Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

MetAP2 inhibition increases energy expenditure through direct action on brown adipocytes.

Inhibitors of methionine aminopeptidase 2 (MetAP2) have been shown to reduce body weight in obese mice and humans. The target tissue and cellular mechanism of MetAP2 inhibitors, however, have not been extensively examined. Using compounds with diverse chemical scaffolds, we showed that MetAP2 inhibition decreases body weight and fat mass and increases lean mass in the obese mice but not in the lean mice. Obesity is associated with catecholamine resistance and blunted ß-adrenergic receptor signaling activities, which could dampen lipolysis and energy expenditure resulting in weight gain. In the current study, we examined effect of MetAP2 inhibition on brown adipose tissue and brown adipocytes. Norepinephrine increases energy expenditure in brown adipose tissue by providing fatty acid substrate through lipolysis and by increasing expression of uncoupled protein-1 (UCP1). Metabolomic analysis shows that in response to MetAP2 inhibitor treatment, fatty acid metabolites in brown adipose tissue increase transiently and subsequently decrease to basal or below basal levels, suggesting an effect on fatty acid metabolism in this tissue. Treatment of brown adipocytes with MetAP2 inhibitors enhances norepinephrine-induced lipolysis and energy expenditure, and prolongs the activity of norepinephrine to increase ucp1 gene expression and energy expenditure in norepinephrine-desensitized brown adipocytes. In summary, we showed that the anti-obesity activity of MetAP2 inhibitors can be mediated, at least in part, through direct action on brown adipocytes by enhancing ß-adrenergic-signaling-stimulated activities.

Development of a novel long-acting antidiabetic FGF21 mimetic by targeted conjugation to a scaffold antibody.

Fibroblast growth factor (FGF)21 improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF21 mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF21 through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF21. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic ß-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF21 protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF21 while preserving full therapeutic functionality.

A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis.

AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid oxidation and glucose uptake. Accordingly, compounds that activate AMPK represent potential drug candidates for the treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library of AMP mimetics identified the phosphonic acid 2 that bears little structural resemblance to AMP but is capable of activating AMPK with high potency (EC50 = 6 nM vs AMP EC50 = 6 µM) and specificity. Phosphonate prodrugs of 2 inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia.