The pleiotropic regulation of cyclin D1 by newly identified sesaminol-binding protein ANT2.

The expression of cyclin D1 is upregulated in various cancer cells by diverse mechanisms, such as increases in mRNA levels, the promotion of the translation by mammalian target of rapamycin complex 1 (mTORC1) signaling and the protein stabilization. We here show that sesaminol, a sesame lignan, reduces the expression of cyclin D1 with decreasing mRNA expression levels, inhibiting mTORC1 signaling and promoting proteasomal degradation. We subsequently generated sesaminol-immobilized FG beads to newly identify sesaminol-binding proteins. As a consequence, we found that adenine nucleotide translocase 2 (ANT2), the inner mitochondrial membrane protein, directly bound to sesaminol. Consistent with the effects of sesaminol, the depletion of ANT2 caused a reduction in cyclin D1 with decreases in its mRNA levels, mTORC1 inhibition and the proteasomal degradation of its protein, suggesting that sesaminol negatively regulates the function of ANT2. Furthermore, we screened other ANT2-binding compounds and found that the proliferator-activated receptor-γ agonist troglitazone also reduced cyclin D1 expression in a multifaceted manner, analogous to that of the sesaminol treatment and ANT2 depletion. Therefore, the chemical biology approach using magnetic FG beads employed in the present study revealed that sesaminol bound to ANT2, which may pleiotropically upregulate cyclin D1 expression at the mRNA level and protein level with mTORC1 activation and protein stabilization. These results suggest the potential of ANT2 as a target against cyclin D1-overexpressing cancers.

Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer.

Resveratrol has various attractive bioactivities, such as prevention of cancer, neurodegenerative disorders, and obesity-related diseases. Therefore, identifying its direct binding proteins is expected to discover druggable targets. Sirtuin 1 and phosphodiesterases have so far been found as the direct molecular targets of resveratrol. We herein identified 11 novel resveratrol-binding proteins, including the DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5, also known as p68), using resveratrol-immobilized beads. Treatment with resveratrol induced degradation of DDX5 in prostate cancer cells. Depletion of DDX5 caused apoptosis by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. Moreover, knockdown of DDX5 attenuated the inhibitory activities of resveratrol against mTORC1 signaling and cancer cell growth. These data show that resveratrol directly targets DDX5 and induces cancer cell death by inhibiting the mTORC1 pathway.

YM-53601, a novel squalene synthase inhibitor, reduces plasma cholesterol and triglyceride levels in several animal species.

The aim of this study was to evaluate the potency of YM-53601 ((E)-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride), a new inhibitor of squalene synthase, in reducing both plasma cholesterol and triglyceride levels, compared with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor and fibrates, respectively. YM-53601 equally inhibited squalene synthase activities in hepatic microsomes prepared from several animal species and also suppressed cholesterol biosynthesis in rats (ED(50), 32 mg kg(-1)). In guinea-pigs, YM-53601 and pravastatin reduced plasma nonHDL-C (=total cholesterol - high density lipoprotein cholesterol) by 47% (P<0.001) and 33% (P<0.001), respectively (100 mg kg(-1), daily for 14 days). In rhesus monkeys, YM-53601 decreased plasma nonHDL-C by 37% (50 mg kg(-1), twice daily for 21 days, P<0.01), whereas the HMG-CoA reductase inhibitor, pravastatin, failed to do (25 mg kg(-1), twice daily for 28 days). YM-53601 caused plasma triglyceride reduction in hamsters fed a normal diet (81% decrease at 50 mg kg(-1), daily for 5 days, P<0.001). In hamsters fed a high-fat diet, the ability of YM-53601 to lower triglyceride (by 73%, P<0.001) was superior to that of fenofibrate (by 53%, P<0.001), the most potent fibrate (dosage of each drug: 100 mg kg(-1), daily for 7 days). This is the first report that a squalene synthase inhibitor is superior to an HMG-CoA reductase inhibitor in lowering plasma nonHDL-C level in rhesus monkeys and is superior to a fibrate in significantly lowering plasma triglyceride level. YM-53601 may therefore prove useful in treating hypercholesterolemia and hypertriglyceridemia in humans.

Species specificity in the blood cholesterol-lowering effect of YM-16638.

