; n = 2. (E) Molecular structures of SNO-CoA, SNO-cysteamine, and NADPH. (F) S-nitrosylation of HO2 by recombinant SCAN and 1 mM SNO-CoA in vitro in the presence of increasing NAPDH. (G) S- nitrosylation of HO2 by recombinant SCAN and 1 mM SNO-CoA in vitro in the presence of increasing NADH. (H) Quantification of SNO-HO2 in (F) and (G); n = 2. (I) Generation of SCAN-knockout HEK cell lines (HEK-SCAN- / -) using CRISPR-Cas9; 6 lines are shown, and lines 2 and 5 (*) were used as knockout. (J) Generation of HEK-SCAN-WT (SCAN-WT) and HEK-SCAN-QTG / NAA (QTG / NAA) re-expressing cell lines from SCAN-deficient parental cells; 2 independent lines each are shown. (K) SNO-SCAN level in eNOS- overexpressing HEK cells in the absence or presence of NOS inhibitor (L-NMMA, 100 mM); n = 2. (L) SNO-SCAN level in LPS / interferon-induced RAW264 cells in the absence or presence of iNOS inhibitor (1400W, 100 mM); n = 2. (M) Truncated HO2 forms, with retained residues indicated. All truncations still contain both SNO sites, Cys265 and Cys282. (N) Interaction between SCAN and truncated HO2 forms. Myc-SCAN and Flag-truncated- HO2 were co-expressed in HEK cells, IP with anti-rabbit myc antibody or non-immune IgG, and IB with anti-mouse Flag antibody. (O) S-nitrosylation of full-length HO2 (1–316) and truncated, non-interacting HO2 (195–316) by recombinant SCAN in vitro with increasing SNO-CoA.

[0040] Figs.9(A-F) illustrate the characterization of SCAN-deficient mice, related to Figure 2 (A) WT BLVRB gene allele and targeting vector for deleting exons 2–4 in ES cells to generate SCAN-deficient mice. (B) Serum bilirubin level in SCAN+ / + and SCAN- / - mice; n = 5 mice per group. (C) The number of white blood cells (WBCs), red blood cells (RBCs), and platelets in 1 mL blood from SCAN+ / + and SCAN- / - mice; n R 13. (D) The percentage of lymphocytes, monocytes, and granulocytes in white blood cells of SCAN+ / + and SCAN- / - mice; n R 13. (E) Free heme concentration in the serum of SCAN+ / + and SCAN- / - mice; n = 9. (G) Hemoglobin concentration in the blood of SCAN+ / + and SCAN- / - mice; n R 13. (H) All results are presented as mean ± SD. Two-tailed Student’s t test was used to detect significance in (B)–(F). *p < 0.05; NS, not significant.

[0041] Figs.10(A-H) illustrate interactions and functions of SCAN in purified systems, cells, and mice, related to Fig.2 (A) NO donor DPTA (200 mM for 20 h) promotes theinteraction between SCAN and INSRb in L6 cells. IP with anti-rabbit SCAN antibody or non-immune IgG; IB with anti-rabbit INSRb antibody; n = 2. (B) Treatment with the NO donor DPTA (200 mM for 20 h) promotes the interaction between SCAN and IRS1. Myc- SCAN and Flag-IRS1 were co-expressed in HEK cells, n = 2. (C)Interaction between SCAN and INSRb after insulin stimulation (100 nM for 30 min). Myc-SCAN and Flag-INSR were co-expressed in HEK cells, n = 2. (D) Interaction between INSRb and SCAN-QTG / NAA or SCAN-C109 / 188R after treatment with the NO donor DPTA (200 mM for 20 h). Flag-INSR and Myc-SCAN, Myc-SCAN-QTG / NAA, or Myc-SCAN-C109 / 188R were co-expressed in HEK cells, n = 2. In (B)–(D), IP with anti-rabbit myc antibody or non-immune IgG; probe with anti-mouse Flag antibody. (E) Body weight of male SCAN+ / +and SCAN- / -mice measured at indicated times during HFD feeding; n = 20. ANOVA with Tukey’s post hoc test was used to detect significance. (F) S-nitrosylation of INSRb by SCAN in vitro (vs. GST control), in the presence of varying SNO-CoA concentration; n = 2. (G) KM of SCAN for SNO-CoA-dependent S-nitrosylation of INSRb; n = 2. (H) KM of SCAN for SNO-CoA- dependent S-nitrosylation of IRS1; n = 3.

[0042] Figs.11(A-Q) illustrate the regulation of INSRb / IRS1 S-nitrosylation by NOS, SCAN, and SCoR, related to Fig.3. (A)Colocalization of SCAN and SCoR in HEK293 cells. Overnight serum-starved HEK cells were treated by insulin for 10 min, fixed, and immunostained with anti-SCAN and anti-SCoR antibodies. (B) Interaction between SCAN and INSRb in SCoR-knockout HEK cells. Myc-SCAN and Flag-INSR were co-expressed in WT HEK cells or SCoR-knockout HEK cells. IP with anti-rabbit myc antibody; probe with anti-mouse Flag antibody; n = 2. (C) SNO-INSRb, SNO-IRS1, and SNO-SCAN in skeletal muscle (gastrocnemius) of 5-h-fasted 12-week-HFD-fed SCoR+ / +and SCoR- / -mice. (D) Quantification of SNO-INSRb, SNO-IRS1, and SNO-SCAN in (C); n = 4 per group. (E) Phosphorylation of AKT (pSer473) and AS160 (pThr642) in skeletal muscle (gastrocnemius) of 5-h-fasted 12-week-HFD-fed SCoR+ / +and SCoR- / -mice. Spliced blots for AKT / p-AKT are indicated. (F) Quantification of phosphorylation of AKT (pSer473) and AS160 (pThr642) in (E); n = 4. (G) Tyrosine kinase activity of purified INSR after SNO-CoA treatment; n = 6. (H) Generation of SCAN-knockout L6 cell lines (L6-SCAN- / -) using CRISPR-Cas9; 7 lines are shown, and lines 4 and 6 (*) were used as knockout. (I–L) Quantification of phosphorylation of INSRb (pTyr1162) (I), IRS1 (pTyr608) (J), AKT (pSer473) (K), and AS160 (pThr642) (L) in overnight-starved L6-WT and L6- SCAN- / -cellsat 1, 30, 60, 120, and 240 min after removal of insulin, following 10-min insulin treatment (100 nM); n = 4. Representative gels are shown in Fig.3F. (M) Phosphorylation of nNOS (pS1412) in overnight serum-starved L6 cells at the indicated time after removal of insulin, following 10-min insulin treatment (100 nM). (N) Amount of SNO-INSRb in eNOS-, nNOS-, or iNOS-overexpressing HEK cells. (O) Amount of SNO-IRS1 in eNOS-, nNOS-, or iNOS-overexpressing HEK cells. (P and Q) Quantification of phosphorylation of AKT (pSer473) (P) and AS160 (pThr642) (Q) in PBS-treated (control) and L-NMMA-treated (100 mM) L6 cells at 1, 30, 60, 120, and 240 min after removal of insulin following 10-min insulin treatment (100 nM); n = 3. Representative gels are shown in Fig.4K. Two-tailed Student’s t test was used to detect significance in (D), (F), and (G). (I)–(L), (P), and (Q) were analyzed by two-way (time 3 treatment) repeated measures analysis of variance followed by Sidak’s multiple comparisons test. *p < 0.05; **p < 0.01; and ***p < 0.001.

[0043] Figs.12(A-K) illustrate levels of SCAN and SNO-INSRb in human tissues, and other SNO-RTKs (receptor tyrosine kinases), related to Figs.5 and 6. (A) Generation of L6- SCAN-WT (SCAN-WT), L6-SCAN-QTG / NAA (QTG / NAA), and L6-SCAN-C109 / 188R (C109 / 188R) re-expressing cell lines from SCAN- deficient parental cells. (B) Modeling of tyrosine kinase domain dimer of INSR. SNO site Cys1083 is indicated in red. (C) Generation of INSR-knockout L6 cell lines (L6-INSR- / -) using CRISPR-Cas9; 6 lines are shown, and line 4 (*) was used as knockout. (D) Generation of L6-INSR-WT (INSR-WT) and L6-INSR- C1083A (C1083A) re-expressing cell lines from INSR-deficient parental cells. (F) Expression of SCAN in 14 human skeletal muscle samples (Hm#) from patients with the indicated BMI, and quantification (lower panel). (F) Expression of SCAN in 14 human visceral adipose samples (Hvad#) with the indicated BMI, and quantification (lower panel). GAPDH is used as internal loading control in (E) and (F), and expression of SCAN was first normalized with expression of internal control GAPDH, and then versus the average expression level of SCAN in the same group of samples. (G) SNO-INSRb level in 14 human skeletal muscle samples from patients with the indicated BMI, and quantification (lower panel). (H) SNO-INSRb level in 12 human visceral adipose samples from patients with the indicated BMI, and quantification (lower). In (G) and (H), SNO-INSRb was first normalized with input of INSRb, and versus the average SNO-INSRb level in the same group of samples. (I) S-nitrosylation of fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor alpha (PDGFRa), and platelet-derived growth factor receptor beta (PDGFRb) inester-CYSNO (100 mM)-treated HEK293 cells (upper), and protein expression by western blot (lower). (J) S-nitrosylation of vascular endothelial growth factor receptor 2 (VEGFR2) in ester-CYSNO (100 mM)-treated HEK293 cells (upper), and protein expression by western blot (lower). (K) S-nitrosylation of human epidermal growth factor receptor 3 (HER3) in wild-type HEK293 cells or eNOS-expressing HEK293 cells (upper), and protein expression by western blot (lower). Minus (-) ascorbate is used as negative control in SNO-RAC assays. DETAILED DESCRIPTION

[0044] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0046] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in the inclusive, open sense, meaning that additional elements may be included. The terms "such as", "e.g.,", as used herein are non-limiting and are for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0047] The term "or" as used herein should be understood to mean "and / or", unless the context clearly indicates otherwise.

[0048] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0049] The term “BLVRB” refers to a Biliverdin reductase B (also called Flavin reductase) gene or SNO-CoA-associated Nitrosyltransferase (SCAN) and its transcriptional and / or translational products. The human BlvrB genetic locus is present on chromosome 19and has a nucleotide sequence that is the reverse complement of nucleotides 40,447,768- 40,465,745 of Accession No. NC_000019.10 of the GENBANK® biosequence database. The human cDNA sequence is disclosed as Accession No. NM_000713.3 of the GENBANK® biosequence database, and encodes a protein having the amino acid sequence disclosed as Accession No. NP_000704.1 of the GENBANK® biosequence database.

[0050] The term "co-administration" means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.

[0051] "Administered concomitantly" refers to the administration of two agents at the same therapeutic time frame, in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration.

[0052] The phrases "parenteral administration" and "administered parenterally" are art- recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0053] The term "treating" is art-recognized and includes inhibiting a disease, disorder or condition in a subject, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

[0054] The term "preventing" is art-recognized and includes stopping a disease, disorder or condition from occurring in a subject, which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it. Preventing a conditionrelated to a disease includes stopping the condition from occurring after the disease has been diagnosed but before the condition has been diagnosed.

[0055] The term "pharmaceutical composition" refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredients (e.g., a formulation of the disclosed compound or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0056] The term "immediate release" is defined as a release of compound from a dosage form in a relatively brief period of time, generally up to about 60 minutes. The term "modified release" is defined to include delayed release, extended release, and pulsed release. The term "pulsed release" is defined as a series of releases of drug from a dosage form. The term "sustained release" or "extended release" is defined as continuous release of a compound from a dosage form over a prolonged period.

[0057] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0058] The phrase "pharmaceutically acceptable carrier" is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0059] The compounds of the application are capable of further forming salts. All of these forms are also contemplated herein.

[0060] "Pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. For example, the salt can be an acid addition salt. One embodiment of an acid addition salt is a hydrochloride salt. The pharmaceutically acceptable salts can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being preferred. Lists of salts are found in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0061] The term "analogue" refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analogue is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.

[0062] A "patient," "subject," or "host" to be treated by the subject method may mean either a human or non-human animal, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder.

[0063] The terms "prophylactic” or “therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0064] The terms “agent’, "therapeutic agent", "drug", "medicament" and "bioactive substance" are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include without limitation pharmaceutically acceptable salts thereof and prodrugs. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.

[0065] The phrase "therapeutically effective amount" or “pharmaceutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amountnecessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.

[0066] A “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. The control may, for example, be examined at precisely or nearly the same time the test cell, tissue, sample, or subject is examined. The control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject. The control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a condition, disease, or disorder for which the test is being performed.

[0067] The term “otherwise identical sample”, as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term “otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term “otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject.

[0068] Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0069] The term "small molecule" is an art-recognized term. In certain embodiments, this term refers to a molecule, which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.

[0070] All percentages and ratios used herein, unless otherwise indicated, are by weight.

[0071] The terms "healthy" and "normal" are used interchangeably herein to refer to a subject or particular cell or tissue that is devoid (at least to the limit of detection) of a disease condition.

[0072] Embodiments described herein relate to compositions for and methods of treating diseases and disorders related to or associated with aberrant or dysregulated S- nitrosylated proteins (SNO-proteins). S-nitrosylation of proteins by NO to form S- nitrosothiols (SNOs) provides control over cellular function. However, in excess, aberrant or dysregulated S-nitrosylation has been implicated in numerous diseases and disorders in humans. In particular, hyper-S-nitrosylation of various SNO-proteins has been implicated in diabetes, heart failure, such as post-myocardial infarction heart failure, and heart failure with preserved ejection fraction (HFpEF), ventricular arrhythmia in individuals with Duchenne muscular dystrophy, Becker muscular dystrophy, sickle cell anemia, neurodegenerative diseases and disorders, such as amyloid lateral sclerosis (ALS), Alzheimer's, Parkinson’s, and Huntington’s disease, cancers, such as cholangiocarcinoma and hepatocellular carcinoma, infections, inflammatory disorders and shock states.

[0073] Compositions and methods described herein are based on the discovery of a novel class of enzyme, SNO-CoA-Associated nitrosyltransferase (SCAN), that catalyzes the S-nitrosylation of proteins, and particularly receptor tyrosine kinases (RTKs), using S- nitroso-Coenzyme A (SNO-CoA) as a cofactor. SCAN activity was identified as residing in fetal biliverdin reductase protein (BLVRB). SCAN or BLVRB (i.e., SCAN / BLVRB) has been identified as a non-specific flavin reductase, but this activity is not linked to physiological functions or targets. We found that SCAN / BLVRB is a protein S-nitrosylase that regulates the insulin pathway via S-nitrosylation of INSRb and IRS1.

[0074] eNOS in skeletal muscle is required for normal insulin responsiveness. By contrast, iNOS-derived NO is deleterious to insulin signaling and contributes to the pathophysiology of type 2 diabetes. Thus, NO can mediate both beneficial and deleterious effects on insulin signaling. In healthy conditions, insulin stimulation of its receptor iscoupled to eNOS / nNOS-mediated S-nitrosylation of INSRb / IRS1, which terminates physiological insulin signaling to avoid hypoglycemia. However, in obesity, iNOS is induced by pro-inflammatory cytokines in skeletal muscle, uncoupling NO generation from insulin signaling. This results in hyper-S-nitrosylation of INSRb / IRS1 and leads to insulin resistance. Importantly, NOS activity is necessary but not sufficient for S-nitrosylation, as NO itself cannot chemically S-nitrosylate proteins. Rather, S-nitrosylation is routed through SNO-CoA by the enzymes SCAN / BLVRB and SCoR, which together determine steady-state levels of SNO-INSRb / IRS1.

[0075] The insulin receptor is a member of the RTK family of 58 cell surface receptors for growth factors, cytokines, and hormones. We found that many RTKs to be S-nitrosylated, implying a class effect with shared regulatory mechanisms. Our finding of insulin-induced S- nitrosylation of INSRb / IRS1 in healthy tissues and hypernitrosylation of INSRb / IRS1 in diabetes provides a working model for enzymatic S-nitrosylation in health and disease: in physiological situations, nitrosylase-catalyzed S-nitrosylation is induced by agonists to regulate cellular signaling. In disease, S-nitrosylation is dysregulated or disrupted by aberrant nitrosylase activity, possibly uncoupled from receptor stimulation.

[0076] Thus, S-nitrosylation by (SCAN / BLVRB) / SNO-CoA defines a new enzyme class and a unique mode of RTK regulation. These findings provide therapeutic opportunities that extend to the human condition and show that modulators of SCAN / BLVRB S- nitrosylation activity can be used to modulate aberrant or dysregulated nitrosylation of proteins associated with a disease or disorder in a subject in need thereof and in particular diseases and disorders associated with SCAN / BLVRB over expression or underexpression.

[0077] Accordingly, a method of treating a disease or disorder characterized by aberrant or dysregulated nitrosylation in a subject in need thereof can include administering to the subject a therapeutically effective amount of an agent that modulates SNO-CoA- associated Nitrosyltransferase / biliverdin IXβ reductase B (SCAN / BLVRB) mediated S- nitrosylation. As used herein, an agent that modulates SCAN / BLVRB mediated S- nitrosylation refers to a composition comprised of a substance that promotes or inhibits SCAN / BLVRB mediated S-nitrosylation and can include any agent that promotes or inhibits SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression in a cell.

[0078] In some embodiments, SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression in a cell can be suppressed, inhibited, and / or blocked in several ways including: direct inhibition of SCAN / BLVRB S-nitrosylation activity (e.g., by using neutralizing antibodies, small molecules, peptidomimetics, or dominant negative polypeptides); inhibition of genes that express the SCAN / BLVRB (e.g., by blocking the expression or activity of the genes and / or proteins); activation of genes and / or proteins that inhibit one or more of SCAN / BLVRB S-nitrosylation activity, S- nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression; introduction of genes and / or proteins that negatively regulate one or more of SCAN / BLVRB S-nitrosylation activity, S- nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression (e.g., by using recombinant gene expression vectors, recombinant viral vectors or recombinant polypeptides); or gene replacement with, for instance, a hypomorphic mutant of the SCAN / BLVRB (e.g., by homologous recombination, overexpression using recombinant gene expression or viral vectors, or mutagenesis).

[0079] In some embodiments, an inhibitor of SCAN / BLVRB S-nitrosylation activity can include a small molecule SCAN / BLVRB inhibitor. The small molecule SCAN / BLVRB inhibitor can include, for example, xanthene dyes and acridine-containing compounds, such as flavin mononucleotide (FMC), a natural SCAN / BLVRB substrate, and similar structures, such as 4-((6-Chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin-1- yl)methyl)phenol. A small molecule screen identified a series of BLVRB inhibitors, most having a tricyclic hydrocarbon core structure, that have potency in the nanomolar range (Nesbitt et al. (2018) J Biol Chem 293(15):5431-5446; Kim et al., (2021) J Med Chem. doi: 10.1021 / acs.jmedchem.lc01664). For example, lumichrome was identified as a lead BLVRB inhibitor compound. Lumichrome is a natural photoproduct of riboflavin (vitamin B2).

