Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

Hypoglycemic prodrugs of 4-(2,2-dimethyl-1-oxopropyl)benzoic acid.

SAH 51-641 (1) is a potent hypoglycemic agent, which acts by inhibiting hepatic gluconeogenesis. It is a prodrug of 4-(2, 2-dimethyl-1-oxopropyl)benzoic acid (2) and 4-(2, 2-dimethyl-1-hydroxypropyl)benzoic acid (3), which sequester coenzyme A (CoA) in the mitochondria, and inhibits medium-chain acyltransferase. 1-3 and 4-tert-butylbenzoic acid all cause testicular degeneration in rats at pharmacologically active doses. 14b (FOX 988) is a prodrug of 3, which is metabolized in the liver at a rate sufficient enough to have hypoglycemic potency (an ED50 of 65 micromol/kg, 28 mg/kg/day, for glucose lowering), yet by avoiding significant escape of the metabolite 3 to the systemic circulation, it avoids the testicular toxicity at doses up to 1500 micromol/kg/day. 14b was selected for clinical studies.

Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.

A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.

Expression of human GLUT4 in mice results in increased insulin action.

Glucose metabolism was evaluated in transgenic mice expressing the human GLUT 4 glucose transporter. Fed GLUT 4 transgenic mice exhibited a 32% and 56% reduction in serum glucose and insulin and a 69% and 33% increase in non-esterified fatty acid and lactate levels, respectively. Transgenic mice exhibited a significant increase in whole-body glucose disposal during a euglycaemic-hyperinsulinaemic clamp. Insulin-stimulated glucose uptake in isolated soleus muscles and adipocytes was greater in transgenic compared to control mice due to increased basal glucose uptake. Transgenic mice displayed increased glycogen levels in liver and gastrocnemius muscle, and increased insulin-stimulated 14C-glycogen accumulation in isolated soleus muscle. We conclude that over-expression of the GLUT 4 glucose transporter in mice results in 1) an increase in whole-body glucose disposal and storage, and 2) an increase in both basal and insulin-stimulated glucose uptake and disposal in vitro. These changes resulted in the reduction of serum glucose and insulin levels. These results provide direct evidence that glucose transport (and GLUT 4 per se) plays a significant role in regulating whole-body glucose homeostasis. Additionally, these data support the idea that pharmacological strategies directed at increasing the expression of GLUT 4 protein may have beneficial (hypoglycaemic) effects in the diabetic state.

Oxygen sensors in vascular smooth muscle.

Inhibition or activation of cellular function due to acute decreases in PO2 can be considered in terms of two different processes: 1) a sensor that monitors PO2 decreases and 2) transduction systems directed from the O2 sensor to reactions that control cellular function. We used the norepinephrine-contracted aortic smooth muscle model to study the nature of the O2 sensor and transduction system during decreased PO2-evoked relaxations. The phosphorylation potential, a measurement of kinetic energy required for ATP hydrolysis, was decreased to 30% of control at the onset of relaxation and progressively fell as muscle relaxed. The free inorganic phosphate intracellular concentration ([Pi]) was experimentally increased approximately 0.6 mM during transients that followed a rapid decrease in PO2. Relaxations to 80% of maximal force were more rapid under conditions of an augmented [Pi] than in control rings, and they occurred at a higher phosphocreatine concentration and phosphocreatine-to-free creatine ratio but at the same phosphorylation potential. Results support the operation of a cytochrome aa3 O2 sensor in the mechanism of decreased PO2-evoked relaxations and implicate an increase in [Pi] and a decrease in kinetic energy in the transduction mechanism directed at rate-limiting reactions that control force.

Smooth muscle guanine nucleotides and receptor-effector coupling following inhibition of oxidative energy production.

We measured total concentrations of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) in rabbit aortic smooth muscle under several different conditions. We computed free [GDP] and free GTP/GDP (using a Keq of 1.0 for the nucleoside diphosphate kinase reduction) under these conditions. At a time when muscle was contracted by 15 microM norepinephrine (NE) for 5 min under normoxia, [GTP], free [GDP], and free GTP/GDP were 0.29 +/- 0.03 mM, 3.5 microM, and 82, respectively. Following rapid inhibition of oxidative energy production during NE-evoked maintained force, which is associated with slow decreases in mean tissue [PCr], and [ATP] and force relaxation, [GTP] and free GTP/GDP were decreased at relaxation threshold to 0.22 +/- 0.02 (SE) mM and 43, respectively, and progressively fell further, paralleling decreases in force and [ATP] and [PCr]. There were marked decreases in the sum of GTP + GDP contents under conditions where muscle energy stores were decreased (i.e., low [PCr] + [ATP]). Similar data were obtained during a 50 mM KCl-evoked contracture. Free [GDP] increased from normoxic values of 3.5 microM to values as great as 6.0 microM at low energy store states. Free GDP was equivalent to 6% of total GDP under normoxia and increased to 16-21% of total GDP under conditions of low energy stores. Evidence was obtained that decreases in [GTP] or free GTP/GDP seen under conditions of low total energy stores were not sufficient to inhibit heterotrimeric G protein function and uncouple receptor-effector coupling.