1. The compound YM-16638, [[5-[[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl] thio]-1,3,4-thiadiazol-2-yl]thio] acetic acid was developed in a series of in vitro and in vivo studies as a leukotriene D4 receptor antagonist. 2. In a clinical trial as a leukotriene antagonist drug, this compound was found to have a potent serum cholesterol lowering effect in normolipidaemic healthy male volunteers. 3. In the present study, we investigated the serum cholesterol lower effect of this compound in various species of experimental animals. 4. Administration of YM-16638 did not cause a significant decrease in serum total cholesterol (TC) in mice (up to 200 mg kg-1, body weight per day for 28 days), rats (200 mg kg-1 for 15 days) or rabbits (90 mg kg-1 for 18 days). In hamsters, administration of YM-16638 orally or by peritoneal injection at 50 mg kg-1 or more daily for 7 days caused a significant decrease in serum TC and the rate of body weight gain. In monkeys, serum TC did not change in YM-16638-administered squirrel monkeys (50 mg kg-1 daily for 3 weeks), but a significant decrease in serum TC was observed in cynomolgus monkeys (33% decrease at 30 mg kg-1 for 4 weeks) and rhesus monkeys (27% decrease at 30 mg kg-1 for 3 weeks) without any serious decrease in body weight. These results were consistent with those in a phase I study with human subjects. In contrast, serum alanine aminotransferase (ALT) level decreased in all animals after YM-16638 treatment. 5. From these results, we conclude that YM-16638 has a potent hypocholesterolaemic effect, but that this effect if species-specific and is only recognized clearly in human subjects and old-world monkeys.

Antithrombotic effect of YM-75466 is separated from its effect on bleeding time and coagulation time.

The antithrombotic effects of YM-75466 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-nap hthyl)methyl]sulfamoyl]acetic acid monomethane sulfonate), a novel orally-active factor Xa inhibitor, and its effects on bleeding time and coagulation time were studied in rats and compared with those of warfarin. Both agents were orally administered. In the venous thrombosis model, YM-75466 and warfarin inhibited thrombus formation dose-dependently, with ID50 values of 3.3 and 0.56 mg/kg, respectively. Ex vivo study showed that both YM-75466 and warfarin prolonged prothrombin time dose-dependently, with doses, causing a two-fold prolongation of prothrombin time in the control group, of 89 and 0.38 mg/kg, respectively. In bleeding time studies, YM-75466 and warfarin prolonged bleeding time dose-dependently, with doses, causing a two-fold prolongation of bleeding time in the control group, of > 100 and 0.43 mg/kg, respectively. These results show that the antithrombotic effects of YM-75466 are markedly separate from its effects on bleeding time and coagulation time compared with warfarin.

Cardiovascular and renal effects of conivaptan hydrochloride (YM087), a vasopressin V1A and V2 receptor antagonist, in dogs with pacing-induced congestive heart failure.

The systemic hemodynamic and renal responses to conivaptan hydrochloride (YM087; 4'-(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine -6-carbonyl)-2-phenylbenzanilide monohydrochloride), a vasopressin V1A and V2 receptor antagonist, were determined in pentobarbital-anesthetized dogs after 2 to 3 weeks of rapid right ventricular pacing. Congestive heart failure, characterized by decreases in first derivative of left ventricular pressure (left ventricular d P/dt(max)) and cardiac output, and increases in left ventricular end-diastolic pressure and total peripheral vascular resistance, was induced by chronic rapid right ventricular pacing at 260-280 beats/min. Intravenous administration of conivaptan (0.1 mg/kg) significantly increased left ventricular dP/dt(max) and cardiac output and significantly decreased left ventricular end-diastolic pressure and total peripheral vascular resistance. Conivaptan also increased urine flow and reduced urine osmolality by markedly increasing free water clearance. These results indicate that conivaptan produced hemodynamic improvement and marked aquaresis in dogs with congestive heart failure. Therefore, conivaptan may find clinical use in treating patients with congestive heart failure.

Comparison of the anticoagulant and antithrombotic effects of YM-75466, a novel orally-active factor Xa inhibitor, and warfarin in mice.

The anticoagulant and antithrombotic effects of YM-75466 (N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naph thyl)methyl]sulfamoyl acetic acid monomethanesulfonate), a novel orally-active factor Xa (FXa) inhibitor, and warfarin were compared in mice. Both agents were orally administered in all studies. In ex vivo studies, the peak effects of YM-75466 occurred 1 hr after administration while the peak of warfarin activity occurred 18 hr after administration. At each peak, both YM-75466 and warfarin prolonged coagulation time dose-dependently. The dose response curve of warfarin for prothrombin time was steeper than that of YM-75466. In a thromboplastin-induced thromboembolism model, administration of 30 mg/kg YM-75466 or 3 mg/kg warfarin significantly improved the lethality ratio. In blood loss studies, YM-75466 did not increase blood loss from the tail even at 30 mg/kg, while warfarin markedly increased blood loss at 3 mg/kg. Agents that interfere with warfarin action did not interfere with YM-75466 action. In conclusion, this study shows that YM-75466 has advantages over warfarin: i) rapid onset of anticoagulant activity, ii) wide therapeutic range, iii) little effect on bleeding and iv) lack of drug interaction with agents that interfere with warfarin. These results suggest that YM-75466 may be promising as a novel oral anticoagulant agent.

