Pharmacological AMPK activation induces transcriptional responses congruent to exercise in skeletal and cardiac muscle, adipose tissues and liver.

Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.

Discovery of MK-8722: A Systemic, Direct Pan-Activator of AMP-Activated Protein Kinase.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of mammalian energy homeostasis and has been implicated in mediating many of the beneficial effects of exercise and weight loss including lipid and glucose trafficking. As such, the enzyme has long been of interest as a target for the treatment of Type 2 Diabetes Mellitus. We describe the optimization of ß1-selective, liver-targeted AMPK activators and their evolution into systemic pan-activators capable of acutely lowering glucose in mouse models. Identifying surrogates for the key acid moiety in early generation compounds proved essential in improving ß2-activation and in balancing improvements in plasma unbound fraction while avoiding liver sequestration.

Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.

BACKGROUND: With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). METHODS: We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. RESULTS: The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. CONCLUSIONS: The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

Tetrahydroindolizinone NK1 antagonists.

A new class of potent NK(1) receptor antagonists with a tetrahydroindolizinone core has been identified. This series of compounds demonstrated improved functional activities as compared to previously identified 5,5-fused pyrrolidine lead structures. SAR at the 7-position of the tetrahydroindolizinone core is discussed in detail. A number of compounds displayed high NK(1) receptor occupancy at both 1 h and 24 h in a gerbil foot tapping model. Compound 40 has high NK(1) binding affinity, good selectivity for other NK receptors and promising in vivo properties. It also has clean P(450) inhibition and hPXR induction profiles.

Potent, brain-penetrant, hydroisoindoline-based human neurokinin-1 receptor antagonists.

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.

Fused bicyclic pyrrolizinones as new scaffolds for human NK1 antagonists.

Previous work on human NK(1) antagonists in which the core of the structure is a substituted pyrrolidine has been disclosed. These compounds showed good binding affinity and functional IP activity, however, many did not exhibit the necessary brain penetration for good in vivo activity. The discovery and preparation of a novel 5,5-fused pyrrolidine core is presented in this paper. This scaffold maintains the excellent binding affinity and functional IP activity of the previously reported compounds, but also exhibits excellent brain penetration as observed in a gerbil foot-tapping assay. The determination of the core structural stereochemistry, which eventually led to the final synthesis of a single active diastereomer, is described.

Pyrrolidine-carboxamides and oxadiazoles as potent hNK1 antagonists.

The preparation and structure-activity-relationships of novel pyrrolidine-carboxamides and oxadiazoles are described. Compounds in this series were found to be potent hNK(1) antagonists in vitro and efficacious in vivo with minimal interactions with P(450) liver enzymes. Oxadiazole analog 22 was determined to have excellent hNK(1) binding affinity, functional activity, and a good PD response in vivo.

The discovery of potent, selective, and orally bioavailable hNK1 antagonists derived from pyrrolidine.

SAR studies on amides, ureas, and vinylogous amides derived from pyrrolidine led to the discovery of several potent hNK(1) antagonists. One particular vinylogous amide (45b) had excellent potency, selectivity, pharmacokinetic profile, and functional activity in vivo. An in vivo rhesus macaque brain receptor occupancy PET study for compound 45b revealed an estimated Occ(90) approximately 300 ng/ml.

NK1 antagonists based on seven membered lactam scaffolds.

A new class of high affinity hNK1R antagonists based on seven-membered ring cores has been identified. This series, with relatively simple, compact structures, includes compounds with high affinity, good selectivity, and promising in vivo properties.

1-Phenyl-8-azabicyclo[3.2.1]octane ethers: a novel series of neurokinin (NK1) antagonists.

1-Phenyl-8-azabicyclo[3.2.1]octane ethers are NK(1) receptor antagonists. Substitution at the 6-exo-position led to high affinity NK(1) antagonists with a prolonged duration of action in vivo. Incorporation of an alpha-methyl substituent in the pendent benzyl ether side chain gave compounds with increased selectivity over the hERG channel.

Synthesis and structure-activity relationships of 8-azabicyclo[3.2.1]octane benzylamine NK1 antagonists.

A series of 8-azabicyclo[3.2.1]octane amine hNK1 antagonists has been investigated and structure-activity relationships of the benzylamine and 6-exo substituents described. Acidic substituents at C6 give a series of high affinity compounds for hNK1 with selectivity over the hERG channel.

Novel lactam NK1 antagonists with anti-emetic activity.

A series of 4,4-disubstituted cyclohexylamine NK(1) antagonists containing a lactam ring is described. The compounds are brain penetrant and activity is demonstrated in a ferret emesis model.

Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608.

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.

Identification, localization and receptor characterization of novel mammalian substance P-like peptides.

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.

Spirocyclic NK(1) antagonists I: [4.5] and [5.5]-spiroketals.

A series of novel spiroketal-based NK(1) antagonists is described. The effect of modifications to the spiroether ring and aromatic substituents are discussed, leading to the identification of compounds with high affinity and excellent CNS penetration.

4,4-Disubstituted cyclohexylamine NK(1) receptor antagonists I.

A series of novel 4,4-disubstituted cyclohexylamine based NK(1) antagonists is described. The effect of changes to the C(1)-C(4) relative stereochemistry on the cyclohexane ring and replacements for the flexible linker are discussed, leading to the identification of compounds with high affinity and good in vivo duration of action.

4,4-Disubstituted cyclohexylamine NK(1) receptor antagonists II.

A series of novel 4,4-disubstituted cyclohexylamines as NK(1) receptor antagonists is described: modifications to the amine moiety retain NK(1) receptor binding affinity whilst disrupting I(Kr) affinity.