Pan AMPK Activation Protects Tubules in Rat Ischemic Acute Kidney Injury.

Acute Kidney Injury (AKI) is characterized by an abrupt decline in kidney function and has been associated with excess risks of death, kidney disease progression, and cardiovascular events. The kidney has a high energetic demand with mitochondrial health being essential to renal function and damaged mitochondria has been reported across AKI subtypes. 5' adenosine monophosphate-activated protein kinase (AMPK) activation preserves cellular energetics through improvement of mitochondrial function and biogenesis when ATP levels are low such as under ischemia-induced AKI. We developed a selective potent small molecule pan AMPK activator, compound 1, and tested its ability to increase AMPK activity and preserve kidney function during ischemia/reperfusion injury in rats. A single administration of 1 caused sustained activation of AMPK for at least 24 hours, protected against acute tubular necrosis, and reduced clinical markers of tubular injury such as NephroCheck and Fractional Excretion of Sodium (FENa). Reduction in plasma creatinine and increased Glomerular Filtration Rate (GFR) indicated preservation of kidney function. Surprisingly, we observed a strong diuretic effect of AMPK activation associated with natriuresis both with and without AKI. Our findings demonstrate that activation of AMPK leads to protection of tubular function under hypoxic/ischemic conditions which holds promise as a potential novel therapeutic approach for AKI. Significance Statement No approved pharmacological therapies currently exist for acute kidney injury. We developed Compound 1 which dose-dependently activated AMPK in the kidney and protected kidney function and tubules after ischemic renal injury in the rat. This was accompanied by natriuresis in injured as well as uninjured rats.

GPR40-Mediated Gα12 Activation by Allosteric Full Agonists Highly Efficacious at Potentiating Glucose-Stimulated Insulin Secretion in Human Islets.

GPR40 is a clinically validated molecular target for the treatment of diabetes. Many GPR40 agonists have been identified to date, with the partial agonist fasiglifam (TAK-875) reaching phase III clinical trials before its development was terminated due to off-target liver toxicity. Since then, attention has shifted toward the development of full agonists that exhibit superior efficacy in preclinical models. Full agonists bind to a distinct binding site, suggesting conformational plasticity and a potential for biased agonism. Indeed, it has been suggested that alternative pharmacology may be required for meaningful efficacy. In this study, we described the discovery and characterization of Compound A, a newly identified GPR40 allosteric full agonist highly efficacious in human islets at potentiating glucose-stimulated insulin secretion. We compared Compound A-induced GPR40 activity to that induced by both fasiglifam and AM-1638, another allosteric full agonist previously reported to be highly efficacious in preclinical models, at a panel of G proteins. Compound A was a full agonist at both the Gαq and Gαi2 pathways, and in contrast to fasiglifam Compound A also induced Gα12 coupling. Compound A and AM-1638 displayed similar activity at all pathways tested. The Gα12/Gα13-mediated signaling pathway has been linked to protein kinase D activation as well as actin remodeling, well known to contribute to the release of insulin vesicles. Our data suggest that the pharmacology of GPR40 is complex and that Gα12/Gα13-mediated signaling, which may contribute to GPR40 agonists therapeutic efficacy, is a specific property of GPR40 allosteric full agonists.

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists.

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.

Discovery of a novel potent GPR40 full agonist.

Compound 12 is a GPR40 agonist that realizes the full magnitude of efficacy possible via GPR40 receptor agonism. In vitro and in vivo studies demonstrated superior glucose lowering by 12 compared to fasiglifam (TAK-875), in a glucose dependent manner. The enhanced efficacy observed with the full agonist 12 was associated with both direct and indirect stimulation of insulin secretion.

Discovery of novel potent imidazo[1,2-b]pyridazine PDE10a inhibitors.

Design and optimization of a novel series of imidazo[1,2-b]pyridazine PDE10a inhibitors are described. Compound 31 displays excellent pharmacokinetic properties and was also evaluated as an insulin secretagogue in vitro and in vivo.

