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Inhibition of DPP-8 and DPP-9 per se does not lead to organ toxicities and mortality in rodents. Thus, a mechanism other than DPP-8/DPP-9 inhibition likely underlies the toxicity previously reported to be associated with a selective DPP-8/DPP-9 inhibitor.

High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented. KEYWORDS: DGAT1, triglyceride synthesis, efflux O rally ingested triglycerides (TG) undergo hydrolysis and then are reassembled within enterocytes into TG-rich chylomicrons destined for systemic circulation. The final committed step in triglyceride biosynthesis is known to be mediated by at least two distinct intracellular acyl-coA diacylglycerol acyltransferases (DGATs), namely DGAT1 1 and DGAT2. 2 Since the development of whole-body knockout models of these enzymes, there has been intense evaluation of pharmacological approaches to modulate their activity. 3−8 For DGAT1, this interest is inspired by the favorable metabolic phenotype of DGAT1 −/− mice, 9 which are resistant to dietinduced body weight gain, 10 are more insulin-sensitive relative to wild-type littermates, 11 and exhibit a reduced rate of chylomicrons formation when challenged with lipid nutrients. 12 Interestingly, all aspects of this phenotype are lost when DGAT1 is reintroduced via a tissue-specific promoter into the intestines of female DGAT1 −/− mice, implying that intestinal DGAT1 plays a crucial role in the effects observed in the whole-body knockout model. 13 Indeed, DGAT1 mRNA expression levels have been shown to be high in regions of the small intestine in mice and humans. 14,15 Selective inhibition of intestinal DGAT1 therefore becomes an intriguing drug discovery approach to recapitulate aspects of the DGAT1 −/− mouse, especially if this gut-specific inhibition reduces the potential risk of on-and off-target activity for candidate molecules. Particularly relevant for DGAT1 pharmacological inhibition is the observation of functional and morphological abnormalities in the fur and sebaceous glands of DGAT1 −/− mice. 16 In this report we describe a novel approach to specifically inhibit intestinal DGAT1, and demonstrate the potential viability of this strategy with regard to efficacy and safety in multiple preclinical models.High-throughput screening efforts using recombinant human DGAT1 enzyme identified the benzimidazole 1 (DGAT1 IC 50 = 1.3 μM; DGAT2 IC 50 > 20 μM; Figure 1) as a potential starting point for optimization. Initial structural modifications demonstrated that both the ethyl carbamate and the 2,6-dichlorophenyl substituents on the benzimidazole core could be replaced without substantial loss of activity (i.e., 2, DGAT1 IC 50 = 1.4 μM), and in fact introducing an additional substituent at the 4-position of the 2,6-dimethylphenyl ring led to an im...

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We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human cells. Qualitative characteristics of the mutational spectra induced by ONOO- differed significantly from those reportedly caused by hydroxyl radical (OH.) in other experimental systems but showed similarities to spectra reportedly produced by singlet oxygen (1O2). The molecular mechanisms of ONOO--mediated DNA damage are unknown. The objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH* and 1O2 to permit direct comparison with mutational spectra induced by ONOO- in this system. Base substitutions were the major form of mutation induced in plasmids replicated in human (AD293) cells by ONOO- (84%) and 1O2 (71%), whereas OH* induced fewer of them (49%). In plasmids replicated in bacteria (Escherichia coli MBL50), frequencies of base substitutions induced by the three treatments were similar. G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%, respectively, in AD293- and MBL50-replicated plasmids) and 1O2 (68% and 71%); they were induced at lower frequencies by OH. (51% and 47%). G:C-to-C:G transversions or G:C-to-A:T transitions were induced at almost equal frequencies by both ONOO- and 1O2, whereas OH* induced these mutations at different frequencies in the AD293 system. Collectively, our results confirm that in several important respects mutational spectra induced by ONOO- have greater similarity to spectra induced by 1O2 than to those induced by OH* and suggest that genotoxic derivatives of ONOO- are likely to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.