Effects of compound-326, a selective delta-5 desaturase inhibitor, in ApoE knockout mice with two different protocols for atherosclerosis development.

PURPOSE: We previously confirmed its anti-atherosclerotic effects by pre-treatment with compound-326, a selective delta-5 desaturase (D5D) inhibitor, in Western diet-fed ApoE knockout mice. In the present study, we evaluated effects of compound-326 in ApoE knockout mice with two different protocols for atherosclerosis development. METHODS: In a post-treatment protocol, where the compound treatment started after 10 weeks pre-feeding of Western diet, compound-326 (1 and 3 mg/kg/day, p.o. for 12 weeks) significantly reduced the atherosclerotic lesion area in the aorta (24% reduction at 3 mg/kg/day). In another protocol using Paigen diet (containing 12.5% cholesterol and 5% sodium cholate), compound-326 (3 and 10 mg/kg/day, p.o. for 7 weeks) also significantly reduced the lesion area (36% reduction at 3 mg/kg/day). RESULTS: In both protocols, Compound-326 significantly reduced the hepatic ratio of arachidonic acid to dihomo-γ-linolenic acid, blood inflammatory eicosanoid production and plasma soluble intercellular adhesion molecule 1 (sICAM-1) levels, similarly to the previous pre-treatment study. CONCLUSIONS: Compound-326 exerted anti-atherosclerotic effects in ApoE knockout mice with the two different protocols for atherosclerosis development further supporting D5D inhibition as a promising strategy in treating atherosclerosis.

A Novel Orally Available Delta-5 Desaturase Inhibitor Prevents Atherosclerotic Lesions Accompanied by Changes in Fatty Acid Composition and Eicosanoid Production in ApoE Knockout Mice.

Delta-5 desaturase (D5D), encoded by fatty acid desaturase 1 (Fads1), is the rate-limiting enzyme for the conversion from dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) in the ω-6 polyunsaturated fatty acid pathway. Several AA-derived eicosanoids (e.g., prostaglandins, thromboxanes, and leukotrienes) and DGLA-derived eicosanoids are reported to promote and/or prevent atherosclerosis progression through, at least in part, its proinflammatory or anti-inflammatory effects. To elucidate the effects of D5D inhibition by a D5D inhibitor on atherosclerosis, we generated a potent, orally available and selective D5D inhibitor, 2-(2,2,3,3,3-Pentafluoropropoxy)-3-[4-(2,2,2-trifluoroethoxy) phenyl]-5,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-4,6-dione, compound-326, and examined its effects on Western-diet fed ApoE knockout (KO) mice. Oral administration of compound-326 (3-10 mg/kg per day for 15 weeks) significantly inhibited the progression of atherosclerotic lesions in the aorta without affecting plasma total cholesterol and triglyceride levels. Compound-326 significantly decreased AA levels, while it increased DGLA levels in the liver and the blood accompanied by decreases in AA-derived eicosanoid production and increases in DGLA-derived eicosanoid production from the blood cells. We conclude that compound-326 prevents the progression of atherosclerosis in Western-diet fed ApoE KO mice by modulating a profile of eicosanoid production, suggesting that D5D inhibitors can be a novel remedy for preventing atherosclerosis and subsequent cardiovascular events. SIGNIFICANCE STATEMENT: This study shows a D5D-specific and orally available potent inhibitor provided the first evidence to support the concept that D5D inhibitors will be a novel remedy for preventing the progression of atherosclerosis.

Discovery of 3,5-Diphenyl-4-methyl-1,3-oxazolidin-2-ones as Novel, Potent, and Orally Available Δ-5 Desaturase (D5D) Inhibitors.

The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3-oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.

A Novel Selective Inhibitor of Delta-5 Desaturase Lowers Insulin Resistance and Reduces Body Weight in Diet-Induced Obese C57BL/6J Mice.

Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. In vivo D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.

Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.

p38 MAPKs regulate the expression of genes in the dopamine synthesis pathway through phosphorylation of NR4A nuclear receptors.

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

Differential requirement of G alpha12, G alpha13, G alphaq, and G beta gamma for endothelin-1-induced c-Jun NH2-terminal kinase and extracellular signal-regulated kinase activation.

In the present study, we examined the roles of G(12), G(13), G(q), and G(i) in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of G alpha(12) and G alpha(13). JNK activation was also inhibited by expression of the G alpha(12)/G alpha(13)-specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the G alpha(q)-specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a G beta gamma-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by G alpha(12), G alpha(13), and G alpha(q) is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-G(i) uncoupler, and GRK2-ct. Thus, ERK was activated by G alpha(i)- and G beta gamma-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK.

G beta gamma counteracts G alpha(q) signaling upon alpha(1)-adrenergic receptor stimulation.

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.

Activation mechanism of Gi and Go by reactive oxygen species.

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.