Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASOR⅐poly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrifugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aortic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL ؉ very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits. Cholesteryl ester transfer protein (CETP)1 is a plasma glycoprotein that catalyzes the transfer of cholesteryl ester and tr...

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

A novel series of RORγ inhibitors was identified starting with the HTS hit 1. After SAR investigation based on a prospective consideration of two drug-likeness metrics, ligand efficiency (LE) and fraction of sp 3 carbon atoms (Fsp 3 ), significant improvement of metabolic stability as well as reduction of CYP inhibition was observed, which finally led to discovery of a selective and orally efficacious RORγ inhibitor 3z.KEYWORDS: Th17, immunological diseases, nuclear receptor, RORγ, ligand efficiency (LE), fraction of sp 3 carbon atoms (Fsp 3 )T wo decades after the discovery of Th1 and Th2 cells, a third subset of T helper cells called Th17 cells was identified and has drawn considerable attention since it was suggested to play a central role in the pathogenesis of various autoimmune diseases such as psoriasis and rheumatoid arthritis. 1,2 Among several regulatory pathways in which Th17 development and function are involved, the one regulated by the nuclear receptor RORγ appears to be crucial for controlling the differentiation and function. 3 Given its validity as an emerging drug target for treatment of immunological diseases, many research groups have made significant efforts in the discovery of RORγ modulators in recent years. 4−19 Since starting our RORγ inhibitor program in 2003, we discovered several structurally diverse hits after a HTS campaign. 20 From these hits we selected compound 1 as the first hit-to-lead series for optimization. In addition to being reasonably potent against RORγ (hLUC EC 50 = 1.7 μM, FRET EC 50 = 0.85 μM), compound 1 also demonstrated >20-fold selectivity over five nuclear receptors (hRORα, hFXR, hRXRα, hPR, and hPPARγ) and was structurally unique in comparison to other nuclear receptor modulators. 16−18 However, this compound has several drawbacks. For example, the microsomal stability in liver microsomes is poor with only 18% remaining at 10 min in human liver microsomes. It also has a modest time-dependent human CYP3A4 inhibition (IC 50 = 4 μM) probably due to some reactive metabolites formed by the oxidation of 1. The ligand efficiency is only 0.25, far below the literature consensus value (0.30) for a drug-like molecule. 21 The concept of ligand efficiency (LE) was first introduced by Kuntz 22 and is widely accepted as a reliable index of drug-like qualities. 23 Improvement of LE inevitably results in lower molecular weight and higher potency. We reasoned that a strategy of increasing LE and lowering the lipophilicity should therefore significantly improve the drug-like properties of compound 1. In addition, compound 1 is a rather flat molecule with a fraction of saturated carbons (Fsp 3 ) of 0.24. Fsp 3 is a newer index representing drug-likeness. 24 Lovering et al. pointed out that a decrease of Fsp 3 value would result in an increased incidence of CYP inhibition. 25 The desired Fsp 3 value is over 0.47 according to the literature. 24 Thus, we considered that improvement of the poor Fsp 3 value of compound 1 would be a rational way to overcome the CYP inhibi...

Retinoid-related orphan receptor gamma (RORγ) directly controls the differentiation of Th17 cell and the production of interleukin-17, which plays an integral role in autoimmune diseases. To obtain insight into RORγ, we have determined the first crystal structure of a ternary complex containing RORγ ligand-binding domain (LBD) bound with a novel synthetic inhibitor and a repressor peptide, 22-mer peptide from silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Comparison of a binary complex of nonliganded (apo) RORγ-LBD with a nuclear receptor co-activator (NCoA-1) peptide has shown that our inhibitor displays a unique mechanism different from those caused by natural inhibitor, ursolic acid (UA). The compound unprecedentedly induces indirect disruption of a hydrogen bond between His479 on helix 11 (H11) and Tyr502 on H12, which is crucial for active conformation. This crystallographic study will allow us to develop novel synthetic compounds for autoimmune disease therapy.

Blockade of T cell activation through ICOS during the immune response phase regulates development of EAM, and therefore, ICOS may be an effective target for treating myocarditis.