Computational design and selections for an engineered, thermostable terpene synthase.

Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme.

Novel tetrahydroisoquinoline derivatives with inhibitory activities against acyl-CoA: cholesterol acyltransferase and lipid peroxidation.

To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity, a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-2,3,5-trimethylphenyl)carbamoyl moiety at the 3-position and an octanoyl moiety at the 2-position (7) was demonstrated to show anti-foam cell formation activity stronger than and anti-lipid peroxidative activity comparable to those of Pactimibe, while it was hardly absorbed orally. To increase its bioavailability, the acyl chain at the 2-position was shortened and various polar or basic moieties were introduced at the 7-position of 7. Among the synthesized derivatives, (S)-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide hydrochloride (21) showed about 16-fold stronger anti-foam cell formation activity, 3-fold stronger hepatic ACAT inhibitory activity, similar anti-low density lipoprotein (LDL) oxidative activity and 2-fold more potent protective activity against macrophage cell death by oxidative stress in comparison with Pactimibe. Compound 21 was efficiently absorbed after oral administration at 10 mg/kg in rats and dogs and its C(max) values were higher than its IC(50) values for in vitro activities. In conclusion, a tetrahydroisoquinoline structure is a useful scaffold for designing a phenolic anti-oxidative ACAT inhibitor, and compound 21 is expected to effectively prevent atherosclerosis.

Novel indoline-based acyl-CoA: cholesterol acyltransferase inhibitor: Effects of introducing a methanesulfonamide group on physicochemical properties and biological activities.

A novel series of indoline-based acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors with a methanesulfonamide group at the 5-position were synthesized and their lipophilicity and biological activities were evaluated. Hepatic ACAT inhibitory and anti-foam cell formation activity increased dependent on lipophilicity of derivatives with various alkyl chains at the 1-position. The logD(7.0)-biological activity curve of the derivatives with a methanesulfonamide group shifted leftward compared to that of Pactimibe derivatives with a carboxymethyl group, and derivatives with no substituent, suggesting that a methanesulfonamide group plays an important role in the interaction with ACAT protein. Among derivatives, N-(1-ethyl-5-methanesulfonylamino-4,6-dimethylindolin-7-yl)-2,2-dimethylpropanamide (1b) had about twofold lower logD(7.0) than Pactimibe, while it showed twofold higher hepatic ACAT inhibition than and the same anti-foam cell formation as Pactimibe, respectively. The C(max) of 1b (10mg/kg, po) was higher than that of Pactimibe in rats. The plasma protein binding ratio of 1b was lower than that of Pactimibe: 64.8% and 97.9%, respectively. Compound 1b showed greater inhibitory effects on hepatic cholesterol secretion in mice than Pactimibe. In conclusion, the introduction of a methanesulfonamide group is effective to design less lipophilic, more efficacious and more bioavailable indoline-based ACAT inhibitors than previous indoline-based inhibitors.

NK1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries.

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK(1) receptors, and endothelium-independent contraction (EIC) via tachykinin NK(2) receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK(1) receptor. Five tachykinin agonists, including septide, a reportedly atypical NK(1) agonist, caused concentration-dependent EDR in the presence of NK2 antagonist (SR-48968) + TXA2 synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK2 antagonist (SR-48968) + nitric oxide synthase inhibitor (l-N(G)-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC(50) values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK(1) agonists to NK(1) receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala(5), beta-Ala(8)]-alpha-neurokinin fragment 4-10 (NK2 agonist) and senktide (NK3 agonist) caused no responses. Two structurally different NK(1) antagonists, CP-99994 and SR-140333, shifted the concentration-EDC and -EDR curves of SPME, a selective NK(1) agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK(1) antagonists to NK1 receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca2+ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca2+ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK1 receptor followed by an increase in intracellular Ca2+, and sensitivity to Ca2+ may be higher in the EDR than EDC pathway.

Impairment of endothelium-dependent ACh-induced relaxation in aorta of diabetic db/db mice--possible dysfunction of receptor and/or receptor-G protein coupling.

Diabetes is a risk factor of ischemic heart disease, cerebral ischemia, and atherosclerosis, in which endothelial dysfunction plays a role in the pathogenesis. We examined vascular responses in the aorta of pre-diabetic db/db mice with normoglycemia, hyperlipidemia, and hyperinsulinemia (6 weeks old), and diabetic db/db mice with hyperglycemia, hyperlipidemia, and hyperinsulinemia (11 weeks old) in comparison with age-matched non-diabetic db/+ mice. Prostaglandin F2alpha (PGF2alpha)-induced contraction was significantly enhanced in the aorta of diabetic but not pre-diabetic db/db mice compared to age-matched non-diabetic db/+ mice. Acetylcholine (ACh), adenosine-5'-diphosphate (ADP), NaF, a G protein activator and A-23187, a Ca-ionophore, caused endothelium-dependent and nitric oxide (NO)-mediated relaxation, and sodium nitroprusside (SNP), an NO donor, caused endothelium-independent relaxation in the pre-contracted aorta of db/db mice. Maximal endothelium-dependent ACh-induced relaxation was reduced in diabetic but not pre-diabetic db/db mice compared to age-matched db/+ mice, while maximal SNP-induced relaxation was not different between diabetic and non-diabetic mice. ACh-induced relaxation in diabetic db/db mice was not affected by ozagrel, a thromboxane A2 (TXA2) synthetase inhibitor, or acetylsalicylic acid (aspirin), a cyclooxygenase inhibitor, suggesting no involvement of endogenous TXA2 or prostanoids in the reduction of relaxation. Maximal endothelium-dependent ADP-, A-23187-, and NaF-induced relaxation was not reduced in diabetic db/db mice. EC50 values for ACh- and SNP-induced relaxation were increased in diabetic but not pre-diabetic db/db mice, suggesting decreases in sensitivity to NO in diabetic mice. Two-week treatment with KV-5070, a PPARgamma agonist, lowered plasma glucose, triglyceride (TG), and insulin but not cholesterol, and reversed the reduced ACh-induced relaxation. In conclusion, ACh-induced endothelium-dependent relaxation is impaired in diabetic db/db mice, probably due to the dysfunction of ACh receptors and/or receptor-G protein coupling. Endothelial dysfunction was not genetic and was considered to be initiated primarily by hyperglycemia, and was improved by anti-diabetic treatment with a PPARgamma agonist.