Pharmacological profile of orally administered YM087, a vasopressin antagonist, in conscious rats.

1. YM087 is a newly synthesized non-peptide arginine vasopressin (AVP) antagonist that shows high affinity for both V1A and V2 receptors. In the present study, the V1A and V2 receptor antagonist effects of orally administered YM087 were assessed in conscious rats. 2. In conscious rats, orally administered YM087 (0.1, 0.3 and 1.0 mg/kg) did not affect basal blood pressure, but YM087 dose-dependently inhibited 30 mU/kg, i.v., AVP-induced pressor responses. This inhibition lasted for over 8 h following the oral administration of the highest dose of YM087 (1 mg/kg). 3. In rats deprived of water and food for 16-18 h, oral administration of YM087 (0.1, 0.3, 1 and 3 mg/kg) dose-dependently increased urine volume and reduced urine osmolality, with associated increases in urinary sodium and potassium excretion. However, these increases in electrolyte excretion were lower than those seen at comparable diuretic doses of furosemide (3, 10, 30 and 100 mg/kg, p.o.). 4. Oral administration of YM087 (0.3, 1 and 3 mg/kg) produced a dose-dependent increase in urine volume in rats allowed free access to water, with the diuretic effect peaking 2-4 h post-dosing at all dose levels. The diuretic effect of YM087 was sustained 8-10 h after a dose of 3 mg/kg; this is in contrast with the transient diuresis seen after furosemide (100 mg/kg, p.o.) dosing. 5. The present results demonstrate that YM087 is an orally active AVP antagonist with potent and long-lasting effects. YM087 suppressed V1A receptor-mediated pressor responses to AVP with minimal effects on basal haemodynamics and exerted a diuretic effect without increased electrolyte excretion by inhibiting V2 receptor-mediated water reabsorption.

[Pharmacology of conivaptan hydrochloride (YM087), a novel vasopressin V1A/V2 receptor antagonist].

Pharmacology of conivaptan hydrochloride (YM087) was investigated in in vitro and in vivo studies. In radioligand binding study, YM087 showed high affinity for both V1A and V2 receptors in animal and human species. Affinity of YM087 for V1A and V2 receptors was comparable to that of vasopressin (AVP). In functional antagonistic activity study, YM087 concentration-dependently inhibited AVP-induced intracellular Ca2+ elevation via human V1A receptors and AVP-stimulated cAMP accumulation via human V2 receptors. Intravenous administration of YM087 dose-dependently inhibited AVP-induced pressor responses and produced a dose-dependent aquaresis in rats and dogs. Oral administration of YM087 showed a potent and long-lasting antagonistic activity on V1A and V2 receptors. YM087 was effective in dogs with heart failure and in heart failure rats with hyponatremia and edema. These results reveal that YM087 is the first orally active V1A/V2 receptor antagonist and suggest that YM087 may be useful in the treatment of congestive heart failure and hyponatremia.

Pharmacological properties of YM17E, an acyl-CoA:cholesterol acyltransferase inhibitor, and diarrheal effect in beagle dogs.

YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl)ureido]methyl]ben zene dihydrochloride) was found to be a potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT) in rabbit liver and intestine microsomes. Dixon plot analysis revealed that YM17E inhibited microsomal ACAT in a non-competitive manner. YM17E induced a marked decrease in serum cholesterol, especially in non-high-density lipoprotein (HDL) fractions, in cholesterol-fed rats and rats fed normal chow. Measurement of bile secretion after oral administration of YM17E in cholesterol-fed rats showed that the drug markedly accelerated the secretion of bile acids and neutral sterols. Furthermore, absorption of [3H]cholesterol from the gut of cholesterol-fed rats was significantly inhibited by YM17E. From these results, the hypocholesterolemic activity of YM17E in these animals resulted from both a decrease in cholesterol absorption from the gut and the stimulation of excretion of cholesterol from the liver into bile. However, YM17E caused secretory diarrhea in beagle dogs at near lipid lowering doses. When YM17E was administered at the same total dosage but divided into 5 daily administrations, the incidence of diarrhea was significantly reduced while its cholesterol lowering effect became stronger. These results suggest that the inhibition of intestinal and/or liver ACAT increases the risk of diarrhea development which, however, can be avoided by controlled drug administration in beagle dogs.