Phosphocreatine, an intracellular high-energy compound, is found in the extracellular fluid of the seminal vesicles in mice and rats.

High levels of phosphocreatine, a compound known to serve as an intracellular energy reserve, were found in the fluid contained in seminal vesicle glands. The concentrations of phosphocreatine in the extracellular fluid in the mouse and rat were found to be 5.6 +/- 1.6 and 2.2 +/- 0.8 mumol/g, respectively, which are higher than the intracellular levels reported for smooth muscles. The creatine concentrations in the seminal vesicular fluid from these two species were 22.8 +/- 3.1 and 13.0 +/- 5.3 mumol/g, respectively. These creatine levels are approximately 100 and 65 times higher than the creatine levels in mammalian blood. Smaller amounts of ATP (phosphocreatine/ATP ratio of 20-40) and traces of ADP were also found. Comparison of the pattern of distribution of macromolecules (proteins and DNA) with the distribution of phosphocreatine between the cells and the fluid of the seminal vesicle indicates that cell lysis did not account for the phosphocreatine in the seminal vesicle fluid. Rather, the available evidence strongly suggests that this high-energy compound is actively secreted. We found that in the testes, the sperm are exposed to the highest known creatine concentration in any mammalian tissue studied. Based on these results and other recent reports, we propose that the extracellular phosphocreatine, ATP, and creatine are involved in sperm metabolism.

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation.

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.

Regulation of actomyosin ATPase in smooth muscle.

Actomyosin in smooth muscle is in a quiescent state. The mechanism or mechanisms by which Ca2+ activates the actomyosin ATPase is not clear. There is sufficient evidence for the presence of enzyme systems which phosphorylate and dephosphorylate myosin light chains. The activity of the kinase that phosphorylates the myosin is regulated by cAMP-dependent protein kinase. Phosphorylated kinase has decreased affinity for calmodulin and lower activity when compared with unphosphorylated myosin light chain kinase. The activity of myosin light chain kinase is also regulated by calcium-calmodulin. In the presence of Ca2+, myosin is phosphorylated. In the absence of Ca2+, the phosphatase activity becomes dominant; the myosin remains in the unphosphorylated form under this condition. The Mg2+-ATPase of the phosphorylated myosin is activated by actin. The maximal activation of the Mg2+-ATPase by actin requires Ca2+ and tropomyosin, a protein located on the thin filament. Hence, the actin-activation of the Mg2+-ATPase requires Ca2+ even after phosphorylation by the calcium-calmodulin dependent kinase. The regulation of actin-activated ATPase activity by myosin light chain phosphorylation is depicted in the schematic diagram. Caldesmon, an actin-binding protein which also binds to calmodulin in the presence of Ca2+, has been shown to be present in thin-filaments isolated from smooth muscle. This protein inhibits actin-activated myosin ATPase activity. The release from this inhibition requires Ca2+ and calmodulin. The possibility that caldesmon is also involved in the calcium regulation of actomyosin in smooth muscle is presently under investigation in a number of laboratories.

Polymer-protomer transition of acetyl-CoA carboxylase occurs in vivo and varies with nutritional conditions.

The protomeric form of purified acetyl coenzyme A carboxylase is inactivated by the binding of avidin to the biotinyl prosthetic group; the catalytically active filamentous form of the enzyme is resistant to avidin. This differential sensitivity to avidin was used to examine the influence of nutritional state on the proportion of polymeric and protomeric carboxylase occurring in avian liver. Hepatic carboxylase was 80% avidin-resistant (polymeric) in the fed chick. Food deprivation for 2 and 6 h reduced the avidin resistance to 54% and 30%, respectively. Similarly, within 1 h after fat intubation, the fraction of polymeric carboxylase had significantly decreased. Accompanying the change in carboxylase transformation was a comparable reduction in 3H2O incorporation into liver fatty acid. These data indicate that the protomer-polymer transition defined for purified acetyl-CoA carboxylase also occurs with the enzyme in vivo and that a lower polymer/protomer ratio is associated with reduced rates of fatty acid synthesis.