[0080] Other SCAN / BLVRB inhibitors can include, for example, phloxine B (disodium 2',4',5',7'- tetrabromo-4,5,6,7-tetrachloro-3-oxo-3H-spiro[[2]benzofuran-l,9'-xanthene]-3',6'- bis(olate); CAS Number 18472-87-2), erythrosin B (3’,6,-dihydroxy-2,,4,,5,,7’- tetraiodospiro[2-benzofuran-3,9’-xanthene]-l-one; CAS Number 15905-32-5), NSC130813 (4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)phenol; CAS Number 500565-15-1), NSC12516 (4-(6-chloro-2-methoxyacridin- 9-ylamino)-2-(pyrrolidin- l-ylmethyl)phenol, hydrochloride), PH001924 (4-[(6-chloro-2- methoxy-9-acridinyl)amino]- 2-[(diethylamino)methyl]phenol; also called ZINC4366439), lumichrome (7,8-dimethyl-benzo[g]pteridine-2,4(lH,3H)-dione; CAS Number 1086-80-2), PH006888 (N-[4-(9- acridinylamino)-3-methoxyphenyl]methanesulfonamide), xanthene, proflavine, alizarin red S, NSC371876 (4-(2-amino-6-naphthalen-l-yl-pyrimidin-4-yl)-6- naphthalen-1-yl-pyrimidin-2- amine), NSC179187 (5’,7, 9, 13-tetramethylspiro[5- oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-ene-6,2’-piperidine]-16-ol; also called purapuridine), NSC53396 (4-[(3S,5R,8R,9S,10S,13R,14S,17S)-14-hydroxy-10,13- dimethyl- 3-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-l,2,3,4,5,6,7,8,9,ll,12,15, 16,17- tetradecahydrocyclopenta[a]phenanthren-17-yl]-2H-furan-5-one; also called actodigin), NSC 10936 (2-(benzylamino)-6-methoxy-2,3-dihydro-lH-inden-1-ol; hydrochloride), NSC 169534 (7-(4-amino-5-hydroxy-6-methyloxan-2-yl)oxy-6,9,ll-trihydroxy-9-(2-hydroxyacetyl)-4- methoxy-8,10-dihydro-7H-tetracene-5,12-dione), NSC 117269 (4-[4,6-diamino-2-(4- chlorophenyl)-2H-l,3,5-triazin-l-yl]-N-pyrimidin-2-ylbenzenesulfonamide), NSC 143491 (7- (4-amino-5-hydroxy-6-methyloxan-2-yl)oxy-6,9,ll-trihydroxy-9-(N-hydroxy-C- methylcarbonimidoyl)-4-methoxy-8,10-dihydro-7H- tetracene-5,12-dione), NSC305821 ([(3E)-3-[(4-chlorophenyl)methylidene]-5,7-dimethyl- l,2-dihydrocyclopenta[b]quinolin-9- yl]-piperidin-2-ylmethanol), NSC 130813 (5- naphthalen-1-yl-1-phenyltetrazol e), ZINC ID ZINC0977089, ZINC27528243 (N-[4- (acridin-9-ylamino)phenyl]-4- methylbenzenesulfonamide), ZINC ID ZINC09330686, ZINC71767103 (1- [[5-[(7-chloro-1 - hydroxy quinolin-4-ylidene)amino]-2-hydroxyphenyl]methyl]piperidin-4-ol), ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC21093196 (l-[(2S)-5-anthracen-9-yl-2-(5- bromo-2-ethoxyphenyl)- 2H-l,3,4-oxadiazol-3-yl]ethenone), ZINC71767097 (l-[[5-[(7- chloroquinolin-4-yl)amino]-2-hydroxyphenyl]methyl]piperidin-4-ol), Asunaprevir (BMS- 650032; tert-butyl N-[(2S)-l-[(2S,4R)-4-(7-chloro-4-methoxyisoquinolin-l-yl)oxy-2- [[(lR,2S)-l- (cyclopropylsulfonylcarbamoyl)-2-ethenylcyclopropyl]carbamoyl]pyrrolidin-l- yl]-3,3- dimethyl-l-oxobutan-2-yl]carbamate), micafungin (5-[(lS,2S)-2-[(3S,6S, 9S,llR,15S,18S,20R,21R,24S,25S,26S)-3-[(lR)-3-amino-l-hydroxy-3-oxopropyl]-ll,20, 21,25-tetrahydroxy-15-[(lR)-l-hydroxyethyl]-26-methyl-2,5,8,14,17,23-hexaoxo-18-[[4- [5- (4-pentoxyphenyl)- 1 ,2-oxazol-3-yl]benzoyl]amino]-1,4,7,13,16,22- hexazatricyclo[22.3.0.09, 13]heptacosan-6-yl]-l,2-dihydroxy ethyl]-2-hydroxyphenyl] hydrogen sulfate), tamibarotene (4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2- yl)carbamoyl]benzoic acid), TSU-68 (SU6668, Orantinib; 3-[2,4-dimethyl-5-[(Z)-(2-oxo- lH- indol-3-ylidene)methyl]-lH-pyrrol-3-yl]propanoic acid), sulfasalazine (2-hydroxy-5- [[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]benzoic acid), febuxostat (2-[3-cyano-4-(2- methylpropoxy)phenyl]-4-methyl-l,3-thiazole-5-carboxylic acid), crenolanib (CP-868596; l- [2-[5-[(3-methyloxetan-3-yl)methoxy]benzimidazol-l-yl]quinolin-8-yl]piperi din-4- amine), olsalazine (5-[(3-carboxy-4-hydroxyphenyl)diazenyl]-2-hydroxybenzoic acid), PTC124 (ataluren; 3-[5-(2-fluorophenyl)-l,2,4-oxadiazol-3-yl]benzoic acid), deferasirox (- [3,5-bis(2- hydroxyphenyl)-l,2,4-triazol-l-yl]benzoic acid), flunixin in combination with meglumine ((2R,3R,4R,5S)-6-(methylamino)hexane-l,2,3,4,5-pentol; 2-[2-methyl-3- (trifluoromethyl)anilino]pyridine-3-carboxylic acid), azelastine (4-[(4- chlorophenyl)methyl]- 2-(l-methylazepan-4-yl)phthalazin-l-one), benzbromarone ((3,5- dibromo-4-hydroxyphenyl)- (2-ethyl-l-benzofuran-3-yl)methanone), triclabendazole (6- chloro-5-(2,3-dichlorophenoxy)- 2-methylsulfanyl-lH-benzimidazole), nifedipine (dimethyl 2,6-dimethyl-4-(2-nitrophenyl)- l,4-dihydropyridine-3,5-dicarboxylate), nisoldipine (3-O-methyl 5-0-(2-methylpropyl) 2,6- dimethyl-4-(2-nitrophenyl)-l,4- dihydropyridine-3,5-dicarboxylate), zafirlukast (cyclopentyl N-[3-[[2-methoxy-4-[(2- methylphenyl)sulfonylcarbamoyl]phenyl]methyl]-l-methylindol-5- yl]carbamate), pyrantel in combination with pamoate (4-[(3-carboxy-2-hydroxynaphthalen-l- yl)methyl]-3- hydroxynaphthalene-2-carboxylic acid;l-methyl-2-[(E)-2-thiophen-2- ylethenyl]-5,6- dihydro-4H-pyrimidine), candesartan cilexetil (1-cy cl ohexyloxycarbonyloxy ethyl 2- ethoxy-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]benzimidazole-4- carboxylate), and azilsartan medoxomil ((5-methyl-2-oxo-l,3-dioxol-4-yl)methyl 2-ethoxy-3- [[4-[2-(5- oxo-4H-l,2,4-oxadiazol-3-yl)phenyl]phenyl]methyl]benzimidazole-4-carboxylate). Phloxine B, for example, is also a dye used in cosmetics and as a food coloring. Erythrosin B (also called erythrosin extra bluish) is a dye used in cosmetics, hair dyes, and color filters.

[0081] Further agents for use as an inhibitor of SCAN / BLVRB S-nitrosylation activity can include a compound selected from:,, aredisclosed in U.S. Patent Publication No.2022 / 0339170, which is incorporated by reference in its entirety.

[0082] It was also shown that the nitric oxide synthase (NOS) inhibitor, L-NMMA, blocks insulin stimulated S-nitrosylation not only of the proteins INSRβ and IRS1 but also SCAN / BLVRB itself. Therefore, in some embodiments, an inhibitor of SCAN / BLVRB S- nitrosylation activity or S-nitrosylation of SCAN / BLVRB can include a nitric oxide synthase (NOS) inhibitor.

[0083] Three isoforms of NOSs exist, originally named according to their first observed cellular / tissue localization or expressional regulation, i.e., neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). A NOS inhibitor can be any agent that reduces or inhibits NOS. In some embodiments, the NOS inhibitor can have an IC50 ≤ 5µM, ≤ 1µM, or ≤100 nM. In still other embodiments, the NOS inhibitor can have a NOS IC50 ≤ 400 nM, ≤ 300 nM, ≤ 200 nM, ≤ 100 nM, ≤ 50 nM, or ≤ 25 nM.

[0084] A NOS inhibitor for use in a composition or method described herein may decrease or inhibit NOS function by interacting with NOS protein thereby reducing production of NOS-derived nitric oxide. The NOS inhibitor may decrease the production of NOS by downregulating expression of NOS. The NOS inhibitor may inhibit or decrease expression of NOS-encoded DNA or RNA.

[0085] A NOS inhibitor may be selective or specific for a given NOS isoform. NOS inhibitors with various degrees of selectivity for NOS isoforms have been described. It will be appreciated that any potential nonselective, selective and / or partially selective NOS inhibitors can be used in the compositions and methods recited herein. In some embodiments, the NOS inhibitor can have a selectivity for either nNOS, iNOS, or eNOS versus other NOSs ≥ 2 times, ≥ 5 times, ≥ 10 times, ≥ 20 times, ≥ 30 times, ≥ 40 times, ≥ 50 times or more times.

[0086] In some embodiments, the NOS inhibitor for use in a method or compositions described herein can include a nonspecific NOS inhibitor selected from the group consisting of ʟ-NMMA (Nγ-Monomethyl-L-arginine acetate), ʟ-NAME (Nω-Nitro-L-arginine Methyl Ester, Hydrochloride), ronopterin (VAS203), 2-Iminobiotin, MTR104, ʟ-NG-benzylarginine, ʟ-NG-aminoarginine, pimagedine (aminoguanidine), OsteoDex, and iminoethylornithine. In certain embodiments, the NOS inhibitor includes ʟ-NMMA.

[0087] In some embodiments, the inhibitor of SCAN / BLVRB mediated S-nitrosylation or S-nitrosylation of SCAN / BLVRB is a nNOS inhibitor. The nNOS inhibitor can be selected from the group consisting of S-methyl-L-thiocitrulline (SMTC), Tat-NR2B9c (NA- 1), ZL006, ARL17477, NXN-462 and NXN-188.

[0088] In some embodiments, the inhibitor of SCAN / BLVRB mediated S-nitrosylation or S-nitrosylation of SCAN / BLVRB is an iNOS inhibitor. The iNOS inhibitor can be selected from the group consisting of GW274150, GW273629, Cindunistat (SD-6010), BYK191023, L-NILTA, ʟ-N iminoethyl lysine (L-NIL), gingivex (guanidinoethyl disulfide), ONO-1714, XQ-1H (Gingko biloba lactone B mesylate), AR-C102222, FR-260330, PPA250, BBS-1, and BBS-2.

[0089] In some embodiments, the inhibitor of SCAN / BLVRB S-nitrosylation or S- nitrosylation of BLVRB is an eNOS inhibitor. The eNOS inhibitor can be selected from the group consisting of L-NIO dihydrochloride, ZZL7 (N-Acetyl-L-alanyl-L-valine methyl ester), Diphenyleneiodonium Chloride, ONO-1714, and a caveolin-1(Cav-1) peptide. In some embodiments, the eNOS inhibitor can include a Cav-1 derived mimetic peptide including the CSD. In some embodiments, at least a portion of the Can-1 peptide can bind directly to eNOS. In some embodiments, the Cav-1 peptide can include the amino acid sequence DGIWKASFTTFTVTKYWFYR (SEQ ID NO.7). In certain embodiments, the Cav-1 peptide consists of an amino acid having SEQ ID NO: 7. In additional embodiments, the Cav-1 peptide may be substantially identical to SEQ ID NO: 7.

[0090] In other embodiments, the inhibitor of SCAN / BLVRB mediated S-nitrosylation can include an agent that reduces or inhibits SCAN / BLVRB expression in tissue or cells of a subject in need thereof. "Expression”, means the overall flow of information from a gene to produce a gene product (typically a protein, optionally post-translationally modified or a functional / structural RNA).

[0091] In some embodiments, the agent can include an RNAi construct that inhibits or reduces expression of the SCAN / BLVRB expression in a cell. RNAi constructs comprise double stranded RNA that can specifically block expression of a target gene, promotes loss of function of the target gene, or mediates RNA interference. "RNA interference" or "RNAi" is a term initially applied to a phenomenon observed in plants and worms where double- stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner.

[0092] The term "loss-of-function," as it refers to genes inhibited by the subject RNAi method, refers to a diminishment in the level of expression of a gene when compared to the level in the absence of RNAi constructs.

[0093] The phrase "mediates RNAi" refers to (indicates) the ability to distinguish which RNAs are to be degraded by the RNAi process, e.g., degradation occurs in a sequence- specific manner rather than by a sequence-independent dsRNA response, e.g., a PKR response.

[0094] The RNAi construct can include, for example, small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species, which can be cleaved in vivo to form siRNAs. RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and / or transcripts which can produce siRNAs in vivo.

[0095] "RNAi expression vector" (also referred to herein as a "dsRNA-encoding plasmid") refers to replicable nucleic acid constructs used to express (transcribe) RNA which produces siRNA moieties in the cell in which the construct is expressed. Such vectors include a transcriptional unit comprising an assembly of (1) genetic element(s) having a regulatory role in gene expression, for example, promoters, operators, or enhancers, operatively linked to (2) a "coding" sequence which is transcribed to produce a double- stranded RNA (two RNA moieties that anneal in the cell to form an siRNA, or a single hairpin RNA which can be processed to an siRNA), and (3) appropriate transcription initiation and termination sequences.

[0096] The choice of promoter and other regulatory elements generally varies according to the intended host cell. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops, which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the application describes other forms of expression vectors that serve equivalent functions and which become known in the art subsequently hereto.

[0097] The RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the "target" gene). The double-strandedRNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, embodiments tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition.

[0098] Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.

[0099] Production of RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. The RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and / or change other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The RNAi construct may be produced enzymatically or by partial / total organic synthesis, a modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.

[0100] Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see for example, Nucleic Acids Res, 25:776-780; J Mol Recog 7:89-98;Nucleic Acids Res 23:2661-2668; Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2'- substituted ribonucleosides, a-configuration).

[0101] The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount, which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.

[0102] In some embodiments, the subject RNAi constructs are inhibitors of SCAN / BLVRB S-nitrosylation activity or expression. For example, an RNAi construct inhibitor of SCAN / BLVRB S-nitrosylation activity or expression can include "small interfering RNAs” (siRNAs) or an antisense oligonucleotide specific to a region in the mRNA of SCAN / BLVRB gene.

[0103] siRNAs are around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, e.g., corresponding in length to the fragments generated by nuclease "dicing" of longer double-stranded RNAs. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.

[0104] The siRNA molecules described herein can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art. For example, short sense and antisense RNA oligomers can be synthesized and annealed to form double-stranded RNA structures with 2-nucleotide overhangs at each end (Proc Natl Acad Sci USA, 98:9742- 9747; EMBO J, 20:6877-88). These double-stranded siRNA structures can then be directlyintroduced to cells, either by passive uptake or a delivery system of choice, such as described below.

[0105] In certain embodiments, the siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence of the enzyme dicer. In one embodiment, the Drosophila in vitro system is used. In this embodiment, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides.

[0106] The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.

[0107] In certain embodiments, the RNAi construct is in the form of a hairpin structure (named as hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Genes Dev, 2002, 16:948-58; Nature, 2002, 418:38-9; RNA, 2002, 8:842-50; and Proc Natl Acad Sci, 2002, 99:6047-52. Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.

[0108] In yet other embodiments, a plasmid is used to deliver the double-stranded RNA, e.g., as a transcriptional product. In such embodiments, the plasmid is designed to include a "coding sequence" for each of the sense and antisense strands of the RNAi construct. The coding sequences can be the same sequence, e.g., flanked by inverted promoters, or can be two separate sequences each under transcriptional control of separate promoters. After the coding sequence is transcribed, the complementary RNA transcripts base-pair to form the double-stranded RNA.

[0109] PCT application WO01 / 77350 describes an example of a vector for bi- directional transcription of a transgene to yield both sense and antisense RNA transcripts of the same transgene in a eukaryotic cell. Accordingly, certain embodiments provide arecombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for an RNAi construct of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene fragment in a host cell.

[0110] In some embodiments, a lentiviral vector can be used for the long-term expression of a siRNA, such as a short-hairpin RNA (shRNA), to knockdown expression of SCAN / BLVRB in a cell. Although there have been some safety concerns about the use of lentiviral vectors for gene therapy, self-inactivating lentiviral vectors are considered good candidates for gene therapy as they readily transfect mammalian cells.

[0111] By way of example, short-hairpin RNA (shRNA) down regulation of the SCAN / BLVRB expression can be created using OligoEngene software (OligoEngine, Seattle, WA) to identify sequences as targets of siRNA. The oligo sequences can be annealed and ligated into linearized pSUPER RNAi vector (OligoEngine, Seattle, WA) and transformed in E coli strain DH5α cells. After positive clones are selected, plasmid can be transfected into 293T cells by calcium precipitation. The viral supernatant collected containing shRNA can then be used to infect mammalian cells in order to down regulate the SCAN / BLVRB.

[0112] In another embodiment, the inhibitor of SCAN / BLVRB mediated S- nitrosylation can include an antisense oligonucleotide, e.g., an antisense oligonucleotide specific to a region in the mRNA of BLVRB gene. Antisense oligonucleotides are relatively short nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of the mRNA encoding a particular protein. Although antisense oligonucleotides are typically RNA based, they can also be DNA based. Additionally, antisense oligonucleotides are often modified to increase their stability.

[0113] The binding of these relatively short oligonucleotides to the mRNA is believed to induce stretches of double stranded RNA that trigger degradation of the messages by endogenous RNAses. Additionally, sometimes the oligonucleotides are specifically designed to bind near the promoter of the message, and under these circumstances, the antisense oligonucleotides may additionally interfere with translation of the message. Regardless of the specific mechanism by which antisense oligonucleotides function, their administration to a cell or tissue allows the degradation of the mRNA encoding a specific protein.Accordingly, antisense oligonucleotides decrease the expression and / or activity of a particular protein (e.g., SCAN / BLVRB enzyme).

[0114] The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups, such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Proc Natl Acad Sci 86:6553-6556; Proc Natl Acad Sci 84:648-652; PCT Publication No. WO88 / 09810, published Dec.15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89 / 10134, published Apr.25, 1988), hybridization-triggered cleavage agents (See, e.g., BioTechniques 6:958-976) or intercalating agents. (See, e.g., Pharm Res 5:539-549). To this end, the oligonucleotide may be conjugated or coupled to another molecule.

[0115] Oligonucleotides described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Proc Natl Acad Sci 85:7448-7451).

[0116] The selection of an appropriate oligonucleotide can be performed by one of skill in the art. Given the nucleic acid sequence encoding a particular protein, one of skill in the art can design antisense oligonucleotides that bind to that protein and test these oligonucleotides in an in vitro or in vivo system to confirm that they bind to and mediate the degradation of the mRNA encoding the particular protein. To design an antisense oligonucleotide that specifically binds to and mediates the degradation of a particular protein, it is important that the sequence recognized by the oligonucleotide is unique or substantially unique to that particular protein. For example, sequences that are frequently repeated across protein may not be an ideal choice for the design of an oligonucleotide that specifically recognizes and degrades a particular message. One of skill in the art can design an oligonucleotide and compare the sequence of that oligonucleotide to nucleic acid sequences that are deposited in publicly available databases to confirm that the sequence is specific or substantially specific for a particular protein.

[0117] In certain embodiments, the subject RNAi constructs are microRNA (miRNA). The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. The term can be used to refer to the single-stranded RNA molecule processed from a precursor or in certain instances the precursor itself. A precursor can include a naturally occurring miRNA precursor or a designed precursor miRNA-like RNA. Individual miRNAs have been identified and sequenced in different organisms, and they have been given names.

