Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors.

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.

The discovery of non-benzimidazole and brain-penetrant prolylcarboxypeptidase inhibitors.

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.

Discovery of benzimidazole pyrrolidinyl amides as prolylcarboxypeptidase inhibitors.

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC(50)'s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.

Potential mechanism of enhanced postprandial glucagon-like peptide-1 release following treatment with a diacylglycerol acyltransferase 1 inhibitor.

Studies have demonstrated that blockade of diacylglycerol acyltransferase 1 (DGAT1) leads to prolonged release of glucagon-like peptide 1 (GLP-1) after meal challenge. The current study was undertaken to investigate the mechanism of action underlying the elevated levels of GLP-1 release following pharmacological inhibition of DGAT1. We utilized a potent, specific DGAT1 inhibitor, compound A, to investigate the changes in intestinal lipid profile in a mouse model after oral administration of the compound and challenge with tracer containing fatty meal. [13C18]-oleic acid and LC-MS were employed to trace the fate of dietary fatty acids provided as part of a meal challenge in lean mice. Lipid profiles in plasma, proximal to distal segments of intestine, and feces were evaluated at various times following the meal challenge to study the kinetics of fatty acid absorption, synthesis into complex lipids, and excretion. Pharmacological inhibition of DGAT1 led to reduction of postprandial total and newly synthesized triglyceride (TG) excursion and significant increases in TG and FFA levels in the distal portion of intestine enriched with enteroendocrine L cells. Enhanced levels of FFA and cholesteryl ester were observed via fecal fat profiling. DGAT1 inhibition leads to enhancement of carbon flow to the synthesis of phosphatidylcholine within the intestine. DGAT1 inhibition markedly increases levels of TG and FFA in the distal intestine, which could be the predominant contributor to the prolonged and enhanced postprandial GLP-1 release. Inactivation of DGAT1 could provide potential benefit in the treatment of dysmetabolic diseases.

Diacylglycerol acyltransferase-1 (DGAT1) inhibition perturbs postprandial gut hormone release.

Diacylglycerol acyltransferase-1 (DGAT1) is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4) inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release--potential insight into mechanism of action.

OBJECTIVE: Investigation was conducted to understand the mechanism of action of diacylglycerol acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L. DESIGN AND METHODS: Biochemical and stable-label tracer approaches were applied to interrogate the functional activities of compounds K and L on TG synthesis and changes of carbon flow. Energy homeostasis and gut peptide release upon DGAT1 inhibition was conducted in mouse and dog models. RESULTS: Compounds K and L, dose-dependently inhibits post-prandial TG excursion in mouse and dog models. Weight loss studies in WT and Dgat1(-/-) mice, confirmed that the effects of compound K on body weight loss is mechanism-based. Compounds K and L altered incretin peptide release following oral fat challenge. Immunohistochemical studies with intestinal tissues demonstrate lack of detectable DGAT1 immunoreactivity in enteroendocrine cells. Furthermore, (13) C-fatty acid tracing studies indicate that compound K inhibition of DGAT1 increased the production of phosphatidyl choline (PC). CONCLUSION: Treatment with DGAT1 inhibitors improves lipid metabolism and body weight. DGAT1 inhibition leads to enhanced PC production via alternative carbon channeling. Immunohistological studies suggest that DGAT1 inhibitor's effects on plasma gut peptide levels are likely via an indirect mechanism. Overall these data indicate a translational potential towards the clinic.

Potent DGAT1 Inhibitors in the Benzimidazole Class with a Pyridyl-oxy-cyclohexanecarboxylic Acid Moiety.

We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.