Transient receptor potential melastatin 8 channel inhibition potentiates the hypothermic response to transient receptor potential vanilloid 1 activation in the conscious mouse.

OBJECTIVES: Mild decrease in core temperature (therapeutic hypothermia) provides lasting neuroprotection following cardiac arrest or cerebral ischemia. However, current methods for producing therapeutic hypothermia trigger a cold-defense response that must be countered by sedatives, muscle paralytics, and mechanical ventilation. We aimed to determine methods for producing hypothermia in the conscious mouse by targeting two transient receptor potential channels involved in thermoregulation, two transient receptor potential (TRP) channels involved in thermoregulation, TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8). DESIGN: Controlled prospective animal study. SETTING: Research laboratory at academic medical center. SUBJECTS: Conscious unrestrained young and aged male mice. INTERVENTIONS: Mice were treated with the TRPV1 agonist dihydrocapsaicin, a TRPM8 inhibitor ("compound 5"), or their combination and the effects on core temperature (Tcore) were measured by implanted thermocouples and wireless transponders. MEASUREMENTS AND MAIN RESULTS: TRPV1 agonist dihydrocapsaicin produced a dose-dependent (2-4 mg/kg s.c.) drop in Tcore. A loading dose followed by continuous infusion of dihydrocapsaicin produced a rapid and prolonged (> 6 hr) drop of Tcore within the therapeutic range (32-34°C). The hypothermic effect of dihydrocapsaicin was augmented in aged mice and was not desensitized with repeated administration. TRPM8 inhibitor "compound 5" (20 mg/kg s.c.) augmented the drop in core temperature during cold exposure (8°C). When "compound 5" (30 mg/kg) was combined with dihydrocapsaicin (1.25-2.5 mg/kg), the drop in Tcore was amplified and prolonged. CONCLUSIONS: Activating warm receptors (TRPV1) produced rapid and lasting hypothermia in young and old mice. Furthermore, hypothermia induced by TRPV1 agonists was potentiated and prolonged by simultaneous inhibition of TRPM8.

Shivering and tachycardic responses to external cooling in mice are substantially suppressed by TRPV1 activation but not by TRPM8 inhibition.

Mild decrease of core temperature (32-34°C), also known as therapeutic hypothermia, is a highly effective strategy of neuroprotection from ischemia and holds significant promise in the treatment of stroke. However, induction of hypothermia in conscious stroke patients is complicated by cold-defensive responses, such as shivering and tachycardia. Although multiple thermoregulatory responses may be altered by modulators of thermosensitive ion channels, TRPM8 (transient receptor potential melastatin 8) and TRPV1 (TRP vanilloid 1), it is unknown whether these agents affect cold-induced shivering and tachycardia. The current study aimed to determine the effects of TRPM8 inhibition and TRPV1 activation on the shivering and tachycardic responses to external cooling. Conscious mice were treated with TRPM8 inhibitor compound 5 or TRPV1 agonist dihydrocapsaicin (DHC) and exposed to cooling at 10°C. Shivering was measured by electromyography using implanted electrodes in back muscles, tachycardic response by electrocardiography, and core temperature by wireless transmitters in the abdominal cavity. The role of TRPM8 was further determined using TRPM8 KO mice. TRPM8 ablation had no effect on total electromyographic muscle activity (vehicle: 24.0 ± 1.8; compound 5: 23.8 ± 2.0; TRPM8 KO: 19.7 ± 1.9 V·s/min), tachycardia (ΔHR = 124 ± 31; 121 ± 13; 121 ± 31 beats/min) and drop in core temperature (-3.6 ± 0.1; -3.4 ± 0.4; -3.6 ± 0.5°C) during cold exposure. TRPV1 activation substantially suppressed muscle activity (vehicle: 25.6 ± 3.0 vs. DHC: 5.1 ± 2.0 V·s/min), tachycardia (ΔHR = 204 ± 25 vs. 3 ± 35 beats/min) and produced a profound drop in core temperature (-2.2 ± 0.6 vs. -8.9 ± 0.6°C). In conclusion, external cooling-induced shivering and tachycardia are suppressed by TRPV1 activation, but not by TRPM8 inhibition. This suggests that TRPV1 agonists may be combined with external physical cooling to achieve more rapid and effective hypothermia.