[0118] In some embodiments, “miRNA” molecules described herein also encompass a region or an additional strand that is partially (between 10 and 50% complementary across length of strand), substantially (greater than 50% but less than 100% complementary across length of strand) or fully complementary to another region of the same single stranded molecule or to another nucleic acid. Thus, nucleic acids may encompass a molecule that comprises one or more complementary or self-complementary strand(s) or “complements” of a particular sequence comprising a molecule. For example, precursor miRNA may have a self-complementary region, which is up to 100% complementary miRNA probes of the invention or can be at least 60, 65, 70, 75, 80, 85, 90, 95 or 100% complementary to their target.

[0119] In some embodiments the inhibitor of SCAN / BLVRB S-nitrosylation activity or expression includes a miRNA that targets the SCAN / BLVRB pathway to decrease SCAN / BLVRB expression by directly binding to its 3’-UTR. In particular embodiments, the miRNA can include miR-127-5p.

[0120] A number of methods have been developed for delivering antisense DNA or RNA to cells, e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.

[0121] However, it may be difficult to achieve intracellular concentrations of the antisense oligonucleotide sufficient to suppress translation on endogenous mRNAs in certain instances. Therefore, another approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.

[0122] Expression of the sequence encoding the antisense RNA can be by a promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to the SV40 early promoter region (Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Cell 22:787-797), the herpes thymidine kinase promoter (Proc Natl Acad Sci 78:1441-1445), the regulatory sequences of the metallothionein gene (Nature 296:39-42), etc. A type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systematically).

[0123] In other embodiments, SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression in a cell can be increased, elevated, and / or promoted in several ways including: introduction of genes (e.g., expression vector or DNA construct) that express the SCAN / BLVRB; activation of genes and / or proteins that promote one or more of SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression; introduction of genes and / or proteins that inhibit genes, enzymes, or proteins that negatively regulate one or more of SCAN / BLVRB S-nitrosylation activity, S- nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression (e.g., by using recombinant gene expression vectors, recombinant viral vectors or recombinant polypeptides.

[0124] In particular embodiments the promoter of SCAN / BLVRB mediated S- nitrosylation is an expression vector or DNA construct that promotes expression or expresses SCAN / BLVRB in a cell of the subject.

[0125] In some embodiments, to increase the nuclear or cellular concentration of SCAN / BLVRB, or fragments or variants thereof, either SCAN / BLVRB or the fragments or variants thereof can be introduced into the cell directly or expressed therein via in vivo cell transformation.

[0126] The SCAN / BLVRB can include any mammalian BVLRB, but preferably human BVLRB (“hBLVRB”). In addition, SCAN / BLVRB from other mammals, including withoutlimitation, rat, mouse pig, and chimp, can be recombinantly expressed and isolated for use in a composition or method described herein. In addition to mammalian BVLRB, non- mammalian BVLRB that is sufficiently homologous to the mammalian BVLRB can be used. Non-mammalian BVLRB sequences can be identified by similar homology search to human BVLRB, particularly using BLAST or motif searches for those regions highly conserved between the two BVLRB sequences.

[0127] SCAN / BVLRB variants and fragments can be substituted for SCAN / BVLRB either in whole or in part. SCAN / BVLRB fragments can include N-terminal, internal, and C- terminal fragments. Subclones of a gene encoding a known BVLRB can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), and Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999 and preceding editions), each of which is hereby incorporated by reference in its entirety. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or polypeptide that can be tested for a particular activity.

[0128] In another approach, based on knowledge of the primary structure of the protein, fragments of a BVLRB gene may be synthesized using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein (Erlich et al., Science 252:1643-51 (1991), which is hereby incorporated by reference in its entirety). These can then be cloned into an appropriate vector for expression of a truncated protein or polypeptide from bacterial cells as described above. For example, oligomers of at least about 15 to 20 nt in length can be selected from a nucleic acid molecule encoding hBVLRB for use as primers.

[0129] In addition, chemical synthesis can also be employed using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, J. Am. Chem. Assoc.85:2149-2154 (1964), which is hereby incorporated by reference in its entirety) or synthesis in homogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E. Wansch, Vol.15, I and II, Thieme, Stuttgart (1987), which is hereby incorporated by reference in its entirety).

[0130] Variants of SCAN / BLVRB proteins or polypeptides can also be expressed. Variants may be made by, for example, the deletion, addition, or alteration of amino acidsthat have either (i) minimal influence on certain properties, secondary structure, and hydropathic nature of the polypeptide or (ii) substantial effect on one or more properties of SCAN / BLVRB. Variants of SCAN / BLVRB can also be fragments of SCAN / BLVRB that include one or more deletion, addition, or alteration of amino acids of the type described above. The SCAN / BLVRB variant can contain a deletion, addition, or alteration of amino acids within one or more functional domains. The substituted or additional amino acids can be either L-amino acids, D-amino acids, or modified amino acids, preferably L-amino acids. Whether a substitution, addition, or deletion results in modification of SCAN / BLVRB variant activity may depend, at least in part, on whether the altered amino acid is conserved. Conserved amino acids can be grouped either by molecular weight or charge and / or polarity of R groups, acidity, basicity, and presence of phenyl groups, as is known in the art.

[0131] Variants may also include, for example, a polypeptide conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, identification, or therapeutic use (i.e., delivery) of the polypeptide.

[0132] Another variant type of SCAN / BLVRB is a fusion polypeptide that includes a fragment of SCAN / BLVRB containing the YMKM motif. The fusion protein can be expressed or synthesized using an in-frame gene fusion according to known techniques in the art. The SCAN / BLVRB fragment can be coupled to a cytoplasmic localization signal. A number of cytoplasmic localization signals have been identified in the art and can be utilized in combination with the fragment of SCAN / BLVRB to obtain the fusion protein.

[0133] It will be appreciated that any mammalian or non-mammalian SCAN / BLVRB sequence can be used in the formation of the chimeric genes and expression systems as described herein. Homologous SCAN / BVR polypeptides from mammals and non-mammals other than those described above can be characterized by an amino acid identity of at least about 60 percent, more preferably at least about 70 percent or 80 percent, most preferably at least about 85 percent or 90 percent or 95 percent as compared to a hBLVRB. Other mammalian and non-mammalian cDNA molecules can be identified based upon their alignment with a hBLVRB cDNA sequence, where such alignment preferably is at least about 60 percent identical (more preferably at least about 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent identical). Alternatively, other mammalian BLVRBencoding cDNA molecules can be identified by the ability of mammalian cDNA sequences to hybridize to the complement of hBLVRB or similar sequence, under stringent hybridization and wash conditions. Exemplary stringent hybridization and wash conditions include, without limitation, hybridization at 50°C or higher (i.e., 55° C., 60° C., or 65° C.) in a hybridization medium that includes 0.9× (or higher, such as 2× or 5×) sodium citrate (“SSC”) buffer, followed by one or more washes at increasing stringency using 0.2×SSC buffer at temperatures from 42°C up to the temperature of the hybridization step. Higher stringency can readily be attained by increasing the temperature for either hybridization or washing conditions or decreasing the sodium concentration of the hybridization or wash medium. Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency.

[0134] The SCAN / BLVRB protein or polypeptide (or fragment or variant thereof) can be recombinantly produced, isolated, and then purified, if necessary. When recombinantly produced, the SCAN / BLVRB protein or polypeptide (or fragment or variant thereof) is expressed in a recombinant host cell, typically, although not exclusively, a prokaryote.

[0135] When a prokaryotic host cell is selected for subsequent transformation, the promoter region used to construct the recombinant DNA molecule (i.e., transgene) should be appropriate for the particular host. The DNA sequences of eukaryotic promoters, as described infra for expression in eukaryotic host cells, differ from those of prokaryotic promoters. Eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.

[0136] Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacentDNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

[0137] Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D- galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

[0138] Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and / or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgamo (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include, but are not limited to, the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

[0139] Mammalian cells can also be used to recombinantly produce SCAN / BLVRB or fragments or variants thereof. Mammalian cells suitable for carrying out the present invention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No.1573), CHOP, and NS-1 cells.

[0140] Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcription and translation control sequences known in the art. Common promoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV- LTR.

[0141] Regardless of the selection of host cell, once the DNA molecule coding for a SCAN / BLVRB protein or polypeptide (or fragment or variant thereof, has been ligated to itsappropriate regulatory regions (or chimeric portions) using well known molecular cloning techniques, it can then be introduced into a suitable vector or otherwise introduced directly into a host cell using transformation protocols well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety).

[0142] When an expression vector is used for purposes of in vivo transformation to induce or inhibit SCAN / BLVRB expression in a target cell, promoters of varying strength and specificity can be employed depending on the degree of enhancement of suppression desired. One of skill in the art can readily select appropriate constitutive mammalian promoters based on their strength as a promoter.

[0143] As an alternative to constitutive promoters, a mammalian tissue-specific promoter can be utilized. Any of a variety of tissue specific promoters are known in the art and can be selected based upon the tissue or cell type to be treated.

[0144] Muscle-specific promoters can be smooth muscle-specific, skeletal muscle- specific, or cardiac muscle-specific. Exemplary muscle-specific promoters include, without limitation, PGC-1α promoter (U.S. Patent Application 20060035849 to Spiegelman et al., which is hereby incorporated by reference in its entirety); creatine kinase promoter (Sun et al., Mol. Ther.11(6):889-98 (2005), which is hereby incorporated by reference in its entirety); mef2c promoter (Heidt et al., Genesis 42(1):28-32 (2005), which is hereby incorporated by reference in its entirety); MuSK promoter (Tang et al., J. Biol. Chem. 281(7):3943-53 (2006), which is hereby incorporated by reference in its entirety).

[0145] Exemplary neuron specific promoters include, without limitation, Thyl promoter (Vidal et al., EMBO J.9:833-840 (1990); Eckenstein et al., Exp Neurol. (online advance publication Feb.15, 2006), each of which is hereby incorporated by reference in its entirety); PrP promoter (Asante et al., Neurobiol Dis.10(1): 1-7 (2002), which is hereby incorporated by reference in its entirety); neuron-specific enolase promoter (Kuhn et al., Eur. J. Neurosci. 22(8):1907-15 (2005), which is hereby incorporated by reference in its entirety); and CaMKIIα promoter (Michalon et al., Genesis 43(4):205-12 (2005), which is hereby incorporated by reference in its entirety).

[0146] Exemplary liver specific promoters include, without limitation, serum amyloid P component promoter (Tanaka et al., Metabolism 54(11):1490-8 (2005), which is hereby incorporated by reference in its entirety); Apo-E promoter (Kakumitsu et al., Leuk Res.29(7):761-9 (2005), which is hereby incorporated by reference in its entirety); alpha 1- antitrypsin (AAT) (Al-Dosari et al., Biochem. Biophys. Res. Commun.339(2):673-8 (2006), which is hereby incorporated by reference in its entirety).

[0147] Exemplary kidney specific promoters include, without limitation, cadherin promoter (Yang et al., Am. J. Physiol. Renal Physiol online advance publication Jan.31, 2006, which is hereby incorporated by reference in its entirety); uromodulin promoter (Huang et al., BMC Biotechnol.5(1):9 (2005); Kim et al., Transgenic Res.12(2):191-201 (2003), each of which is hereby incorporated by reference in its entirety); CLC-K1 and CLC-K2 promoters (Uchida et al., Kidney Int.60(2):416-21 (2001), which is hereby incorporated by reference in its entirety); P1-PTHR promoter (Amizuka et al., Endocrinology.138(1):469-81 (1997), which is hereby incorporated by reference in its entirety).

[0148] Other tissue-specific promoters are known in the art and can be utilized in the present invention to obtain a tissue-specific recombinant gene that encodes SCAN / BLVRB (or fragment or variant thereof).

[0149] Whether the promoter is tissue-specific or not, the promoter can also be made inducible for purposes of controlling when expression of SCAN / BLVRB is desired. One of skill in the art can readily select appropriate inducible mammalian promoters from those known in the art. One exemplary inducible promoter includes a Tet-O response element (Farson et al., Hum. Gene Ther.12(8):981-97 (2001), which is hereby incorporated by reference in its entirety). When used in combination with a tissue-specific promoter, the Tet- O response elements can render a tissue-specific promoter inducible to tetracycline and its derivatives (see, e.g., Michalon et al., Genesis 43(4):205-12 (2005), which is hereby incorporated by reference in its entirety).

[0150] The recombinant molecule can be introduced into host cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. The host cells, when grown in an appropriate medium, are capable of expressing the biliverdin reductase (or fragment or variant thereof), which can then be isolated therefrom and, if necessary, purified. The SCAN / BLVRB, or fragment or variant thereof, is preferably produced in purified form (preferably at least about 60%, more preferably 80%, pure) by conventional techniques.

[0151] The cell in which the nuclear or cellular concentration of SCAN / BLVRB, or fragments or variants thereof, is to be modified can be located in vivo or ex vivo. The modification of SCAN / BLVRB nuclear or cellular concentrations can also be used as one part of a multi-component approach for treating diseases or disorders (i.e., generally, conditions) that are characterized by aberrant or dysregulated nitrosylation. Such complimentary treatments can be any suitable therapy, whether now known or hereafter developed.

[0152] The nuclear or cellular concentration of SCAN / BLVRB (or fragments or variants thereof) can be modified according to a number of approaches, either by delivering the SCAN / BLVRB (or fragments or variants thereof) that affords the protein or polypeptide to be active within the cell, or by delivering DNA encoding SCAN / BLVRB (or fragments or variants thereof) into the cell in a manner effective to induce the expression thereof in the cell.

[0153] When SCAN / BLVRB (or fragments or variants thereof) is delivered into target cells, it may be desirable that such delivery be effective to cause nuclear uptake of the SCAN / BLVRB (or fragments or variants thereof). For example, SCAN / BLVRB or fragments or variants may contain the native SCAN / BLVRB nuclear localization signal or a chimeric nuclear localization signal. In another embodiment, a variant SCAN / BLVRB can be prepared so that it lacks a functional nuclear localization signal, in which case the variant will remain in the cytoplasmic fraction of a cell into which it is introduced or expressed.

[0154] One approach for delivering therapeutic protein or polypeptides or nucleic acid molecules into cells involves the use of liposomes. Basically, this involves providing a liposome which includes that protein or polypeptide or nucleic acid to be delivered, and then contacting the target cell with the liposome under conditions effective for delivery of the protein or polypeptide or nucleic acid into the cell.

[0155] Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature. Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through theaction of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.

[0156] In contrast to passive drug release, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), each of which is hereby incorporated by reference in its entirety). When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.

[0157] Alternatively, the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane, which enzyme slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve “on demand” drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release.

[0158] This liposome delivery system can also be made to accumulate at a target organ, tissue, or cell via active targeting (e.g., by incorporating an antibody or hormone on the surface of the liposomal vehicle). This can be achieved according to known methods.

[0159] Different types of liposomes can be prepared according to Bangham et al., J. Mol. Biol.13:238-252 (1965); U.S. Pat. No.5,653,996 to Hsu et al.; U.S. Pat. No.5,643,599 to Lee et al.; U.S. Pat. No.5,885,613 to Holland et al.; U.S. Pat. No.5,631,237 to Dzau et al.; and U.S. Pat. No.5,059,421 to Loughrey et al., each of which is hereby incorporated by reference in its entirety.

[0160] Like liposomes, micelles have also been used in the art for drug delivery. A number of different micelle formulations have been described in the literature for use in delivery proteins or polypeptides, and others have been described which are suitable for delivery of nucleic acids. Any suitable micelle formulations can be adapted for delivery of the therapeutic protein or polypeptide or nucleic acids of the present invention. Exemplary micelles include without limitation those described, e.g., in U.S. Pat. No.6,210,717 to Choi etal.; and U.S. Pat. No.6,835,718 to Kosak, each of which is hereby incorporated by reference in its entirety.

[0161] An alternative approach for delivery of proteins or polypeptides or nucleic acids involves the conjugation of the desired therapeutic agent to a polymer that is stabilized to avoid enzymatic degradation of the conjugated protein or polypeptide. Conjugated proteins or polypeptides of this type are described in U.S. Pat. No.5,681,811 to Ekwuribe, which is hereby incorporated by reference in its entirety.

[0162] Yet another approach for delivery of proteins or polypeptides involves preparation of chimeric proteins according to U.S. Pat. No.5,817,789 to Heartlein et al., which is hereby incorporated by reference in its entirety. The chimeric protein can include a ligand domain and, e.g., SCAN / BLVRB or a fragment or variant thereof as described above. The ligand domain is specific for receptors located on a target cell. Thus, when the chimeric protein is delivered intravenously or otherwise introduced into blood or lymph, the chimeric protein will adsorb to the targeted cell, and the targeted cell will internalize the chimeric protein.

[0163] When it is desirable to achieve heterologous expression of a desirable protein or polypeptide in a target cell, DNA molecules encoding the desired protein or polypeptide can be delivered into the cell. Basically, this includes providing a nucleic acid molecule encoding the protein or polypeptide, and then introducing the nucleic acid molecule into the cell under conditions effective to express the protein or polypeptide in the cell. Preferably, this is achieved by inserting the nucleic acid molecule into an expression vector before it is introduced into the cell.

[0164] Any suitable viral or infective transformation vector can be used. Exemplary viral vectors include, without limitation, adenovirus, adeno-associated virus, and retroviral vectors (including lentiviral vectors).

[0165] Adenovirus gene delivery vehicles can be readily prepared and utilized given the disclosure provided in Berkner, Biotechniques 6:616-627 (1988) and Rosenfeld et al., Science 252:431-434 (1991), WO 93 / 07283, WO 93 / 06223, and WO 93 / 07282, each of which is hereby incorporated by reference in its entirety. Additional types of adenovirus vectors are described in U.S. Pat. No.6,057,155 to Wickham et al.; U.S. Pat. No.6,033,908 to Bout et al.; U.S. Pat. No.6,001,557 to Wilson et al.; U.S. Pat. No.5,994,132 to Chamberlain et al.; U.S. Pat. No.5,981,225 to Kochanek et al.; U.S. Pat. No.5,885,808 toSpooner et al.; and U.S. Pat. No.5,871,727 to Curiel, each of which is hereby incorporated by reference in its entirety.

[0166] Adeno-associated viral gene delivery vehicles can be constructed and used to deliver into cells a recombinant gene encoding a desired nucleic acid. The use of adeno- associated viral gene delivery vehicles in vitro is described in Chattedee et al., Science 258:1485-1488 (1992); Walsh et al., Proc. Nat'l Acad. Sci. USA 89:7257-7261 (1992); Walsh et al., J. Clin. Invest.94:1440-1448 (1994); Flotte et al., J. Biol. Chem.268:3781-3790 (1993); Ponnazhagan et al., J. Exp. Med.179:733-738 (1994); Miller et al., Proc. Nat'l Acad. Sci. USA 91:10183-10187 (1994); Einerhand et al., Gene Flier.2:336-343 (1995); Luo et al., Exp. Hematol.23:1261-1267 (1995); and Zhou et al., Gene Thier.3:223-229 (1996), each of which is hereby incorporated by reference in its entirety. In vivo use of these vehicles is described in Flotte et al., Proc. Nat'l Acad. Sci. USA 90:10613-10617 (1993); and Kaplitt et al., Nature Genet.8:148-153 (1994), each of which is hereby incorporated by reference in its entirety.

[0167] Retroviral vectors which have been modified to form infective transformation systems can also be used to deliver a recombinant gene encoding a desired nucleic acid product into a target cell. One such type of retroviral vector is disclosed in U.S. Pat. No. 5,849,586 to Kriegler et al., which is hereby incorporated by reference in its entirety. Lentivirus vectors can also be utilized, including those described in U.S. Pat. No.6,790,657 to Arya, and U.S. patent Application Nos.20040170962 to Kafri et al. and 20040147026 to Arya, each of which is hereby incorporated by reference in its entirety.

