Kenneth Ellsworth

Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase–1

Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase-1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.

Inhibition of rat α-reductases by finasteride: Evidence for isozyme differences in the mechanism of inhibition

The mechanism of inhibition of the rat types 1 and 2 5alpha-reductase by finasteride was investigated using recombinantly expressed enzymes. These studies revealed that finasteride is a potent, reversible inhibitor of the rat type 1 5alpha-reductase with Ki=10.2+/-1.3 nM. Finasteride is a potent inhibitor of the rat type 2; however, in this case the compound binds to the type 2 isozyme-NADPH complex to form a ternary complex with Ki=1.19+/-0.10 nM, which then rearranges to a high affinity complex (E:I) with a pseudo first order rate constant of 1.62+/-0.22 x 10(-3)/s. The second order rate constant is k3/Ki=1.37+/-0.31 x 10(6) M/s. Heat denaturation of the (type 2 enzyme:inhibitor) complex releases dihydrofinasteride and presumably the NADP+-adduct previously identified with the human 5alpha-reductases. The effects of finasteride were also studied in intact COS cells transiently expressing the rat types 1 and 2 5alpha-reductase. Results with whole cell assays confirm differences in mechanism of inhibition of rat types 1 and 2 5alpha-reductase by finasteride.

Antidiabetic and antisteatotic effects of the selective fatty acid synthase (FAS) inhibitor platensimycin in mouse models of diabetes

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

Glucose-lowering in a db/db mouse model by dihydropyridine diacid glycogen phosphorylase inhibitors.

The synthesis of a series of novel dihdyropyridine diacid glycogen phosphorylase inhibitors is presented. SAR and functional assay data are discussed, along with the effect of a single inhibitor on blood glucose in a diabetic animal model.

Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo

Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.

MK386 : a potent, selective inhibitor of the human type 1 5α-reductase

Abstract Steroid 5α-reductase is required for the conversion of testosterone to dihydrotestosterone. Localization of type 1 5α-reductase in the sebaceous gland of skin offers the possibility for selective inhibition of this isozyme as a treatment for acne. The goals of these studies are to demonstrate the mechanism of inhibition of MK386 and its selectivity for type 1 5α-reductase. The apparent potency of MK386 differed depending on the source of the enzyme (i.e. recombinant vs. native), yet selectivity for type 1 5α-reductase was unchanged. Our results indicate that the apparent potency of MK386 is modulated by the membrane concentration of the assay. These results suggest that MK386 has a high affinity for the lipid-rich membrane environment of 5α-reductase. MK386 was also found to be a slow binding inhibitor of type 1 5α-reductase. However, the cause of this time-dependent inhibition is unrelated to partitioning of the inhibitor into the membrane because similar studies with type 2 5α-reductase indicate that MK386 is a reversible, competitive inhibitor. A number of counterscreens were developed to demonstrate that MK386 is a poor inhibitor of other steroid metabolizing enzymes.

Evidence that inhibition of insulin receptor signaling activity by PC-1/ENPP1 is dependent on its enzyme activity

Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.

Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-Reductase: evidence for mechanism-Based inhibition by finasteride

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus SaRs shared 94% and 96% identity with the human type 1 and 2 isozymes, respectively. Despite a four amino acid insertion at the N-terminal region of rhesus 5aR1, the biochemical properties of rhesus and human homologs are very similar with respect to pH optimum, Km values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expected, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays time dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M(-1) s(-1) and 5.2 x 10(5) M(-1)s(-1). Inhibition of rhesus 5aR2 with 3H-finasteride resulted in 3H bound to the enzyme which is not released by dialysis. Heat denaturation of the [rhesus SaR2:inhibitor] complex releases dihydrofinasteride, a breakdown product presumably related to the NADP+-adduct previously identified with the human SaRs (Bull et al., Mechanism-based inhibition of human steroid 5alpha-reductase by finasteride: Enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J. Amer. Chem. Soc., 1996, 118, 2359-2365). Taken together, these results provide good evidence that the rhesus macaque is a suitable model to evaluate the pharmacological properties of finasteride and other 5aR inhibitors.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.