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors.

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.

Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia.

Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model of neuropathic pain is described.

Developments in factor Xa inhibitors for the treatment of thromboembolic disorders.

Thromboembolic diseases are the leading causes of morbidity and mortality in the developed world. Anticoagulants provide effective treatment for venous or arterial thromboembolism. Two coagulation factors, factor Xa (fXa) and thrombin, are the primary targets under active investigation for anticoagulant therapy. fXa, in contrast to the multifunctional roles of thrombin in the coagulation cascade, converts prothrombin to thrombin collectively at the junction of the intrinsic and extrinsic pathway of coagulation. The effectiveness of fXa inhibitors as antithrombotic agents and their potentially reduced bleeding risks may offer superior therapeutic profiles with respect to thrombin inhibitors. After decades of research, many fXa inhibitors are now in the advanced stages of clinical trials. Unlike most reviews, which only provide incremental updates, this review provides an overview of fXa and the medicinal chemistry of its inhibitors. Overviews on coagulation models, antithrombotic therapy, and fXa will be provided, followed by the evolution of the medicinal chemistry of fXa inhibitors over the past few decades.

Reducing ion channel activity in a series of 4-heterocyclic arylamide FMS inhibitors.

During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity.

Discovery of a GPR40 Superagonist: The Impact of Aryl Propionic Acid α-Fluorination.

GPR40 is a G-protein-coupled receptor which mediates fatty acid-induced glucose-stimulated insulin secretion from pancreatic beta cells and incretion release from enteroendocrine cells of the small intestine. GPR40 full agonists exhibit superior glucose lowering compared to partial agonists in preclinical species due to increased insulin and GLP-1 secretion, with the added benefit of promoting weight loss. In our search for potent GPR40 full agonists, we discovered a superagonist which displayed excellent in vitro potency and superior efficacy in the Gαs-mediated signaling pathway. Most synthetic GPR40 agonists have a carboxylic acid headgroup, which may cause idiosyncratic toxicities, including drug-induced-liver-injury (DILI). With a methyl group and a fluorine atom substituted at the α-C of the carboxylic acid group, 19 is not only highly efficacious in lowering glucose and body weight in rodent models but also has a low DILI risk due to its stable acylglucuronide metabolite.

7-fluoroindazoles as potent and selective inhibitors of factor Xa.

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.

Design and synthesis of a pyrido[2,3-d]pyrimidin-5-one class of anti-inflammatory FMS inhibitors.

A series of pyrimidinopyridones has been designed, synthesized and shown to be potent and selective inhibitors of the FMS tyrosine kinase. Introduction of an amide substituent at the 6-position of the pyridone core resulted in a significant potency increase. Compound 24 effectively inhibited in vivo LPS-induced TNF in mice greater than 80%.

Discovery of piperidine carboxamide TRPV1 antagonists.

A series of piperidine carboxamides were developed as potent antagonists of the transient receptor potential vanilloid-1 (TRPV1), an emerging target for the treatment of pain. A focused library of polar head groups led to the identification of a benzoxazinone amide that afforded good potency in cell-based assays. Synthesis and a QSAR model will be presented.

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif.

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

Synthesis and evaluation of novel 3,4,6-substituted 2-quinolones as FMS kinase inhibitors.

A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.

Discovery of novel FMS kinase inhibitors as anti-inflammatory agents.

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.

Structure-based optimization of a potent class of arylamide FMS inhibitors.

An anti-inflammatory 1,2,4-phenylenetriamine-containing series of FMS inhibitors with a potential to form reactive metabolites was transformed into a series with equivalent potency by incorporation of carbon-based replacement groups. Structure-based modeling provided the framework to efficiently effect this transformation and restore potencies to previous levels. This optimization removed a risk factor for potential idiosyncratic drug reactions.

Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury.

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 µM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 µM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.