[0168] Regardless of the type of infective transformation system employed, it should be targeted for delivery of the nucleic acid to a specific cell type. For example, for delivery of the nucleic acid into a cluster of cells, a high titer of the infective transformation system can be injected directly within the site of those cells so as to enhance the likelihood of cell infection. The infected cells will then express the desired product, in some embodiments SCAN / BLVRB (or fragments or variants thereof), to modify the expression of cell cycle or cell signaling proteins.

[0169] The agents that modulate SCAN / BLVRB mediated S-nitrosylation described herein can be provided in pharmaceutical compositions with at least one pharmaceutically acceptable carrier. Suitable carriers are described in "Remington: The Science and Practice, Twentieth Edition," published by Lippincott Williams & Wilkins, which is incorporatedherein by reference. Pharmaceutical compositions according to the invention may also comprise one or more non-inventive compound active agents.

[0170] The compositions comprising an agent that modulates SCAN / BLVRB mediated S-nitrosylation can be utilized in any pharmaceutically acceptable dosage form, including, but not limited to injectable dosage forms, liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release formulations, extended-release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, etc. Specifically, the SCAN / BLVRB mediated S-nitrosylation modulators can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, intravenous, intra-arterial, intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal, intraperitoneal, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, tablets, sachets, and capsules; (c) into a dosage form selected from the group consisting of lyophilized formulations, dry powders, fast melt formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination thereof.

[0171] For respiratory disorders, an inhalation formulation can be used to achieve high local concentrations. Formulations suitable for inhalation include dry power or aerosolized or vaporized solutions, dispersions, or suspensions capable of being dispensed by an inhaler or nebulizer into the endobronchial or nasal cavity of infected patients to treat upper and lower respiratory bacterial infections.

[0172] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can comprise one or more of the following components: (1) a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; (2) antibacterial agents such as benzyl alcohol or methyl parabens; (3) antioxidants such as ascorbic acid or sodium bisulfite; (4) chelating agents such as ethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates, or phosphates; and (5) agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A parenteralpreparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0173] Pharmaceutical compositions suitable for injectable use may comprise sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The pharmaceutical composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0174] The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol or sorbitol, and inorganic salts such as sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0175] Sterile injectable solutions can be prepared by incorporating the active reagent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating at least one compound of the invention into a sterile vehicle that contains a basic dispersion medium and any other required ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze-drying, both of which yield a powder of a compound of the invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0176] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed, for example, in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compound of the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition.

[0177] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, a nebulized liquid, or a dry powder from a suitable device. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active reagents are formulated into ointments, salves, gels, or creams as generally known in the art. The reagents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0178] In one embodiment, the SCAN / BLVRB mediated S-nitrosylation modulators are prepared with carriers that will protect against rapid elimination from the body. For example, a controlled release formulation can be used, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0179] Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0180] Additionally, suspensions of the agents of the invention may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also include suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[0181] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the compound of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the compound of the invention and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.

[0182] Pharmaceutical compositions that include the SCAN / BLVRB mediated S- nitrosylation modulators can comprise one or more pharmaceutical excipients. Examples of such excipients include, but are not limited to binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Exemplary excipients include: (1) binding agents which include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, silicified microcrystalline cellulose, gum tragacanth and gelatin; (2) filling agents such as various starches, lactose, lactose monohydrate, and lactose anhydrous; (3) disintegrating agents such as alginic acid, Primogel, corn starch, lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof; (4) lubricants, including agents that act on the flowability of a powder to be compressed, include magnesium stearate, colloidal silicon dioxide, talc, stearic acid, calcium stearate, and silica gel; (5) glidants such as colloidal silicon dioxide; (6) preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride; (7) diluents such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and / or mixtures of any of the foregoing; examples of diluents include microcrystalline cellulose; lactose suchas lactose monohydrate, and lactose anhydrous; dibasic calcium phosphate, mannitol; starch; sorbitol; sucrose; and glucose; (8) sweetening agents, including any natural or artificial sweetener, such as sucrose, saccharin sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9) flavoring agents, such as peppermint, methyl salicylate, orange flavoring, bubble gum flavor, fruit flavors, and the like; and (10) effervescent agents, including effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0183] In some embodiments, a pharmaceutical composition including a modulator of SCAN / BLVRB mediated S-nitrosylation can be used in therapy, such as in methods for preventing or treating (e.g., alleviating one or more symptoms of) medical conditions. The methods encompass a therapeutic regimen that results in a clinically desirable outcome and comprise administering a therapeutically effective amount of a modulator of SCAN / BLVRB mediated S-nitrosylation modulator to a patient or subject in need thereof. The compositions can also be used for prophylactic therapy, e.g., preventing diabetes in a subject.

[0184] A therapeutically effective amount of a modulator of SCAN / BLVRB mediated S-nitrosylation for treatment of a subject in need thereof is a modulating amount in vivo that causes amelioration of the disorder being treated or protects against a risk associated with the disorder. For example, lowering of S-nitrosylation levels by administrating an inhibitor of SCAN / BLVRB mediated S-nitrosylation to a subject in need thereof can (i) reduce SCAN / BLVRB mediated S-nitrosylation by at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or more relative to pre-dose level. In another example, promoting S-nitrosylation levels by administering a promoter of SCAN / BLVRB mediated S-nitrosylation to a subject in need thereof can (i) increase SCAN / BLVRB mediated S-nitrosylation by at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or more relative to pre-dose level.

[0185] In some embodiments, the modulator SCAN / BLVRB mediated S-nitrosylation activity can be an inhibitor of SCAN / BLVRB mediated S-nitrosylation activity that is used in a method of treating a hyper-S-nitrosylation associated disease or disorder in a subject in need thereof. The method can include administering to the subject an amount of an agent that inhibits SCAN / BLVRB mediated S-nitrosylation as described herein. In some embodiments, INSRβ / IRS1 in the subject is hypernitrosylated and the inhibitor of SCAN / BLVRB activity is administered at an amount effective to inhibit hypernitrosylation.

[0186] For example, it has been shown that inhibition of insulin signaling by (SCAN / BLVRB) / SNO-CoA defines a novel mode of receptor tyrosine kinase (RTK) regulation causing diabetes. As illustrated in Fig.4, S-nitrosylation of INSRβ / IRS1 by (SCAN / BLVRB) / SNO-CoA is induced by insulin as a feedback mechanism to turn off insulin signaling and avoid hypoglycemia. In obesity, sustained hyper-S-nitrosylation of INSRβ / IRS1 by (SCAN / BLVRB) / SNO-CoA blocks insulin signaling and leads to insulin resistance and an inhibitor of SCAN / BLVRB mediated S-nitrosylation can be used to regulate insulin resistance in the treatment of diabetes. Therefore, in particular embodiments, a pharmaceutical composition including an inhibitor of SCAN / BLVRB mediated S- nitrosylation described herein can be used in a method of preventing or treating diabetes in a subject in need thereof. In a particular embodiment, the diabetes is type 2 diabetes.

[0187] Hyper-S-nitrosylation of proteins that is causal in insulin resistance (Fig.4) is also an established feature of other diseases including cholesterol related disorders, heart failure, neurodegenerative disorders, muscular dystrophy, infection, malignant hyperthermia, inflammatory disorders, cell proliferative disorders (e.g., cancer) and shock states and therefore an inhibitor of SCAN / BLVRB mediated S-nitrosylation can be useful in treating and / or preventing cholesterol related disorders, heart failure, neurodegenerative disorders, muscular dystrophy, infection, malignant hyperthermia, inflammatory disorders, cell proliferative disorders (e.g., cancer) and shock states associated with hyper-S-nitrosylation in a subject in need thereof.

[0188] The cholesterol related disorder (which includes serum cholesterol related disorders) can include any one or more of the following: familial hypercholesterolemia, non- familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular diseases, Alzheimer's disease and generally dyslipidemias, which can be manifested, for example, by an elevated total serum cholesterol,elevated LDL, elevated triglycerides, elevated VLDL, and / or low HDL. Some non-limiting examples of primary and secondary dyslipidemias that can be treated using an SCAN / BLVRB inhibitor, either alone, or in combination with one or more other agents include the metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemias, including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial defective apoplipoprotein B-100; polygenic hypercholesterolemia; remnant removal disease, hepatic lipase deficiency; dyslipidemia secondary to any of the following: dietary indiscretion, hypothyroidism, drugs including estrogen and progestin therapy, beta-blockers, and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia.

[0189] In some embodiments, a pharmaceutical composition comprising an inhibitor of SCAN / BLVRB mediated S-nitrosylation can be used in a therapeutic method for the reduction of LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments, the therapy is the treatment of hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, acute coronary syndrome, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type I diabetes, type 2 diabetes, type 2 diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease, peripheral vascular disease and stroke. In additional embodiments, the therapy is the reduction of CHD risk. In certain aspects, the therapy is treatment or prevention of atherosclerosis. In certain embodiments, the therapy is treatment or prevention of atherosclerosis-induced ischemic stroke (AIIS) in a subject. In certain embodiments, the therapy is the prevention of coronary heart disease. In other embodiments, a pharmaceutical composition comprising an inhibitor of SCAN / BLVRB mediated S-nitrosylation can be used in a therapeutic method for promoting platelet generation.

[0190] In some embodiments, the neurodegenerative disease treated using an inhibitor of SCAN / BLVRB mediated S-nitrosylation containing pharmaceutical composition isselected from the group consisting of amyloid lateral sclerosis (ALS), Alzheimer's, Parkinson’s, and Huntington’s disease.

[0191] The term "cell proliferative disorder" refers to conditions in which the unregulated and / or abnormal growth of cells can lead to the development of an unwanted condition or disease, which can be cancerous or non-cancerous, for example a psoriatic condition. As used herein, the term "psoriatic condition" refers to disorders involving keratinocyte hyperproliferation, inflammatory cell infiltration, and cytokine alteration. The cell proliferative disorder can be a precancerous condition or cancer. The cancer can be primary cancer or metastatic cancer, or both.

[0192] As used herein, the term "cancer" includes solid tumors, such as lung, breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamous carcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma or hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, malignant melanoma, non-melanoma skin cancers, as well as hematologic tumors and / or malignancies, such as leukemia, childhood leukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia such as acute lymphoblastic, acute myelocytic, or chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm, and cancers associated with AIDS.

[0193] In addition to psoriatic conditions, the types of proliferative diseases which may be treated using the compositions of the present invention are epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, and other dysplastic masses, and the like. In one embodiment, proliferative diseases include dysplasias and disorders of the like.

[0194] In some embodiments, treating cancer can include a reduction in tumor size, decrease in tumor number, a delay of tumor growth, decrease in metastatic lesions in other tissues or organs distant from the primary tumor site, an improvement in the survival of patients, or an improvement in the quality of patient life, or at least two of the above.

[0195] In another embodiment, treating a cell proliferative disorder comprises a reduction in the rate of cellular proliferation, reduction in the proportion of proliferating cells, a decrease in size of an area or zone of cellular proliferation, or a decrease in the number orproportion of cells having an abnormal appearance or morphology, or at least two of the above.

[0196] In certain embodiments, the cancer treated using a pharmaceutical composition described herein can be selected from the group consisting of a liver cancer, such as hepatocellular carcinoma (HCC) or cholangiocarcinoma, prostate cancer, and acute lymphoblastic leukemia (ALL).

[0197] In some embodiments, a therapeutically effective amount of an inhibitor of SCAN / BLVRB mediated S-nitrosylation activity for treatment of a subject afflicted with pathologically proliferating cells in vivo can include an antiproliferative effective amount. Such antiproliferative effective amount as used herein means an amount causing reduction in rate of proliferation of at least about 20%, at least about 10%, at least about 5%, or at least about 1%.

[0198] In certain embodiments, an inhibitor of SCAN / BLVRB mediated S-nitrosylation can be co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the inhibitor of SCAN / BLVRB mediated S-nitrosylation. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the inhibitor of SCAN / BLVRB mediated S-nitrosylation. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of inhibitor of SCAN / BLVRB mediated S-nitrosylation. In certain embodiments, one or more pharmaceutical compositions described herein are co- administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent.

[0199] In certain embodiments pharmaceutical agents that may be co-administered with the inhibitor of SCAN / BLVRB mediated S-nitrosylation herein include lipid-lowering agents. For example, lipid-lowering agents can be administered in combination with an inhibitor of SCAN / BLVRB mediated S-nitrosylation containing composition for the treatment or prevention of cholesterol related disorder. In certain such embodiments, pharmaceutical agents that may be co-administered with an inhibitor of SCAN / BLVRB mediated S- nitrosylation include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to, atthe same time as, or following administration of an inhibitor of SCAN / BLVRB mediated S- nitrosylation.

[0200] In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid- lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid- lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.

[0201] In certain embodiments, a co-administered lipid-lowering agent is an HMG- CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments, the statin is selected from, for example, atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.

[0202] In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe.

[0203] In certain embodiments, a co-administered lipid-lowering agent is a co- formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe / simvastatin.

[0204] In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).

[0205] In certain embodiments, a co-administered lipid-lowering agent is an oligonucleotide targeted to ApoB.

[0206] In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.

[0207] In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended-release nicotinic acid, and sustained release nicotinic acid.

[0208] In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

[0209] Further examples of pharmaceutical agents that may be co-administered with an inhibitor of SCAN / BLVRB mediated S-nitrosylation described herein, include, but are not limited to, corticosteroids, including but not limited to prednisone; LXR agonists; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non- steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.

[0210] In other embodiments, the inhibitor of SCAN / BLVRB mediated S-nitrosylation can be used in combination with surgical procedures such as angioplasty for cardiovascular diseases. Angioplasty is often accompanied by the placement of a reinforcing a metallic tube-shaped structure known as a "stent" into a damaged coronary artery. For more serious conditions, open heart surgery such as coronary bypass surgery may be required. These surgical procedures entail using invasive surgical devices and / or implants and are associated with a high risk of restenosis and thrombosis. Accordingly, the inhibitors of SCAN / BLVRB mediated S-nitrosylation may be used as coatings on surgical devices (e.g., catheters) and implants (e.g., stents) to reduce the risk of restenosis and thrombosis associated with invasive procedures used in the treatment of cardiovascular diseases.

[0211] An inhibitor of SCAN / BLVRB mediated S-nitrosylation may also be co- administered with a phosphodiesterase inhibitor (e.g., rolipram, cilomilast, roflumilast, VIAGRA (sildenifil citrate), CIALIS (tadalafil), LEVITRA (vardenifil), etc.), a β-agonist, a steroid, or a leukotriene antagonist (LTD-4). Those skilled in the art can readily determine the appropriate therapeutically effective amount depending on the disorder to be ameliorated.

[0212] The inhibitor of SCAN / BLVRB mediated S-nitrosylation may be used as a means to improve β-adrenergic signaling. In particular, the SCAN / BLVRB inhibitor alone or in combination with β-agonists could be used to treat or protect against heart failure, or other vascular disorders such as hypertension and asthma. The inhibitor of SCAN / BLVRBmediated S-nitrosylation can also be used to modulate G protein coupled receptors (GPCRs) by potentiating Gs G-protein, leading to smooth muscle relaxation (e.g., airway and blood vessels), and by attenuating Gq G-protein, and thereby preventing smooth muscle contraction (e.g., in airway and blood vessels).

[0213] In yet another embodiment, where the disorder to be treated is a cell proliferative disorder, such as cancer, the inhibitor of SCAN / BLVRB mediated S- nitrosylation can be administered in combination with a second chemotherapeutic agent. The second chemotherapeutic agent can include, for example, tamoxifen, raloxifene, anastrozole, exemestane, letrozole, cisplatin, carboplatin, paclitaxel, cyclophosphamide, lovastatin, minosine, gemcitabine, araC, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristin, vinblastin, nocodazole, teniposide, etoposide, epothilone, navelbine, camptothecin, daunonibicin, dactinomycin, mitoxantrone, amsacrine, doxorubicin, epirubicin, idarubicin imatanib, gefitinib, erlotinib, sorafenib, sunitinib malate, trastuzumab, rituximab, cetuximab, or bevacizumab.

[0214] In one embodiment, the inhibitor of SCAN / BLVRB mediated S-nitrosylation can be administered in combination with an agent that imposes nitrosative or oxidative stress. Agents for selectively imposing nitrosative stress to inhibit proliferation of pathologically proliferating cells in combination therapy with the SCAN / BLVRB inhibitor and dosages and routes of administration therefor include those disclosed in U.S. Pat. No.6,057,367, which is incorporated herein.

[0215] In some embodiments, the expression level of SCAN / BLVRB can be determined prior to administration of the SCAN / BLVRB inhibitor and / or other inhibitor of SCAN / BLVRB mediated S-nitrosylation described herein. It was found that expression of the SCAN / BLVRB is upregulated both in skeletal muscle and in visceral and subcutaneous adipose samples from patients with a higher body mass index (BMI). A significant linear correlation between amounts of SNO-INSRβ and the expression of SCAN / BLVRB protein in both human skeletal muscle and human adipose tissue implies that S-nitrosylation of INSRβ is likely mediated by SCAN / BLVRB in human tissues. Collectively, the multiple linear correlations among SCAN / BLVRB expression, BMI, and SNO-INSRb (taken together with animal data) suggests that overexpression of SCAN / BLVRB in patients, patients and particularly obese patients, contributes to insulin resistance through hypernitrosylation of INSRb and over expression in of SCAN / BLVRB in skeletal muscle or adipose tissue can beused as a marker for selecting patients who would benefit from treatment with the SCAN / BLVRB inhibitor and / or other inhibitor of SCAN / BLVRB mediated S-nitrosylation described herein. For example, the skeletal or adipose tissue expression of SCAN / BLVRB may be used as a marker for identifying or selecting suitable diabetic patient who would benefit from the SCAN / BLVRB inhibitor and / or other inhibitor of SCAN / BLVRB mediated S-nitrosylation described herein.

[0216] Although the methods of inhibition SCAN / BLVRB mediated S-nitrosylation described herein including assaying SCAN / BLVRB expression are described as comprising the selection of a patient that is, or is not, suitable for the SCAN / BLVRB inhibitor and / or other inhibitor of SCAN / BLVRB mediated S-nitrosylation described herein, it should be understood that these methods apply generally to the selection of a patient with over or under expression of SCAN / BLVRB expression. Further, in any the methods comprising the measurement of SCAN / BLVRB expression in a test tissue sample, it should be understood that the step comprising the provision of a test tissue sample obtained from a patient is an optional step. That is, in certain embodiments the method includes this step, and in other embodiments, this step is not included in the method. It should also be understood that in certain preferred embodiments the "assessing" step to identify, or determine the number or proportion of, cells in the test tissue sample that express SCAN / BLVRB is performed by a transformative method of assaying for SCAN / BLVRB expression, for example by performing an immunoassay or reverse transcriptase-polymerase chain reaction (RT-PCR) assay. In certain other embodiments, no transformative step is involved and SCAN / BLVRB expression is assessed by, for example, reviewing a report of test results from a laboratory. In certain embodiments, the steps of the methods up to, and including, assessing SCAN / BLVRB expression provides an intermediate result that may be provided to a physician or other medical practitioner for use in selecting a suitable candidate for SCAN / BLVRB inhibitor therapy and / or administering the SCAN / BLVRB inhibitor to the patient. In certain embodiments, the steps that provide the intermediate result may be performed by a medical practitioner or someone acting under the direction of a medical practitioner. In other embodiment, these steps are performed by an independent person or laboratory.

[0217] The disclosure further provides a method for treatment of a subject afflicted with a disorder potentially associated with the SCAN / BLVRB overexpression, such asdiabetes, which method comprises: (a) selecting a subject that is not suitable for treatment with the SCAN / BLVRB inhibitor, the selecting comprising (i) optionally providing a test tissue sample obtained from a patient with diabetes of the tissue, the test tissue sample comprising SCAN / BLVRB expressing cells; (ii) assessing the proportion of cells in the test tissue sample that SCAN / BLVRB; and (iii) selecting the subject as not suitable for therapy with a SCAN / BLVRB inhibitor, based on an assessment that the proportion of cells in the test tissue sample that express SCAN / BLVRB is less than a predetermined threshold level; and (b) administering a standard-of-care therapeutic other than the SCAN / BLVRB to the selected subject.

