Structure-Based Design and Preclinical Characterization of Selective and Orally Bioavailable Factor XIa Inhibitors: Demonstrating the Power of an Integrated S1 Protease Family Approach.

The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.

Cannabinoid 1 G protein-coupled receptor (periphero-)neutral antagonists: emerging therapeutics for treating obesity-driven metabolic disease and reducing cardiovascular risk.

INTRODUCTION: Obesity and related cardiometabolic derangements are spiraling global health problems urgently in need of safe, effective and durable pharmacotherapy. AREAS COVERED: As an orexigenic and anabolic biosignaling network, the endocannabinoid system interacts with other information-transducing pathways to help ensure metabolic homeostasis. Hyperphagia stimulates reinforcing neuronal circuits favoring energy intake and conservation, inviting overweight/obesity and cardiometabolic risk factors ('metabolic syndrome'). Associated increases in cannabinoid 1 G protein-coupled receptor (CB1R) activity/expression further exacerbate food consumption and the metabolic shift toward fat production and accumulation. The role of CB1R activity in hyperphagia and weight gain spurred the development of rimonabant (SR141716; Acomplia), the first-in-class CB1R antagonist/inverse agonist weight-loss drug. Rimonabant and similar CB1R inverse agonists also exert pleiotropic actions in addition to weight-loss effects that help correct obesity-related metabolic derangements and reduce cardiovascular risk in humans. The medicinal utility of these agents was crippled by clinically significant central and peripheral adverse effects that appear to reflect CB1R inverse agonists as a class. Consequently, increased attention is being given to CB1R neutral antagonists, CB1R blockers with intrinsically weak, if any, functional potency to elicit the negative-efficacy responses associated with inverse agonists. Laboratory studies demonstrate that CB1R neutral antagonists - whether readily accessible to the central nervous system or not (i.e., 'periphero-neutral' antagonists) - retain the salient therapeutic effects of CB1R inverse agonists on hyperphagia, weight-gain, and obesity-driven metabolic abnormalities with the distinct advantage of being associated with significantly less preclinical adverse events than are conventional CB1R inverse agonists such as rimonabant. EXPERT OPINION: CB1R (periphero-)neutral antagonists merit continued analysis of their molecular pharmacology and evaluation of their therapeutic significance and translational potential as new-generation medicines for obesity-related derangements, including nonalcoholic fatty liver disease and type 2 diabetes, if not obesity itself.

Estrogen-related receptor gamma is a key regulator of muscle mitochondrial activity and oxidative capacity.

Estrogen-related receptor gamma (ERRgamma) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRgamma expression in skeletal muscle by exercise. Muscle-specific VP16ERRgamma transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRgamma exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRgamma in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRbeta/gamma can increase mitochondrial function in mouse myotubes. Our data indicate that ERRgamma plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle.

Estrogen-related receptor alpha is essential for the expression of antioxidant protection genes and mitochondrial function.

Estrogen-related receptor alpha (ERRalpha) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERRalpha null mice. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) stimulated mitochondrial gene expression program in control cells, but not in the ERRalpha null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1alpha levels was dependent on ERRalpha. Furthermore, we found that the PGC-1alpha-mediated induction of estrogen-related receptor gamma and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERRalpha. Basal levels of NRF-2 were decreased in the absence of ERRalpha. The absence of ERRalpha resulted in a decrease in citrate synthase enzyme activity in response to PGC-1alpha overexpression. Our results indicate an essential role for ERRalpha as a key regulator of oxidative metabolism.

Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1.

Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of alpha-secretase-derived APP (APPs alpha). Increased beta-amyloid (A beta) generation was also observed in cells showing the FE65-dependent increase in APPs alpha. To understand the mechanism for the observed increase in both A beta and APPs alpha given that alpha-secretase cleavage of a single APP molecule precludes A beta generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates gamma-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A beta 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on gamma-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.

Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE --). IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE -- mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.