[0218] In certain embodiments of any of the methods described herein, the proportion of cells that express SCAN / BLVRB is assessed by performing an assay to determine the presence of SCAN / BLVRB RNA. In further embodiments, the presence of SCAN / BLVRB RNA is determined by RT-PCR, in situ hybridization or RNase protection. In other embodiments, the proportion of cells that express SCAN / BLVRB is assessed by performing an assay to determine the presence of SCAN / BLVRB. In further embodiments, the presence of SCAN / BLVRB is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry.

[0219] In certain embodiments, the SCAN / BLVRB inhibitor can be administered to a subject where the SCAN / BLVRB expression level exceeds a predetermined threshold value. The predetermined threshold value relating to, for example, SCAN / BLVRB expression in skeletal or adipose tissue. The predetermined threshold is based on a level of SCAN / BLVRB expression in a test tissue sample. In addition, in any method where administration of a SCAN / BLVRB inhibitor is selected or administered based on an assessment that the measured level of SCAN / BLVRB in a test tissue sample from the subject is below a predetermined threshold level, it follows that a complementary method of treatment may be performed wherein a standard-of-care treatment other than the SCAN / BLVRB inhibitor is selected or administered to the subject.

[0220] This disclosure further provides a method for predicting the therapeutic effectiveness of an SCAN / BLVRB inhibitor for treating a subject afflicted with a disorder potentially associated with the SCAN / BLVRB overexpression, such as diabetes, which method comprises: (a) optionally providing a test tissue sample obtained from a patient with the disorder, such as diabetes, the test tissue sample comprising skeletal or adipose tissue; (b)assaying the test tissue sample to determine the level of SCAN / BLVRB expression; (c) comparing the level of SCAN / BLVRB with a predetermined threshold value; and (d) predicting the therapeutic effectiveness of the SCAN / BLVRB inhibitor in treating the disorder, e.g., diabetes, wherein if the level of SCAN / BLVRB exceeds the threshold level the SCAN / BLVRB inhibitor is predicted to be effective in treating the patient, and wherein if the proportion of cells that express SCAN / BLVRB is below the threshold level the SCAN / BLVRB inhibitor is predicted to not be effective in treating the patient.

[0221] In other embodiments, the modulator of SCAN / BLVRB mediated S- nitrosylation can be a promoter of SCAN / BLVRB mediated S-nitrosylation that is used in a method of treating a hyponitrosylation associated disease or disorder in a subject in need thereof. In some embodiments, the promoter of SCAN / BLVRB mediated S-nitrosylation can be administered to a subject at an amount effective to promote S-nitrosylation of INSRβ / IRS1 in a subject having the hyponitrosylation associated disease or disorder.

[0222] Administration of a promoter of SCAN / BLVRB mediated S-nitrosylation to a subject in need thereof can treat diseases and disorders associated with NO / SNO deficiency or disruptions in protein S-nitrosylation. For example, it has been shown that storage of red blood cells (RBCs) leads to a rapid depletion of S-nitrosylated hemoglobin (SNO-Hb), a principal regulator of tissue oxygen delivery. In addition, heart disease, diabetes, Cystic Fibrosis, asthma, sickle cell disease, pulmonary hypertension, stroke, multiple sclerosis, and ischemia are among the many conditions characterized by diminished SNOs. Loss of SNO- Hb also impairs the ability of banked blood to dilate blood vessels after transfusion, resulting in exacerbation rather than correction of anemia-induced reduction in tissue oxygenation.

[0223] Accordingly, in some embodiments, a promoter of SCAN / BLVRB mediated S- nitrosylation can be administered to a subject to raise SNO levels and increase S-nitrosylation of proteins in the subject and treat disorders associated with NO / SNO deficiency or disruptions in protein S-nitrosylation, promote maintenance (or restoration) of SNO-Hb levels ("renitrosylation"), lower cholesterol levels, treat ischemia, and treat disorders associated with NO / SNO deficiency, such as cystic fibrosis, asthma, inflammatory bowel disease, hypertension, heart failure, acute coronary syndromes, impotence, stroke, septic shock, as well as promote liver regeneration, stem cell enhancement, antimicrobial activity, and protect against ischemic injury, including renal ischemia and cardiac ischemia.

[0224] In some embodiments, a promoter of SCAN / BLVRB mediated S-nitrosylation can be used in a method of treating a subject afflicted with a disorder ameliorated by NO donor therapy. Such disorders can include pulmonary disorders associated with hypoxemia and / or smooth muscle constriction in the lungs and airways and / or lung infection and / or lung inflammation and / or lung injury (e.g., pulmonary hypertension, ARDS, asthma, pneumonia, pulmonary fibrosis / interstitial lung diseases, cystic fibrosis, COPD); cardiovascular disease and heart disease (e.g., hypertension, ischemic coronary syndromes, atherosclerosis, heart failure, glaucoma); diseases characterized by angiogenesis (e.g., coronary artery disease); disorders where there is risk of thrombosis occurring; disorders where there is risk of restenosis occurring; inflammatory diseases (e.g., AIDS related dementia, inflammatory bowel disease (IBD), Crohn's disease, colitis, and psoriasis); functional bowel disorders (e.g., irritable bowel syndrome (IBS)); diseases where there is risk of apoptosis occurring (e.g., heart failure, atherosclerosis, degenerative neurologic disorders, arthritis, and liver injury (ischemic or alcoholic)); impotence; sleep apnea; diabetic wound healing; cutaneous infections; treatment of psoriasis; obesity caused by eating in response to craving for food; stroke; reperfusion injury (e.g., traumatic muscle injury in heart or lung or crush injury); and disorders where preconditioning of heart or brain for NO protection against subsequent ischemic events is beneficial, central nervous system (CNS) disorders (e.g., anxiety, depression, psychosis, and schizophrenia); and infections caused by bacteria (e.g., tuberculosis, C. difficile infections, among others).

[0225] In other embodiments, a promoter of SCAN / BLVRB mediated S-nitrosylation can be used to treat a subject that exhibits at least one symptom of an ischemic tissue or tissue damaged by ischemia. In some embodiments, the subject is a human who is has or who is at risk of having an ischemic tissue or tissue damaged by ischemia, e.g., a subject that has diabetes, peripheral vascular disease, thromboangiitis obliterans, vasculitis, cardiovascular disease, coronary artery disease or heart failure, or cerebrovascular disease, cardiovascular disease, or cerebrovascular disease.

[0226] Illustrative examples of genetic disorders, syndromic conditions, traumatic injuries, chronic conditions, medical interventions, or other conditions that cause or are associated with ischemia, or increase the risk of ischemia in a subject, or cause a subject to exhibit more or more symptoms of ischemia, and thus, suitable for treatment or amelioration using the methods described herein, include, but are not limited to, acute coronary syndrome,acute lung injury (ALI), acute myocardial infarction (AMI), acute respiratory distress syndrome (ARDS), arterial occlusive disease, arteriosclerosis, articular cartilage defect, aseptic systemic inflammation, atherosclerotic cardiovascular disease, autoimmune disease, bone fracture, bone fracture, brain edema, brain hypoperfusion, Buerger's disease, bums, cancer, cardiovascular disease, cartilage damage, cerebral infarct, cerebral ischemia, cerebral stroke, cerebrovascular disease, chemotherapy-induced neuropathy, chronic infection, chronic mesenteric ischemia, claudication, congestive heart failure, connective tissue damage, contusion, coronary artery disease (CAD), critical limb ischemia (CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcer healing, delayed wound -healing, diabetes (type I and type II), diabetic neuropathy, diabetes induced ischemia, disseminated intravascular coagulation (DIC), embolic brain ischemia, graft-versus-host disease, frostbite, hereditary hemorrhagic telengiectasiaischemic vascular disease, hyperoxic injury, hypoxia, inflammation, inflammatory bowel disease, inflammatory disease, injured tendons, intermittent claudication, intestinal ischemia, ischemia, ischemic brain disease, ischemic heart disease, ischemic peripheral vascular disease, ischemic placenta, ischemic renal disease, ischemic vascular disease, ischemic-reperfusion injury, laceration, left main coronary artery disease, limb ischemia, lower extremity ischemia, myocardial infarction, myocardial ischemia, organ ischemia, osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease, peripheral arterial disease (PAD), peripheral artery disease, peripheral ischemia, peripheral neuropathy, peripheral vascular disease, pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder, renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers, solid organ transplantation, spinal cord injury, stroke, subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissue ischemia, transient ischemic attack (TIA), traumatic brain injury, ulcerative colitis, vascular disease of the kidney, vascular inflammatory conditions, von Rippel-Lindau syndrome, and wounds to tissues or organs.

[0227] Other illustrative examples of genetic disorders, syndromic conditions, traumatic injuries, chronic conditions, medical interventions, or other conditions that cause or are associated with ischemia, or increase the risk of ischemia in a subject, or cause a subject to exhibit more or more symptoms of ischemia suitable for treatment or amelioration using the methods of the present invention, include, ischemia resulting from surgery, chemotherapy, radiation therapy, or cell, tissue, or organ transplant or graft.

[0228] In some embodiments, a promoter of SCAN / BLVRB mediated S-nitrosylation can be used for treating cerebrovascular ischemia, myocardial ischemia, limb ischemia (CLI), myocardial ischemia (especially chronic myocardial ischemia), ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, intestinal ischemia, and the like.

[0229] In various embodiments, pharmaceutical compositions described herein can be used to treat an ischemic tissue in which it is desirable to increase the blood flow, oxygen supply, glucose supply, or supply of nutrients to the tissue.

[0230] Still other embodiments described herein relate to use of a promoter of SCAN / BLVRB mediated S-nitrosylation for treating a subject afflicted with pathologically proliferating cells. The pathologically proliferating cells can be pathologically proliferating microbes. The microbes involved can be those where an alcohol dehydrogenase (ADH), such as S-nitrosoglutathione reductase (GSNOR), is expressed to protect the microbe from nitrosative stress or where a host cell infected with the microbe expresses the enzyme, thereby protecting the microbe from nitrosative stress. The term "pathologically proliferating microbes" is used herein to mean pathologic microorganisms including, but not limited to, pathologic bacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa, pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata. More detail on the applicable microbes is set forth at columns 11 and 12 of U.S. Pat. No.6,057,367. The term "host cells infected with pathologic microbes" includes not only mammalian cells infected with pathologic viruses but also mammalian cells containing intracellular bacteria or protozoa, e.g., macrophages containing Mycobacterium tuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi (typhoid fever).

[0231] In another embodiment, the pathologically proliferating cells can be pathologic helminths. The term "pathologic helminths" is used herein to refer to pathologic nematodes, pathologic trematodes and pathologic cestodes. More detail on the applicable helminths is set forth at column 12 of U.S. Pat. No.6,057,367.

[0232] In another embodiment, the pathologically proliferating cells can be pathologically proliferating mammalian cells. The term "pathologically proliferating mammalian cells" as used herein means cells of the mammal that grow in size or number in said mammal so as to cause a deleterious effect in the mammal or its organs. The term includes, for example, the pathologically proliferating or enlarging cells causing restenosis,the pathologically proliferating or enlarging cells causing benign prostatic hypertrophy, the pathologically proliferating cells causing myocardial hypertrophy, and proliferating cells at inflammatory sites such as synovial cells in arthritis or cells associated with a pre-cancerous, cancerous, or a non-cancerous cell proliferation disorder, for example a psoriatic condition.

[0233] In some embodiments, a promoter of SCAN / BLVRB mediated S-nitrosylation can be administered in combination with a second therapeutic agent such as, but not limited to a chemotherapeutic agent, an agent that imposes nitrosative or oxidative stress, vasodilators, prostanoid agonists, antiandrogens, cyclosporins and their analogues, antimicrobials, triterpenes, a phosphodiesterase inhibitor (e.g., rolipram, cilomilast, roflumilast, VIAGRA (sildenifil citrate), CLAUS (tadalafil), LEVITRA (vardenifil), etc.), a β-agonist, a steroid, or a leukotriene antagonist (LTD-4). In some embodiments, a promoter of SCAN / BLVRB mediated S-nitrosylation can be administered in combination with one or more ADH inhibitors, AKR inhibitors, and / or SNO-CoAR inhibitors.

[0234] The invention is further illustrated by the following example, which is not intended to limit the scope of the claims. Example

[0235] In this Example, we describe the prototype for a class of enzyme, SCAN (SNO- CoA-assisted nitrosylase), that uses SNO-CoA as its cofactor to S-nitrosylate specific target proteins, and we reveal core mechanistic parallels with acyltransferases, firmly establishing enzymatic function. Separate domains in SCAN are involved in SNO-CoA and target protein binding, allowing SCAN to selectively catalyze NO group transfer from SNO-CoA to SCAN to multiple target proteins, including the insulin receptor b-subunit (INSRb) and insulin receptor substrate 1 (IRS1). SCAN activity acts physiologically to regulate insulin signaling on the one hand, while aberrant activity contributes to diabetes on the other hand. While SCAN activity is dependent on NO derived from nitric oxide synthase (NOS), the major role of NOS is to generate the LMW SNO cofactor. Thus, our characterization of SCAN unveils enzymatic principles shared with other enzymatic mediators of signal transduction and supports a new paradigm for NO biology.Materials and Methods Mice

[0236] Mouse studies were approved by the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC). Housing and procedures complied with the Guide for the Care and Use of Laboratory Animals and with the American Veterinary Medical Association Guidelines on Euthanasia. All mice were housed in a specific pathogen-free barrier facility on a 12:12 light:dark cycle with ad libitum access to food and water. The standard chow diet was Teklad P3000 (Envigo).

[0237] SCAN+ / -mice were generated by Model Animal Research Center, Nanjing University. Briefly, the inactivated Blvrb allele in ES cells was first created by insertion of a LacZ-Neo cassette in place of exons 2, 3 and 4 of the Blvrb gene, disrupting in-frame translation of BLVRB (Fig.9A). Targeted ES cells were injected into blastocysts to generate chimeric mice, and male chimeras were bred to C57BL / 6J females to produce BLVRB+ / -F1 mice. BLVRB+ / -F1 males and females were used as breeders to generate control mice (BLVRB+ / +) and homozygous mutant mice (BLVRB- / -), which were maintained by intercrossing BLVRB+ / +males and BLVRB+ / +females or BLVRB- / -males and BLVRB- / -females, respectively. Genotyping of Blvrb used the following PCR primers: Blvrb-FRT-tF1 5’-AGAGTTTGGGTCCTCCCTTTCCT-3’ (SEQ ID NO: 1) and common-En2-R: 5’- CCAACTGACCTTGGGCAAGAACAT-3’ (SEQ ID NO: 2).

[0238] To induce dietary obesity, 6–7-week-old male mice (body weight: 20-23g) were fed high-fat diet (60 kcal% fat diet D12492, Research Diets, Inc.) for 16 weeks before use in experiments.

[0239] Male ob / ob mice (Jax #000632), homozygous for the obese spontaneous mutation, were purchased from Jackson Labs and fed standard chow diet for 16 weeks before use in experiments. Cell culture and generation of CRISPR-knockout and replacement cell lines

[0240] HEK293 and L6 cells were obtained from ATCC and cultured according to the ATCC protocols at 37oC under 5% CO2in complete DMEM (10% FBS, 1% Pen-Strep). To induce the production of nitric oxide in HEK293 cells, pcDNA-eNOS was co-transfected into HEK293 cells using PolyJet transfection reagent. HEK-BLVRB knockout cell lines (HEK- BLVRB- / -), L6-SCAN knockout cell lines (L6-SCAN- / -), and L6-INSR knockout cell lines(L6-INSR- / -) were created using CRISPR-Cas9 with specific guide RNAs. The human BLVRB CRISPR / Cas9 KO plasmid and BLVRB HDR plasmid were from Santa Cruz Biotechnology; the rat eSpCas9-rBLVRB and eSpCas9-rINSR plasmids were from GenScript. The eSpCas9-rSCAN contained gRNAs 5’-CCCCTCTGACGGTAACCTGC-3’(SEQ ID NO: 3) and 5’-TCGGTGCCACCGGAAGGACC-3’ (SEQ ID NO: 4) targeting rat BLVRB gene. The eSpCas9-rINSR plasmids contained gRNAs 5’- TATCGACTGGTCCCGCATCCTGG-3’(SEQ ID NO: 5) and 5’- GCCTGATTATCAACATCCGAGGG-3’(SEQ ID NO: 6) targeting rat INSR gene. HEK- 293 or L6 cells were transfected with BLVRB CRISPR / Cas9 KO / BLVRB HDR plasmid, eSpCas9-rBLVRB or eSpCas9-rINSR using PolyJet transfection reagent per manufacturer’s instructions. Twenty-four hours after transfection, the media was replaced, and cells were grown for another 24 h. Cells were then split into growth media. After 48 h, media containing 3 mg / mL puromycin was added to select for candidate knockout cell colonies. Single colonies were picked and cultured in 96-well plates with growth media containing 3 mg / mL puromycin. Knockout cell lines were confirmed by Western blot. Puromycin- resistant colonies containing normal endogenous expression of BLVRB (or INSR) were used for negative controls. To build stable HEK-BLVRB-WT, BLVRB-QTG / NAA, L6-SCAN- WT and L6-SCAN-QTG / NAA cell lines, HEK-BLVRB- / -or L6-SCAN- / -knockout cells were transfected with pcDNA-hBLVRB-WT or pcDNA-hBLVRB-QTG / NAA using PolyJet transfection reagent per manufacturer’s instructions. To build stable L6-INSR-WT and L6- INSR-C1083 cell lines, L6-INSR- / -were transfected with pcDNA-hINSR-WT or pcDNA- hINSR-C1083. Twenty-four hours after transfection, media was changed and cells grown for another 24 h before being split into growth media. After 48 h, G418 was added into the media at 400 mg / mL to select for cell lines expressing the proteins of interest. Expression in the stable cell lines was confirmed by Western blot. Human samples

[0241] Acquisition of deidentified human skeletal muscle samples, human visceral adipose and human subcutaneous adipose tissue samples was approved by the Institutional Review Board (IRB) for the protection of human subjects at Case Western Reserve University under a protocol waiver.

[0242] Deidentified frozen human samples with pathological characterization were provided by the Tissue Resource Core at University Hospitals Cleveland. Human tissue was snap-frozen in liquid nitrogen immediately after collection from surgical procedures. Average age of patients for adipose tissues is 55.3 ± 13.4 years (ranging from 34 to 87 years). Average age of patients for skeletal muscle is 61.2 ± 12.6 years (ranging from 37 to 85 years). The SCAN expression and SNO levels of INSR in human samples were quantified by semi-quantitative analysis from western blots. SCAN expression was normalized relative to GAPDH detected on the same gel to control for any variation of protein loading among samples. These values were then normalized to the mean value (mean for all samples in same gel) for each gel to create a normalized measure to allow samples from two replicate gels to be directly compared. Method Details Plasmids, cloning and mutagenesis

[0243] Human cDNA of HO2 and SCAN were cloned through reverse transcriptase– polymerase chain reaction (RT-PCR). The mammalian cell expression plasmids pCS2-flag- hHO2 and pcDNA-myc-hSCAN were generated through inserting cDNA of HO2 and SCAN into vector pCS2-flag and pcDNA3.1-myc, respectively. The pcDNA-flag-hIRS1 and pcDNA-flag-hINSR were obtained from GenScript. pCS2-flag-hHO2-C127R, pCS2-flag- hHO2-C265R, pCS2-flag-hHO2-C282R, pcDNA-myc-hSCAN-QTG / NAA, pcDNA-myc- hSCAN-C109R, pcDNA-myc-hSCAN-C188R and pcDNA-myc-hSCAN-C109 / 188R, pcDNA-flag-hINSR-C825A, pcDNA-flag-hINSR-C834A and pcDNA-flag-hINSR-C1083 mutants were generated by QuikChange II Site-Directed Mutagenesis Kit (Agilent). The mammalian cell expression plasmids containing truncated hHO2(1–296), hHO2(65–316), hHO2(130–316) or hHO2(195–316) were generated subcloning PCR bands of HO2 truncations into pCS2-flag. For purification of recombinant hHO2, hHO2(195–316), hSCAN-WT or hSCAN-QTG / NAA and hSCAN-C109 / 188R, cDNA encoding human HO2, hHO2(195– 316), SCAN-WT, SCAN-QTG / NAA and hSCAN-C109 / 188R gene were subcloned into the pET21b vector to introduce a C-terminal 6xHis tag on the expressed protein. FGFR1 in pDONR221 vector was purchased from DNASU. All others (VEGFR2 / KDR #23925, PDGFRa #23892, PDGFRb #23893, HER3 #23874) in pDONR223 vector were purchased from Addgene. These entry vectors were cloned into pcDNA-DEST40 Gateway destination vectors (Invitrogen) using Gateway LR clonase II reaction, generating mammalian expression plasmids with terminal V5 tag. All plasmids and mutations were confirmed by DNA sequencing. SNO-CoA agarose bead pull down

[0244] Coenzyme A–agarose (50% slurry) was prepared by suspending Coenzyme A– agarose powder (Sigma) in water overnight at 4oC. To generate SNO-CoA beads, CoA beads were washed twice with 30 volumes of 10 mM HCl and supernatant was aspirated. Pelleted CoA beads were resuspended in 0.5 mL of 10 mM HCl, and 0.5 mL of 10 mM NaNO2 was added to the suspension to generate SNO- CoA. Immediately following NaNO2 addition, 10 mL of washing buffer (150 mM NaCl, 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine (Sigma) and 50 mM borate buffer, pH 8.0) was added to dilute the SNO-CoA beads. The SNO-CoA beads were washed three times with washing buffer. For pulldown experiments, bovine liver tissue (5g) was suspended in 25 mL of lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine, 50 mM borate buffer, pH 8.0, 1 mM PMSF and protease inhibitor mixture) and lysed in a blender, followed by homogenization with a Dounce homogenizer (Wheaton). Following centrifugation twice at 20,0003 g for 45 min, the supernatant was collected. The low-molecular weight co-factor NADPH was removed from the lysate using Amicon Ultra 3K Cen- trifugal Filter Devices. To pre-clear the CoA- binding proteins in the lysate, 10 mL of lysate (30 mg / mL) was incubated with 0.3 mL CoA– agarose beads for 2 h in the cold room in the dark. The pre-cleared lysate was incubated with 0.1 mL SNO-CoA beads for 2 h at 4oC in the dark. After incubation, the beads were washed six times with washing buffer (150 mM NaCl, 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine, and 50 mM borate buffer, pH 8.0), and SNO-CoA-binding proteins were eluted with excess SNO-CoA (20 mM, prepared as for SNO-CoA beads).

[0245] For in vitro binding experiments, 1 mg of recombinant BLVRB protein diluted in 1 mL of binding buffer (150 mM NaCl, 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine, and 50 mM borate buffer, pH 8.0) was incubated with 30 mL of 50% amylose resin (BioLabs), acti- vated thiol Sepharose 4B (Cytiva GE Healthcare), thiopropyl Sepharose 6B (GE healthcare), glutathione–agarose (Invitrogen), SNO-modified activated thiol (GSNO)– Sepharose 4B, SNO-CoA–agarose or CoA–agarose for 2 h at 4oC in the dark. For in vivo binding experiments, 1 mL of HEK cell lysate (1 mg / mL) was incubated with 30 mL of 50%GSH–agarose, GSNO–Sepharose 4B, CoA–agarose, Acetyl-CoA–agarose, SNO-CoA– agarose or Palmitoyl-Coenzyme A–agarose (Sigma) for 2 h at 4oC in the dark. Beads were washed six times with binding buffer, and bound proteins were eluted with 50 mL of 1X SDS loading dye (Invitrogen) containing 5% 2-mercaptoethanol (Sigma). SNO-RAC

[0246] SNO-RAC was carried out as described previously. Mouse skeletal muscle and human skeletal muscle were manually homogenized in liquid nitrogen with mortar and pestle. Ground skeletal muscle or human adipose tissue were homogenized in lysis buffer (1 mg / 5 mL lysis buffer) containing 100 mM HEPES, 1 mM EDTA, 100 mM neocuproine (HEN), 50 mM NaCl, 0.1% (v / v) Nonidet P-40, 0.2% S-methylmethanethiosulfonate (MMTS) as a free thiol-blocking agent, 1 mM PMSF and protease inhibitors using Bead-beater (BioSpec). After centrifugation (20,000 x g, 4oC, 20 min, 32), SDS and MMTS were added to the supernatants to 2.5% and 0.2% respectively, and incubated at 50oC for 20 min. Proteins were precipitated with -20oC acetone, and re-dissolved in 1 mL of HEN, 1% SDS. Precipitation of proteins were repeated with -20oC acetone and the final pellets were resuspended in HEN, 1% SDS buffer, and protein concentrations were determined using the Bicinchoninic Acid (BCA) method. Total lysates (2 mg) were incubated with freshly prepared 50 mM ascorbate and 50 mL thiopropyl-Sepharose (50% slurry) and rotated end-over-end in the dark for 4 h. The bound SNO-proteins were sequentially washed with HEN, 1% SDS and then 10% HEN, 0.1% SDS buffers; SNO-proteins were eluted with 10% HEN, 1% SDS, 10% b- meracaptoethanol and analyzed by SDS / PAGE and immunoblotting. In vitro S-nitrosylation assay

[0247] For in vitro assay of S-nitrosylation of HO2, two master tubes were prepared. The control reaction tube contained 5 mg of HO2-6xHis, 5 mg GST and 2000 mL assay buffer (phosphate buffer pH 7.0 supplemented with 100 mM EDTA, 100 mM DTPA and 0.1% NP-40). The enzyme reaction tube contained 5 mg of HO2-6xHis, 5 mg SCAN and 2000 mL assay buffer. The master mixture was separated into 5 tubes (400 mL / tube).20 mL of SNO-CoA dilutions (0, 2 mM, 20 mM, 200 mM or 2 mM) were added into pairs of control and enzyme tubes.

[0248] For in vitro assay of S-nitrosylation of HO2 mediated by mutant SCAN, two master tubes were prepared. The control reaction tube contained 4 mg of HO2-6xHis, 4 mg GST and 1600 mL assay buffer. The enzyme reaction tube contained 4 mg of HO2-6xHis, 4 mg SCAN-QTG / NAA, SCAN-C109 / 188R and 1600 mL assay buffer. Master mixture was split into 4 assay tubes (400mL / each tube), and 20mL SNO-CoA dilutions were added into individual tubes to final concentrations of 1 to 100 mM. For in vitro S-nitrosylation of IRS1 and INSR, two master tubes were prepared. Control reaction tube contained 1 mg of IRS1- FLAG or 1 mg of INSR-FLAG, 5 mg GST and 2000 mL assay buffer. Enzyme reaction tube contained 1 mg IRS1-FLAG or 1 mg INSR-FLAG, 5 mg SCAN and 2000 mL assay buffer. The master mixture was equally separated into 5 tubes (400 mL / each tube). 20 mL SNO- CoA (0, 20 mM, 200 mM, 2 mM or 4 mM) was respectively added into the tubes. Reaction tubes were incubated at 37oC for 30 min in the dark. Reactions were quenched by the addition of 3 volumes ice-cold 100% acetone. Following quenching, 50 mL2 mg / mL BSA was added to samples. Proteins were precipitated at -20oC for 30 min, pelleted at 4500 g for 8 min, and washed 4X with 70% acetone. Protein pellets were resuspended in 300 mL HEN buffer containing 2.5% SDS and 0.3% MMTS. Resuspended proteins were processed by SNO-RAC as described above. Glucose uptake measurement

[0249] In vitro glucose uptake measurement was performed as previously described. Naive SCAN+ / +and SCAN- / -mice (5 male and 5 female) per group were euthanized by isoflurane anesthesia. Soleus muscles were dissected tendon-to-tendon and rapidly rinsed in ice-cold 1X Krebs–Henseleit buffer. Muscles were recovered in vials including the recovery buffer (1X Krebs–Henseleit buffer, 0.1% BSA, 2 mM sodium pyruvate, 32 mM mannitol, 8 mM glucose supplemented with human-effective insulin dose (12 nM) or without insulin (basal)), shaken at 45 revolutions per minute while continuously gassed (95% O2–5% CO2) in a heated water bath (35oC) for 60 min. Muscles were subsequently washed twice for 10 min with at 35oC in washing buffer (1X Krebs–Henseleit buffer, 0.1% BSA, 2mM sodium pyruvate and 32 mM Mannitol). Muscles were transferred to a vial containing 5 mL incubation buffer (1X Krebs–Henseleit buffer, 0.1% BSA, 2 mM sodium pyruvate, 32 mM mannitol, 4 mM 2-deoxyglucose, 2 mCi / mL3H-2-deoxyglucose, and 0.3 mCi / mL14C- mannitol). Separate incubations were performed with insulin (12 nM) or without insulin(basal). Vials were shaken at 45 revolutions per minute while continuously gassed (95% O2– 5% CO2) in a heated water bath (35oC) for 20 min. Following this step, muscles were rinsed quickly in 1X Krebs–Henseleit buffer once and dried on filter paper and weighed. Muscle was digested with 1:10 (1 mg / 10 mL) 1 M NaOH at 60oC for 1 h. The samples were centrifuged 15,000 x g for 15 min at 4oC. Supernatants (150 mL) were transferred to new tubes and neutralized with 150 mL of 1M HCl. Fifty mL of each neutralized sample was placed in a scintillation vial and the3H and14C counts determined in a scintillation counter (PerkinElmer). Extracellular volume was calculated using disintegrations per minute (DPM) of the14C-mannitol, which is not membrane permeant. Intracellular 2-DG levels were then determined after accounting for3H DPM in the extracellular space, and 2-DG uptake rates were expressed as nmol 2-DG / 100 mg muscle / 20 min. Intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT) and acute hypoglycemia

[0250] IPGTT and IPITT were performed according to the standard protocol of the International Mouse Phenotyping Consortium. Mice were fasted for 16 h for IPGTT or 5 h for IPITT in clean cages with no food or feces in the bedding, in the standard light–dark cycle and with free access to water. Blood was collected from the tail tip directly onto a glucose test strip and read immediately using a calibrated glucometer. For IPGTT, glucose was injected i.p. at 2 g of glucose / kg of body weight. Blood drawn from sequential tail tip cuts was used to measure glucose at 15, 30, 60, 90 and 120 min after glucose injection. For IPITT, insulin was injected i.p at 1 unit insulin / kg of body weight. Glucose levels in tail tip blood were measured at 15, 30, 60, 90 and 120 min after insulin injection. Insulin-induced acute hypoglycemia (‘‘severe’’ ITT) in mice was produced according to a published protocol. Insulin (2.5 units / kg body weight) was injected i.p. into 3 h-fasted mice, producing blood glucose <40 mg / dL when measured at 90 min after injection and without leading to unconsciousness, seizures, or death. Blood glucose was measured before insulin injection and after 30, 90, 120,180 and 240 min via tail tip bleed. Insulin, bilirubin, free hemin, and blood tests

[0251] Blood (50 mL) was collected from the mouse facial vein. To measure insulin, bilirubin and free hemin in serum, blood was placed in BD Microtainer tubes with Serum Separator, and centrifuged at 4oC at 20003 g for 10 min to obtain serum. Insulinconcentration was measured following the protocol of Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem). Mice were fasted for 5 h prior to basal insulin measurement. Hematology analyses were performed using HESKA HemaTrue System. Blood was placed in heparin-treated 1.5mL microtube. Twenty mL blood was used for measurement of 17 parameters including total red blood cell count, total white blood cell count, total platelet count, hemoglobin concentration, lymphocyte percentage, mid-sized cells (e.g., monocytes) percentage, and granulocyte percentage. Insulin treatment

[0252] For L6 cell treatment, L6 cells at 50–70% confluence on 10 cm plates were starved in DMEM medium (+5% BSA) for 16 h. Human insulin was added into medium to 100 nM final. Following 10-min insulin treatment, cells were washed three times using warm 1X PBS, and complete DMEM medium (+10% FBS and 1% Pen-Strep) was added. L6 cells were collected at 1, 30, 60, 120 and 240 min after media change and stored -80oC. For mouse treatment, human insulin (1 U / kg body weight) was intraperitoneally injected into mice that had been fasted for 5 h. After 30 or 60 min, mice were euthanized by isoflurane anesthesia. Skeletal muscle (lateral gastrocnemius) was collected and quickly frozen in liquid nitrogen. iTRAQ-coupled SNO-RAC

[0253] iTRAQ-Coupled SNO-RAC was carried out as described previously. HEK- BLVRB- / -and HEK-BLVRB+ / +(control) lysates were prepared, and SNO-RAC (4 mg of protein per sample) was carried out as described above. SDS / PAGE gels were Coomassie- blue stained, and lanes were separated into eight segments top-to-bottom and collected in two 1.5 mL tubes. Five hundred ml of 50% acetonitrile (ACN) / 50% 100 mM ammonium bicarbonate was used to wash gel bands for more than 5 h while vortexing. After removal of washing buffer, 400 mL of 100% acetonitrile was added to gel pieces and vortexed for 10 min. After removal of ACN, gel pieces were dried in a speed vacuum dryer for 10 min. Two hundred ml of 10 mM dithiothreitol (DTT) was added to dry gel pieces and vortexed for 45 min. After removal of DTT buffer, 200 mL of 55 mM iodoacetamide (IAA) was added to the gel pieces and incubated for 45 min in the dark. After removal of IAA buffer, 400 mL of 1x iTRAQ dissolution solution and 400 mL ACN were used to wash the gel pieces twice. Gel pieces were dried for 10 min in a speed vacuum dryer. 500 ng trypsin in 150 mL 1x iTRAQbuffer was added to dried gel pieces on ice for 30 min to rehydrate, and then incubated overnight at 37oC. Supernatant from the digested protein solution was transferred to a 1.5 mL tube using gel-loading tips.200 mL extraction buffer (60% ACN / 5% formic acid) were added to gel pieces, vortexed for 30 min, and sonicated for 15 min. The supernatant containing peptide extracts was transferred to the same 1.5 mL tube, and extractions were repeated two more times. The final digested solution pool was dried completely in a speed vacuum dryer.

[0254] iTRAQ labeling was performed according to the instructions of iTRAQ Reagents - 4plex Applications Kit. Briefly, 30 mL of iTRAQ dissolution buffer (10x) was added to each sample tube (pH > 7), and then one iTraq labeling reagent (mass 114, 115, 116 or 117) was added to individual sample tubes. Reactions were incubated for more than 5 h at room temperature with vortexing to ensure com- plete labeling. The four labeled samples were mixed together and dried. One hundred sixty mL of 5% ACN containing 0.5% TFA was added to the labeled sample mix, and the solution cleaned using C18 ziptips. Briefly, C18 tips were wetted 5 times with 20 mL of 50% ACN, and equilibrated with 100 mL of 5% ACN containing 0.5% TFA. Samples were then loaded to the tip by drawing and expelling 50 cycles to ensure complete binding. The tips were then washed with 20 mL of 5% ACN containing 0.5% TFA 10 times. Peptides were eluted from tips in 3320 mL of 60% ACN containing 0.1% formic acid, and eluates combined and dried for LC-MS / MS Analysis. LC-MS / MS analysis

[0255] Digested peptides were separated by UPLC (Waters, Milford, MA) with a Nano- ACQUITY UPLC BEH300 C18 column. Separated peptides were continuously injected into an Orbitrap Elite hybrid mass spectrometer (Thermo Finnigan, San Jose, CA) by a nanospray emitter (10 mm, New Objective). Peptides were eluted using a linear gradient using mobile phase A (0.1% formic acid in water) and B (100% acetonitrile) was used at a flow rate of 0.3 mL / min, starting with 1% mobile phase B and increasing to 40% B at 65 min for protein interaction identification, or increasing to 40% B at 130 min for iTRAQ experiments. All mass spectrometry data were acquired in positive ion mode. For protein interaction identification, a full MS scan (m / z 350–1800) at resolution of 120,000 was conducted, twenty MS2 scans (m / z 350–1800) were selected using the twenty most intense peptide peaks of full MS scans. CID cleavage mode was performed at normalized collision energy of 35%. ForiTRAQ experiments, a full MS scan (m / z 300–1800) at resolution of 120,000 was conducted, and ten MS2 scans (m / z 100–1600) were activated from the five most intense peptide peaks of full MS scans. CID and HCD cleavage modes were performed alternatively of the same peptides selected from full MS scans. MS2 resolution of HCD is 15,000. Bioinformatic software MassMatrix was used to search MS data against a database composed of sequences of mouse proteins from Uniprot and their reversed sequences as a decoy database. Modifications including oxidation of methionine and labeling of cysteine (IA modifications) were selected as variable modifications in searching. For iTRAQ labeling searching, MS tag of N terminus, Lys and / or Tyr were selected as variable modification to test labeling efficiency and fixed modification for iTRAQ quantitation analysis. Trypsin was selected as the in-silico enzyme to cleave proteins after Lys and Arg. Precursor ion searching was within 10 ppm mass accuracy and product ions within 0.8 Da for CID cleavage mode and 0.02 Da for HCD cleavage mode.95% confidence interval was required for protein identification. Immunoprecipitation

[0256] To investigate the interaction of SCAN and IRS1, Myc-SCAN and IRS1-Flag were co-expressed in HEK293 cells. To investigate the effect of insulin stimulation, disruption of SNO-CoA binding, mutation of SNO site C109 / 188 and of SCoR, Myc-SCAN, Myc-SCAN-QTG / NAA or Myc-SCAN-C109 / 188R were co-expressed with INSR-Flag in HEK or SCoR-knockout HEK cells or in wild-type or SCoR- knockout L6 cells. HEK cells or L6 cells were treated with NO donor DPTA (200mM) for 20 h as required. Anti-rabbit C- Myc Agarose Affinity Gel (Sigma) was used for immunoprecipitation. To investigate the interaction of endogenous SCAN with HO2 or INSR, 10 mg of BLVRB Rabbit monoclonal antibody (Sino Biological) was incubated with 50 mL of Protein G Sepharose (GE) (1:1 slurry) at 4oC overnight, then washed with NETN buffer (150 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5% (v / v) Nonidet P-40) three times to prepare for immunoprecipitation. HEK cells or L6 cells were homogenized in EBC lysis buffer (120 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5% (v / v) NP-40, 1 mM PMSF and protease inhibitor cocktail). After centrifugation (20,000 x g, 4oC, 20 min, x 2), 2 mL (2 mg / mL) supernatant was pre-cleared by incubation with 50 mL Protein G Sepharose (1:1 slurry) for 1 h at 4oC. After spinning at 1000 x g for 1 min, the supernatant was transferred into new tubes and incubated with 50 mL anti BLVRB antibody-Protein G Sepharose or withanti c-Myc-Agarose (1:1 slurry) for 5 h at 4oC. Beads were washed by NETN buffer and proteins were eluted with 50 mL 1X SDS loading dye containing 5% 2-mercaptoethanol. Eluted proteins were electrophoresed in 4–20% Criterion Precast Midi Protein Gels (Bio Rad), transferred to PVDF membranes, and membranes were blotted with mouse Flag antibody, HO2 antibody or INSR antibody. Western blot analysis

[0257] Proteins were extracted from cells using sonication in RIPA buffer (Sigma) supplemented with 1 mM PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail. Muscle samples were ground with a mortar and pestle under liquid nitrogen, then the powder added to RIPA buffer as above, and mechanically homogenized using a bead beater. Extracts were clarified by centrifugation (20,000 x g, 4oC, 20 min, x2), and protein concentration was determined by bicinchoninic acid assay. The extracts were electrophoresed in 4-20% Criterion Precast Midi Protein Gels and transferred to PVDF membranes. Membranes were incubated overnight at 4oC with primary antibodies, washed with PBS containing 0.1% Tween 20, incubated with HRP-conjugated secondary antibody for 1 h, washed, and detected by chemiluminescent detection (ECL) using X-ray film developer system (JPI, Filmprocessor) or a digital imager system (Kwik- quant). Antibodies employed in western blotting included: rabbit polyclonal rabbit Anti-BLVRB (13151-R009, Sino Biological Inc), mouse monoclonal Anti-HO2 (H00003163, Abnova), rabbit polyclonal Anti-IRS1 (06–248, EMD Millipore), rabbit polyclonal Anti-INSRb (sc-711, Santa Cruz), rabbit monoclonal Anti-INSRb (phospho-Y1163,1164) (MA5-15148, Invitrogen), rabbit Anti-IRS1 phospho-Tyr608 (09432, EMD Millipore), rabbit monoclonal Anti-NOS2 (D9A5L, Cell Signaling), rabbit Anti-eNOS (phospho-S1177) (PA597371, Invitrogen), rabbit monoclonal Anti-AKT (C67E7, Cell Signaling), rabbit Anti-AKT (phospho-S473) (9271, Cell Signaling), mouse monoclonal p97 (10R- P104A, Fitzgerald), rabbit monoclonal GAPDH (Ab181602, abcam), rabbit monoclonal Anti-AS160 (MA5-14840, Invitrogen), rabbit Anti-AS160 (phospho-T642) (44-1071G, Invitrogen) and mouse monoclonal FLAG- M2 (F3165, Sigma) (see Key Resources). Protein levels were quantified by semi-quantitative analysis from western blots using ImageJ (NIH).Identification of SNO site of INSR by mass spectrometry

[0258] The pcDNA-INSR-FLAG plasmid (GenScript) was transfected into HEK293 cells using PolyJet. Cells were harvested and lysed in EBC lysis buffer. INSR-FLAG was purified from lysate with anti-FLAG M2 affinity gel (Sigma) and eluted with 100 mM phosphate buffer, pH 7.4 containing 100 mg / mL FLAG peptide (Sigma), 100 mM EDTA and 100 mM DTPA. Purified INSR was subsequently treated with 100 mM freshly prepared SNO-CoA for 20 min at room temperature in the dark. Following treatment with SNO-CoA, protein was supplemented with 100 mg bovine serum albumin carrier, mixed with 3 volumes ice-cold 100% acetone, and kept at -20oC for 30 min. Following cold incubation, the sample was spun at 14000 x g for 15 min to precipitate protein. Pelleted protein was washed 3 times with ice-cold 70% acetone, air dried, and resuspended in 400 mL of HENS buffer. Unreacted SNO-CoA was removed using Zeba desalting spin columns (Thermo Fisher) preequilibrated with HENS buffer. Following filtration, 1 M MMTS and 25% SDS were added into the protein solution (final 20mM MMTS and 2.5% SDS) and the solution was vortexed. The solution was incubated for 20 min at 50oC to block free thiols. Protein was again precipitated with 3 volumes ice-cold 100% acetone with incubation for 1 h at -20oC. Following incubation, precipitated proteins were collected by centrifugation at 10000 x g for 10 min at 4oC. Supernatant was removed from precipitated protein pellets, and pellets allowed to dry at room temperature for 10 min. Proteins were resuspended in HENS buffer and labeled with iodoTMT (Thermo Fisher) in the presence of 20 mM sodium ascorbate for 1 h at room temperature in the dark. Reactions were quenched with 20 mM DTT for 15 min at room temperature in the dark. Protein was precipitated with 3 volumes ice-cold 100% acetone at - 20oC for 1 h, then centrifuged at 10000 x g for 10 min at 4oC. Supernatant was removed, and the protein pellet dried for 10 min. Protein was resuspended in HENS buffer and treated with 16.67 mM iodoacetamide for 1 h at room temperature in the dark. Protein was again precipitated as above, supernatant removed, and protein pellet dried. Precipitated protein was then resuspended in 50 mM ammonium bicarbonate buffer, pH 8.0. Proteins were then serially digested with 25 mg / mg protein using Lys-c (4 h at 37oC) then 20 mg / mg protein using trypsin overnight at 37oC. Following digestion, samples were acidified with 25 mL of 10% TFA. Peptides were processed through a C18 SPE column and then frozen and lyophilized. Lyophilized peptides were resuspended in TBS and enriched using anti-TMTresin (Thermo Fisher) following the manufacturer’s instructions, and subsequently frozen and lyophilized. Samples were resuspended in 5% acetonitrile, 0.1% formic acid and run through a 0.22 mm filter to remove any excess anti-TMT resin. Peptides were then injected into LC- MS / MS system for analysis. Purification of proteins

[0259] The recombinant HO2, SCAN-WT or SCAN-QTG-NAA proteins were purified from BL21-CodonPlus Competent E. coli cells (Agilent). Overnight E. coli cultures were sub-cultured into 1 L of LB medium at 5%. At OD600 of 0.5, cultures were induced with 100 mM IPTG and grown 4 h at 28oC. Cultures were centrifuged at 4000 x g for 10 min to harvest the cells. Cell pellets from 1 L cultures were lysed by sonication in 10 mL of PBS buffer containing 1 mM PMSF and protease-inhibitor cocktail. After centrifugation at 14500 x g for 20 min, the supernatant was collected and diluted (up to 30 mL) with PBS buffer containing 1 mM PMSF and protease-inhibitor cocktail. This lysate was incubated with 1 mL of Ni-NTA agarose at 4oC for 1 h with rotation. The slurry was then poured into an empty PD-10 column (GE Healthcare), and the beads washed with 100 mL of 50 mM NaH2PO4, 300 mM NaCl buffer containing 20 mM imidazole. Elution was done with 2 mL of 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole. Eluate buffer was exchanged with modified Roeder D [(20mM HEPES (pH 7.9), 20% (v / v) glycerol, 0.1M KCL, 0.2mM EDTA)] using a Microcon centrifugal filter device (Millipore).

[0260] IRS1-FLAG and INSR-FLAG were purified from HEK cells. Briefly, HEK cells were transfected with pcDNA-Flag-hIRS1 or pcDNA-Flag-hINSR plasmid using PolyJet transfection reagent (SignaGen) per manufacturer’s instructions and grown for 48 h. After 48 h, cells were harvested in IP wash buffer (as above) supplemented with 0.5% Triton X-100 and protease inhibitor cocktail. Cells were lysed by sonication and lysate clarified by centrifugation at 15000 x g for 15 min. Anti-FLAG agarose affinity gel was equilibrated into IP wash buffer, and clarified lysate was applied to anti-FLAG agarose affinity gel. After 4 h incubation in 4oC, beads were washed 5 times with IP wash buffer. Proteins were eluted in 300mL 1xPBS containing 100 mg / mL 3X FLAG peptide. Protein concentration was determined using BCA.Tyrosine kinase activity

[0261] Tyrosine kinase activity of INSR was measured using InsR Kinase Enzyme System-coupled with ADP-Glo Assay (Promega). To purify the INSR-FLAG from HEK cells, the same procedure was used in above ‘‘Purification of proteins’’ except INSR-FLAG complex was eluted in 200mL reaction buffer A (provided in InsR Kinase Enzyme System) containing 100 mg / mL 3X FLAG peptide and 2mM MnCl2. Ten mL (100 ng / mL) purified INSR-FLAG complex was treated with control Tris buffer or 500 mM SNO-CoA (5 mL) for 30 min at 37oC. Ten mL AxLtide (substrate, 1 mg / mL)+ATP (125 mM) mixture was added into the reaction. After 60 min incubation at 25oC, 25 mL ADP-Glo Reagent was added into the reaction. Fifty mL kinase detection reagent was added after 40 min incubation at 25oC, and the luminescence read after 30 min incubation at 25oC in a Promega GloMax Luminometer. Immunostaining

[0262] HEK293 cells were grown in 2 mL culture medium to approximately 50% confluency on cover slips in 6-well plates. For studying insulin stimulation, cells were starved in DMEM medium (+5% BSA) for 16 h. Human insulin was added into medium to 100 nM final concentration for 10min. Media was aspirated, and 1mL cold 4% PFA in PBS was used to fix cells for 15 min at room temperature. After each incubation step, cells were washed 3x with PBS. Cells were permeabilized with PBS +0.05% Triton X-100 for 5 min, washed, blocked with 8% BSA in PBS for 20min, washed and incubated with primary antibodies diluted in blocking buffer for 2 h. Primary antibodies rabbit anti-SCAN (Sino Biological Inc), mouse anti-Cytochrome C (Cell Signaling) and mouse anti-AKR1A1 (Or- igene TA500740 clone 9F1) were used in immunostaining. After washing, secondary antibodies were added for 2 h at room temperature. Secondary antibodies used were A11001 (Invitrogen, AF488 anti-mouse IgG at 1:1000) and A11036 (AF568 anti-rabbit IgG at 1:1000). DAPI (Biotium, 5 mg / mL) was used to stain nuclei. Samples were washed once again, mounted with Fluoromount G onto slides and imaged with a Nikon Eclipse Ti2 microscope using a 60x objective. TIFF images were processed in FIJI / ImageJ with the JaCoP plugin used to calculate Pearson’s and M1 / M2 coefficients.Quantification and Statistical Analysis

[0263] Statistics were analyzed using GraphPad Prism 8. Comparisons between continuous characteristics of two subject groups were analyzed with two-tailed Student’s t test. For comparisons among more than two groups, one-way ANOVA with Tukey post hoc or two-way ANOVA with Sidak’s multiple comparisons test were used. Simple linear regression was performed in comparing human samples. Bar graphs with the corresponding dot plots were created using GraphPad Prism. Results are presented as mean ± SD, and sample sizes or number of replicates are indicated in individual figure legends. Results

[0264] By analogy to acyltransferases that use acetyl-CoA to acetylate proteins, we considered that SNO-CoA might serve as a cofactor for unknown nitrosylase enzymes that target S-nitrosylation of specific substrate proteins. We therefore purified SNO-CoA binding proteins from bovine liver to identify candidate SNO-CoA- dependent nitrosylases. Eight proteins that bound SNO-CoA, but not CoA, were identified (Fig.1A). Biliverdin reductase B (BLVRB) and CBR1 were the two most enriched SNO-CoA binding proteins. CBR1 is known to be an LMW SNO reductase; however, the function of BLVRB is unclear. BLVRB converts fetal b-biliverdin to bilirubin during development, but retains expression in adults, where its substrate is not present.

[0265] Using HEK293 cell lysates and recombinant BLVRB protein, we first confirmed that BLVRB bound SNO-CoA with relatively high affinity versus a panel of both LMW SNO- and CoA-conjugates (Figs.1B, 7A, and 7B). Despite being known as an NADPH- dependent reductase enzyme, BLVRB was unable to reduce SNO-CoA (Figs.7C and 7D). To assess whether BLVRB contributed in some other manner to SNO-protein metabolism, we used SNO-protein quantitative mass spectrometry in lysates from HEK-BLVRB- / -cells compared with parental wild-type HEK (HEK-WT) cells. We found 50 proteins whose S- nitrosylation was significantly diminished in the absence of BLVRB expression, representing potential targets of BLVRB acting as a nitrosylase (Table 1). Since protein S-nitrosylation typically operates within multiprotein complexes, we isolated the BLVRB interactome by immunoprecipitation from HEK293 cell lysates and identified 47 proteins (Table 2). Notably, six of these BLVRB-associated cytoplasmic proteins (BLVRB is mainly localized in the cytoplasm [Figs.7E and 7F]) overlapped with the BLVRB nitrosoproteome (Fig.1C),including heme oxygenase 2 (HO2), which is known to function together with BLVRB in heme metabolism. Table 1 - BLVRB-dependent nitrosoproteome. SNO-proteins enriched in HEK-BLVRB- / - vs. HEK-BLVRB+ / +. Related to Fig.1 e 5 4 2 9 7 5 0 0 9 2 1 0 0 7 6 4 8 5 1 0 9 7 4 2 9 8 64 3 2 8 6 4 4 1 0 8 2 1 1 0 9 4 4 9 6 5 3 1 1Hit order Accession Description Symbol in MS UniProtK Binan endothelial NOS (eNOS)- or inducible NOS (iNOS)-dependent manner (Figs.8A and 8B) at two cysteine sites (Cys265 and Cys282), as mutation of both Cys265 and Cys282 to arginine (HO2-C265 / 282R) blocked S-nitrosylation of HO2 (Fig.8C). We confirmed that BLVRB directly interacted with HO2, and this was enhanced by an NO donor (Fig 8D), and we verified that S-nitrosylation of HO2 (SNO-HO2) was markedly reduced in HEK-BLVRB- / -cells compared with HEK-WT cells (Figs.1D and 1F). Recombinant BLVRB catalyzed S- nitrosylation of purified HO2 by SNO-CoA (Michaelis-Menten constant [KM] of 4 mM),while SNO-CoA itself (in the presence of GST [glutathione S-transferase] protein control) S- nitrosylated HO2 only at very high concentrations (Figs.1E, 1G, and 1H). We conclude that BLVRB functions as an SNO-CoA-assisted nitrosylase (SCAN).

[0267] SNO-CoA and NADPH are structurally related (Fig.8E), and BLVRB / SCAN is a known NADPH-binding protein. We investigated whether the NADPH-binding motif in SCAN might be required for SNO-CoA binding. Addition of NADPH, but not SNO- cysteamine (SNO-CoA analog), inhibited SCAN binding to SNO-CoA resin (Fig.1I) in a dose-dependent manner (Fig.1J), suggesting that NADPH and SNO-CoA share the same binding site. Although NADPH competes with SCAN binding to SNO-CoA in vitro, SCAN can utilize SNO-CoA (1 mM) to S-nitrosylate HO2 in the presence of high excess concentrations of NADPH (250 mM) or NADH (1 mM) (Fig.8F–8H), concentrations that far exceed endogenous cytosolic NADPH (3 mM) or NADH (50–110 mM). Mutation of three critical amino acids, Q14N, T15A, and G16A (SCAN-QTG / NAA), within the NADPH- binding motif prevented SCAN binding to SNO-CoA resin, con- firming that the NADPH- binding site in SCAN is required for SNO- CoA binding (Fig.1K). Overexpression of SCAN-QTG / NAA in HEK-SCAN- / -cells was unable to rescue S-nitrosylation of HO2 (Figs. 1L, 1M, 8I, and 8J), and recombinant SCAN-QTG / NAA could not efficiently S-nitrosylate HO2 in the presence of SNO-CoA in an in vitro assay (Figs.1N and 1O). Thus, SNO-CoA binding to SCAN is required for SCAN-mediated S-nitrosylation of its protein substrate HO2.

[0268] To explore the mechanism of SCAN-mediated S-nitrosylation, we investigated whether SNO-Cys within SCAN is required for transnitrosylation (i.e., whether SNO-SCAN exists as an intermediate in transfer of NO groups to substrate). In HEK293 cells or RAW 264.7 cells, SCAN was endogenously S-nitrosylated in an eNOS- or iNOS-dependent manner (Figs.8K and 8L). There are two cysteine residues (Cys109 and Cys188) within SCAN. Mutation of both Cys188 and Cys109 to arginine eliminated SNO modification of SCAN (Fig.1P). SCAN-C109 / 188R expressed in HEK-BLVRB- / -cells could not rescue HO2 S- nitrosylation (Fig.1Q), and recombinant SCAN-C109 / 188R cannot S-nitrosylate the substrate HO2 in vitro (Figs.1R and 1S), indicating that SNO-SCAN is required for S- nitrosylation of HO2. To investigate if interaction between BLVRB and substrate HO2 is required for BLVRB-mediated S-nitrosylation, we generated a truncated HO2 protein (HO2- 195-316), which retains the two SNO sites (Cys265 / Cys282), but is unable to interact withBLVRB (Figs.8M and 8N). In vitro assays indicated that BLVRB / SCAN cannot efficiently nitrosylate HO2-195-316 (Fig.8O). These results suggest that SCAN utilizes a ‘‘ping-pong’’ mechanism to catalyze S-nitrosylation. SCAN associates with both SNO-CoA and HO2 substrate; NO groups are then transferred from SNO-CoA to SCAN-Cys109 / 188 (forming SNO-SCAN) and from SNO-SCAN to the substrate (Fig.1T), yielding product (SNO-HO2).

[0269] To investigate the physiological role of SCAN in mammals, we utilized global SCAN-knockout mice (SCAN- / -) (Figs.2A and 9A). The level of bilirubin in serum and whole blood cell counts are indistinguishable in SCAN- / -and wild-type (SCAN+ / +) mice, confirming that BLVRB / SCAN is not involved in biliverdin degradation or hematopoiesis in adult mice under normal conditions (Figs.9B–9F). Because insulin receptor substrate 4 (IRS4), the IRS family member predominantly expressed in HEK293 cells, was identified in the SCAN-dependent nitrosoproteome (Fig.1C), and because SCAN contains an insulin receptor (INSR)-interacting motif with unknown function, we explored a role for SCAN / SNO-CoA in insulin signaling.

[0270] We first confirmed that SCAN interacts with both INSR and IRS1 and found that interactions are greatly increased by NO donor treatment or insulin stimulation (Fig.10A–10C). Mutation of the SCAN SNO-CoA binding site (QTG / NAA) or its SNO sites (C109 / 188R) reduced the association of SCAN with the INSR (Fig.10D), consistent with a ping-pong mechanism (Fig.1T). Expression of SCAN was dramatically increased in skeletal muscle from 12-week-old genetically obese mice (ob / ob) and from wild-type mice fed a high-fat diet (HFD) for 12 weeks and was paralleled by increases in iNOS expression (Fig.2B). SCAN- / -mice gained weight more slowly than SCAN+ / +mice on HFD (Fig.10E). SCAN- / -mice fed a HFD for 16 weeks displayed reduced blood glucose after 5 h of fasting, compared to SCAN+ / +mice (Fig.2C), but blood insulin levels were normal (Fig.2D), indicating that insulin signaling, not pancreatic insulin production / secretion, is responsible for protection from hyperglycemia. Insulin tolerance tests showed higher sensitivity to insulin in HFD-fed SCAN- / -mice than in SCAN+ / +mice (Fig.2E). Although basal glucose levels are much lower in HFD-fed SCAN- / -mice than WT, glucose tolerance tests demonstrated a similar pattern in glucose uptake between HFD-fed SCAN+ / +and SCAN- / -mice (Fig.2F), which may reflect elevated insulin levels in HFD-fed WT mice (Fig.2D). That is, higher insulin levels may alleviate glucose intolerance in WT mice resulting from hyper- S- nitrosylation of IRS1 / INSR. Using the14C-labeled non-metabolizable glucose analog 2-deoxyglucose as a tracer, we found that knockout of SCAN in HFD-fed mice improved insulin-stimulated glucose transport in ex vivo soleus muscle (Fig.2G). Therefore, SCAN contributes to insulin resistance in HFD-obese mice, whereas SCAN deletion improves insulin sensitivity.

[0271] Excessive S-nitrosylation of INSRb / IRS1 by iNOS-derived NO is implicated in insulin resistance and provides a model for pathological S-nitrosylation in disease. In this model, S-nitrosylation was proposed to be mediated chemically by high amounts of reactive NO. Thus, we sought to determine if S-nitrosylation of INSRb / IRS1 is in fact mediated enzymatically by SCAN / SNO-CoA. Amounts of SNO-INSRb and SNO-IRS1 were much higher in skeletal muscle of HFD- or chow-fed WT mice than in SCAN- / -mice (Figs.2H and 2I). Further, amounts of SNO-INSRb and SNO-IRS1 were higher in HFD-fed than chow-fed WT mice, but diet had no effect on SNO levels in SCAN- / -mice. Thus, SCAN is required for S-nitrosylation of INSRb and IRS1 in situ. Further, in in vitro assays, purified SCAN efficiently S-nitrosylated purified INSR and IRS1 (KM of 0.96 mM and 4.57 mM, respectively) (Figs.2J, 2K, and 10F–10H). To investigate the role of SNO-CoA per se in S- nitrosylation of INSR / IRS1, we utilized SNO-CoA reductase (SCoR) knockout mice and cells, which cannot effectively metabolize SNO-CoA. Both SCoR and SCAN are localized mainly in the cytoplasm (Fig.11A). Deletion of SCoR in HEK cells increased association of SCAN with INSRb (Fig.11B). HFD-fed SCoR-knockout mice (SCoR- / -) demon- strated increased S-nitrosylation of INSRb / IRS1 and reduced phosphorylation of the insulin effectors AKT and AS160 (Figs.11C–11F). Taken together, our data support the notion that S- nitrosylation of INSRb and IRS1 is enzymatically mediated, and that hypernitrosylation of proteins after iNOS induction may require SCAN and SNO-CoA.

[0272] To determine if SCAN-mediated S-nitrosylation of INSRb / IRS1 regulates insulin signaling, we measured the phosphorylation of INSRb, IRS1, AKT, and AS160 in skeletal muscle of HFD-fed mice after intraperitoneal injection of insulin. The insulin- stimulated phosphorylation levels of INSRb (Tyr1162), IRS1 (Tyr608), AKT (Ser473), and AS160 (Thr642) were all higher in SCAN- / -mice than SCAN+ / +mice, indicating that S- nitrosylation by SCAN inhibits INSRb / IRS1-dependent insulin signaling, at least in significant part through inhibition of tyrosine kinase activity of INSRb (Figs.2L and 11G).

[0273] To further explore the physiological role of SCAN-mediated inhibition of insulin signaling, we generated SCAN-deficient rat myoblast L6 cell lines (L6-SCAN- / -)(Fig.11H). While L6-SCAN- / -cells and wild-type parental L6 cells (L6-WT) have similarly low basal levels of SNO-INSRb and SNO-IRS1, insulin increased S-nitrosylation of INSRb / IRS1 in L6-WT cells, but not in L6-SCAN- / -cells. Insulin-stimulated S-nitrosylation of INSRb / IRS1 was dynamic in L6-WT cells, reaching a peak 60–120 min after removal of insulin (after a 10-min treatment) and gradually sub- siding (Figs.3A–3C). Similarly, intraperitoneal insulin injection in chow-fed SCAN+ / +mice induced significant S- nitrosylation of INSRb / IRS1, but not in SCAN- / -mice (Figs.3D and 3E). We investigated the function of insulin-induced S-nitrosylation of INSRb / IRS1 in L6 cells and in healthy mice. We found that deletion of SCAN in L6 cells prolongs insulin-stimulated phosphorylation of IRS1, AKT, and AS160 (Figs.3F and 11I–11L). This suggests that agonist-stimulated S-nitrosylation of INSRb / IRS1 is part of a negative feedback loop that turns off insulin signaling. To validate this idea in mammals, we performed an insulin tolerance test in chow-fed SCAN+ / +and SCAN- / -mice. Recovery of glucose levels after insulin injection (1 U / kg or 2.5 U / kg) was delayed in SCAN- / -mice compared to SCAN+ / +mice (Figs.3G and 3H), consistent with higher S-nitrosylation of INSRb in SCAN+ / +than in SCAN- / -skeletal muscle (Figs.3I and 3J). These results confirm that insulin-induced S- nitrosylation mediated by SCAN acts to turn off insulin signaling in skeletal muscle, thereby slowing glucose uptake to prevent hypoglycemia.

[0274] Insulin treatment in both WT mice and L6-WT cells markedly increased phosphorylation of eNOS at Ser1177 and neuronal NOS (nNOS) at Ser1412, respective measures of eNOS and nNOS activity (Figs.4A–4F and 11M). Taken together, our results indicate that SCAN mediates insulin-stimulated S-nitrosylation of INSRb / IRS1 in both L6 cells and skeletal muscle of healthy mice, likely using NO generated by eNOS or nNOS (Figs.11N and 11O). This was confirmed by showing that in L6 cells, the NOS inhibitor L- NMMA blocked insulin-stimulated S-nitrosylation not only of INSRb and IRS1 but also of SCAN itself (Figs.4G–4J). Accordingly, phosphorylation of AKT and AS160 was increased in the presence of L-NMMA (Figs.4K, 11P and 11Q). Thus, eNOS and nNOS may provide the source of SNO for SCAN activity under physiological conditions.

[0275] As a confirmatory measure, we re-expressed SCAN-WT (L6-SCAN-WT), the SCAN-QTG / NAA mutant unable to bind SNO-CoA (L6-SCAN-QTG / NAA) or the SCAN- C109 / 188R mutant unable to form SNO (L6-SCAN-C109 / 188R) in SCAN- deficient rat myoblast L6 cell lines (L6-SCAN- / -) (Fig.12A). Re-expression of SCAN-WT, but notSCAN-QTG / NAA or L6-SCAN-C109 / 188R, rescued the S-nitrosylation of INSRb and IRS1 and inhibited insulin-stimulated phosphorylation of IRS1 and AKT (Figs.5A–5D). Thus, the S-nitrosylation of INSRb / IRS1 and subsequent inhibition of insulin signaling requires SCAN and SNO-CoA. To dissect the role of SNO-INSRb in insulin signaling, we first mapped the SNO sites in INSR. Four peptides containing four candidate cysteine SNO sites were identified (Fig.5E); Cys825, Cys834, and Cys1083 are present in INSRb, while Cys462 is in INSRa. Using mutagenesis, we determined that Cys1083 is the primary SNO site in INSRb; in HEK293 cells, mutation of Cys1083 to alanine (INSR-C1083A) reduced the S- nitrosylation of INSRb by ~70% (Figs.5F and 5G). The INSRb SNO site (Cys1083) is localized in the tyrosine kinase catalytic domain (IRK). Insulin-stimulated IRK cross- phosphorylates the activation loop of the other INSR in the dimer, triggering its intrinsic tyrosine kinase activity. Using PyMOL, we determined that the SNO site Cys1083 resides at the interface between two IRKs of dimeric INSR, suggesting that S-nitrosylation may affect cross-phosphorylation (Fig.12B). To investigate the role of INSR-C1083A in insulin signaling, endogenous INSR in L6 cells was deleted and then replaced by INSR-WT or INSR-C1083A (Figs.12C and 12D). Following stimulation with insulin, cells expressing mutant receptor refractory to S-nitrosylation (INSR- C1083A) showed increased and sustained phosphorylation of AKT and AS160 (vs. WT INSR) (Figs.5H–5J). Thus, SCAN turns off insulin signaling by S-nitrosylation of INSRb. SNO-INSRb may be considered a marker of insulin receptor desensitization.

[0276] To determine if S-nitrosylation of INSRb by SCAN is involved in obesity- related insulin resistance in humans, we quantified the expression of SCAN and amount of SNO-INSRb in 14 human skeletal muscle samples and in 26 human adipose depot samples from patients with a range of body mass indexes (BMIs). Notably, we found that expression of SCAN is upregulated both in skeletal muscle and in visceral and subcutaneous adipose samples from patients with a higher BMI (Figs.6A– 6C, 12E, and 12F). A significant linear relationship between the amount of SNO-INSRβ and BMI in human tissues suggests that S- nitrosylation of INSRβ is an important factor in insulin resistance in humans (Figs.6D–6F, 12G, and 12H). Further, a significant linear correlation between amounts of SNO-INSRβ and the expression of SCAN protein in both human skeletal muscle and human adipose tissues (Figs.6G and 6H) implies that S-nitrosylation of INSRβ is likely mediated by SCAN in human tissues. Collectively, the multiple linear correlations among SCAN expression, BMI,and SNO-INSRb (taken together with animal data) suggests that overexpression of SCAN in obese patients contributes to insulin resistance through hypernitrosylation of INSRb.

[0277] INSRβ is one of multiple receptor tyrosine kinases (RTKs) that have been reported to be S-nitrosylated, including insulin-like growth factor 1 receptor (IGF-1) and epidermal growth factor receptor (EGFR), and our survey of papers reporting SNO-protein identification from untargeted mass spectroscopy identified 20 of the 58 RTK family members as S-nitrosylated. We find that four additional RTKs (FGFR1, PDGFRb, VEGFR2, and HER3) are S-nitrosylated in human HEK293 cells (Figs.12I–12K). S-nitrosylation of other RTKs by additional SCAN-like enzymes therefore seems likely.

[0278] Protein-dependent S-nitrosylation has been convincingly demonstrated for multiple SNO-proteins, including hemoglobin, GAPDH, thioredoxin, and S100A8 / A9. However, strict enzymatic criteria (that would justify a ‘‘nitrosylase’’ designation) have not been met previously, as these SNO-proteins are consumed in a single reaction cycle that generates product (whereas a catalyst is not consumed in a chemical reaction). Thus, the concepts of catalyst and substrate are blended. By contrast, SCAN fully meets enzymatic criteria, including demonstrated specificity, catalytic activity, and conversion of substrate to product. SCAN thus turns over in vitro with classic enzyme kinetics. Moreover, SCAN reveals a unifying catalytic mechanism for transnitrosylases entailing NO group transfer chemistry from SNO substrate (e.g., SNO-CoA) to SNO product (e.g., SNO- INSR, SNO- IRS, SNO-HO2), and suggests at least two classes of transnitrosylases based on source of SNO: LMW SNO (as in SCAN) and Protein SNO (as in GAPDH), where a dedicated SNO synthase provides the SNO substrate, as shown in E. coli.

[0279] Our findings thus fundamentally revise the conception of LMW SNO action, from being only nonspecific NO donors to acting as specific enzyme cofactors. More broadly, SCAN activity can be understood in terms of a catalytic mechanism shared by HATs, PATs, and ubiquitin ligases: group transfer chemistry from activated thiyl-donors (thionitrites and thioesters) to target nucleophiles, including cysteine and lysine. Particularly, SCAN utilizes a ping-pong mechanism to catalyze S-nitrosylation. The NO group is first transferred from SNO-CoA to SCAN, and then from S-nitrosylated SCAN to its substrate; therefore, SCAN may require only transient binding to SNO-CoA to induce SCAN auto-S- nitrosylation. This may help explain why SNO-CoA can compete with endogenous NADPH to efficiently nitrosylate substrates (despite sharing a binding site in SCAN). By the sametoken, our results refine the mechanism of disease caused by S-nitrosylation from that of excessive NO (e.g., derived from iNOS) modifying proteins indiscriminately to that of dedicated enzymes directing elevated NO to specific targets just as they do at normal levels of NO (e.g., from eNOS). It should be noted in this regard that the hyper-S-nitrosylation of proteins that is known to be causal in insulin resistance is also an established feature of other diseases, including heart failure, Alzheimer’s disease, muscular dystrophy, malignant hyperthermia, and liver cancer. SCAN-like enzymes may therefore represent attractive therapeutic targets in many diseases.

[0280] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Claims: Having described the invention, we claim:

1. A method of treating a disease or disorder characterized by aberrant or dysregulated nitrosylation in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates SNO-CoA-associated Nitrosyltransferase / biliverdin IXβ reductase B (SCAN / BLVRB) mediated S-nitrosylation.

2. The method of claim 1, wherein the disease or disorder is characterized by hypernitrosylation and the agent is an inhibitor of SCAN / BLVRB mediated S-nitrosylation.

3. The method of claim 2, wherein the inhibitor of SCAN / BLVRB mediated S- nitrosylation inhibits SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

4. The method of claim 2 or 3, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity is selected from phloxine B, erythrosin B, NSC130813, NSC12516, PH001924, Lumichrome, PH006888, ZINC4366439 / NSC 12516, xanthene, proflavine, alizarin red S, NSC ID 371876, NSC ID 179187, NSC ID 53396, NSC ID 10936, NSC ID 169534, NSC ID 117269, NSC ID 143491, NSC ID 305821, NSC ID 130813, ZINC ID ZINC0977089, ZINC ID ZINC27528243, ZINC ID ZINC09330686, ZINC ID ZINC71767103, ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC ID ZINC21093196, ZINC ID ZINC71767097, asunaprevir (BMS-650032), micafungin, tamibarotene, TSU-68 (SU6668, Orantinib), sulfasalazine, febuxostat, crenolanib (CP- 868596), olsalazine, PTC124 (ataluren), deferasirox, flunixin in combination with meglumin, azelastine, benzbromarone, triclabendazole, nifedipine, nisoldipine, zafirlukast, pyrantel in combination with pamoate, candesartan cilexetil, and azilsartan medoxomil, prodrugs thereof, metabolites thereof, and / or pharmaceutically acceptable salts thereof, or any combination thereof.

5. The method of claim 3, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity or expression comprises a small interfering RNA (siRNA) molecule or an antisense oligonucleotide specific to a region in the mRNA of BLVRB gene.

6. The method of claim 3, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity or expression is a microRNA (miRNA), preferably, miR-127-5p.

7. The method of claim 3, wherein the inhibitor of SCAN / BLVRB S- nitrosylation or S-nitrosylation of SCAN / BLVRB comprises an NOS inhibitor.

8. The method of claim 7, wherein the NOS inhibitor is selected from an nNOS inhibitor, an iNOS inhibitor, an eNOS inhibitor, or combinations thereof.

9. The method of claim 7, wherein the NOS inhibitor is selected from L-NMMA (Nγ-Monomethyl-L-arginine acetate), L-NIO dihydrochloride, ZZL7 (N-Acetyl-L-alanyl-L- valine methyl ester), Diphenyleneiodonium Chloride, Nω-Nitro-L-arginine Methyl Ester, Hydrochloride, (L-NAME), a caveolin-1(Cav-1) peptide, or combinations thereof.

10. The method of claim 9, wherein the NOS inhibitor is L-NMMA.

11. The method of any of claims 1 to 10, wherein the disease or disorder treated is associated with insulin resistance and the inhibitor of SCAN / BLVRB mediated S- nitrosylation is administered at an amount effective to inhibit hyper S-nitrosylation of INSRβ / IRS1.

12. The method of any of claims 2 to 11, wherein the disease or disorder characterized by hyper-S-nitrosylation includes at least one of diabetes, heart failure, atherosclerosis, atherosclerosis-induced ischemic stroke, ventricular arrhythmia in individuals with Duchenne muscular dystrophy, sickle cell anemia, neurodegenerative diseases and disorders, cancers, infections, inflammatory disorders, or shock states.

13. The method of any of claims 1 to 12, wherein the disease or disorder treated is diabetes and the inhibitor of SCAN / BLVRB mediated S-nitrosylation is administered at an amount effective to inhibit hyper S-nitrosylation of INSRβ / IRS1.

14. The method of claim 12, wherein the heart failure is selected from post- myocardial infarction heart failure and heart failure with preserved ejection fraction (HFpEF).

15. The method of claim 12, wherein the neurodegenerative disease or disorder is selected from the group consisting of ALS, Alzheimer's, Parkinson’s, and Huntington’s disease.

16. The method of claim 12, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), cholangiocarcinoma, prostate cancer, and acute lymphoblastic leukemia.

17. The method of claim 16, further comprising co-administering to the subject one or more additional cancer therapeutic agents with the inhibitor of SCAN / BLVRB mediated S-nitrosylation.

18. The method of claim 17, the one or more additional cancer therapeutic agents comprising a chemotherapeutic agent.

19. The method of claim 1, wherein the disease or disorder is characterized by hyponitrosylation and the agent is a promoter of SCAN / BLVRB mediated S-nitrosylation.

20. The method of claim 19, wherein the promoter of SCAN / BLVRB mediated S- nitrosylation promotes SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

21. The method of claim 20, wherein the promoter of SCAN / BLVRB mediated S- nitrosylation comprises an expression vector or DNA construct that promotes expression or expresses SCAN / BLVRB in a cell of the subject.

22. A method of modulating S-nitrosylation of INSRβ / IRS1 in a subject in need thereof, the method comprising: administering to the subject an amount of an agent that modulates SCAN / BLVRB mediated S-nitrosylation.

23. The method of claim 22, wherein the INSRβ / IRS1 is hypernitrosylated and the agent is an inhibitor of SCAN / BLVRB mediated S-nitrosylation that is administered at amount effective to inhibit hypernitrosylation.

24. The method of claim 23, wherein the inhibitor of SCAN / BLVRB mediated S- nitrosylation inhibits SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

25. The method of claim 24, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity is selected from phloxine B, erythrosin B, NSC130813, NSC12516, PH001924, Lumichrome, PH006888, ZINC4366439 / NSC 12516, xanthene, proflavine, alizarin red S, NSC ID 371876, NSC ID 179187, NSC ID 53396, NSC ID 10936, NSC ID 169534, NSC ID 117269, NSC ID 143491, NSC ID 305821, NSC ID 130813, ZINC ID ZINC0977089, ZINC ID ZINC27528243, ZINC ID ZINC09330686, ZINC ID ZINC71767103, ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC ID ZINC21093196, ZINC ID ZINC71767097, asunaprevir (BMS-650032), micafungin, tamibarotene, TSU-68 (SU6668, Orantinib), sulfasalazine, febuxostat, crenolanib (CP- 868596), olsalazine, PTC124 (ataluren), deferasirox, flunixin in combination with meglumin, azelastine, benzbromarone, triclabendazole, nifedipine, nisoldipine, zafirlukast, pyrantel in combination with pamoate, candesartan cilexetil, and azilsartan medoxomil, prodrugs thereof, metabolites thereof, and / or pharmaceutically acceptable salts thereof, or any combination thereof.

26. The method of claim 24, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity or expression comprises a small interfering RNA (siRNA) molecule or an antisense oligonucleotide specific to a region in the mRNA of BLVRB gene.

27. The method of claim 24, wherein the inhibitor of SCAN / BLVRB S- nitrosylation activity or expression is a microRNA (miRNA), preferably, miR-127-5p.

28. The method of claim 27, wherein the inhibitor of SCAN / BLVRB S- nitrosylation or S-nitrosylation of SCAN / BLVRB comprises an NOS inhibitor.

29. The method of claim 28, wherein the NOS inhibitor selected from an nNOS inhibitor, an iNOS inhibitor, an eNOS inhibitor, or combinations thereof.

30. The method of claim 28 or 29, wherein the NOS inhibitor is selected from L- NMMA (Nγ-Monomethyl-L-arginine acetate), L-NIO dihydrochloride, ZZL7 (N-Acetyl-L- alanyl-L-valine methyl ester), Diphenyleneiodonium Chloride, Nω-Nitro-L-arginine Methyl Ester, Hydrochloride, (L-NAME), a caveolin-1(Cav-1) peptide, or combinations thereof.

31. The method of claim 30, wherein the NOS inhibitor is L-NMMA.

32. The method of any of claims 22 to 31, wherein the subject in need of S- nitrosylation of INSRβ / IRS1 modulation has an elevated or increased body mass index.

33. The method of any of claims 22 to 32, wherein the subject is obese.

34. The method of any of claims 22 to 33, wherein the subject has type 2 diabetes.

35. The method of claim 22, wherein the INSRβ / IRS1 is hyponitrosylated and the agent is a promoter of SCAN / BLVRB mediated S-nitrosylation that is administered at an amount effective to inhibit hyponitrosylation or promote SCAN / BLVRB mediated S- nitrosylation.

36. The method of claim 35, wherein the promoter of SCAN / BLVRB mediated S- nitrosylation promotes SCAN / BLVRB S-nitrosylation activity, S-nitrosylation of SCAN / BLVRB, or SCAN / BLVRB expression.

37. The method of claim 38, wherein the promoter of SCAN / BLVRB mediated S- nitrosylation is an expression vector or DNA construct that promotes expression or expresses SCAN / BLVRB in a cell of the subject.

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