Terrance D. Barrett

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

Functional pharmacology: the drug discovery bottleneck?

Abstract It is our contention that functional pharmacology, particularly in vivo, must play a greater role in drug discovery if the promises of reductionist techniques are to be brought to full fruition. Despite the advent of high-throughput screening, combinatorial chemistry and informatics, the number of new chemical entities (NCEs) coming on the market has fallen. While the advent of these methodologies makes it possible to move very quickly from molecular target to lead compound, the problem of demonstrating therapeutic utility remains. Has proof of concept (in vivo) become the bottleneck in drug discovery? This article focuses on this problem, and discusses what can be done about it.

Canagliflozin and Heart Failure in Type 2 Diabetes Mellitus: Results From the CANVAS Program (Canagliflozin Cardiovascular Assessment Study)

Background: Canagliflozin is a sodium glucose cotransporter 2 inhibitor that reduces the risk of cardiovascular events. We report the effects on heart failure (HF) and cardiovascular death overall, in those with and without a baseline history of HF, and in other participant subgroups. Methods: The CANVAS Program (Canagliflozin Cardiovascular Assessment Study) enrolled 10 142 participants with type 2 diabetes mellitus and high cardiovascular risk. Participants were randomly assigned to canagliflozin or placebo and followed for a mean of 188 weeks. The primary end point for these analyses was adjudicated cardiovascular death or hospitalized HF. Results: Participants with a history of HF at baseline (14.4%) were more frequently women, white, and hypertensive and had a history of prior cardiovascular disease (all P<0.001). Greater proportions of these patients were using therapies such as blockers of the renin angiotensin aldosterone system, diuretics, and &bgr;-blockers at baseline (all P<0.001). Overall, cardiovascular death or hospitalized HF was reduced in those treated with canagliflozin compared with placebo (16.3 versus 20.8 per 1000 patient-years; hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.67–0.91), as was fatal or hospitalized HF (HR, 0.70; 95% CI, 0.55–0.89) and hospitalized HF alone (HR, 0.67; 95% CI, 0.52–0.87). The benefit on cardiovascular death or hospitalized HF may be greater in patients with a prior history of HF (HR, 0.61; 95% CI, 0.46–0.80) compared with those without HF at baseline (HR, 0.87; 95% CI, 0.72–1.06; P interaction =0.021). The effects of canagliflozin compared with placebo on other cardiovascular outcomes and key safety outcomes were similar in participants with and without HF at baseline (all interaction P values >0.130), except for a possibly reduced absolute rate of events attributable to osmotic diuresis among those with a prior history of HF (P=0.03). Conclusions: In patients with type 2 diabetes mellitus and an elevated risk of cardiovascular disease, canagliflozin reduced the risk of cardiovascular death or hospitalized HF across a broad range of different patient subgroups. Benefits may be greater in those with a history of HF at baseline. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifiers: NCT01032629 and NCT01989754.

Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2α (HRI) kinase

A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.

Targeting gastrin for the treatment of gastric acid related disorders and pancreatic cancer

Gastrin, acting through peripheral cholecystokinin (CCK) 2 receptors, is a major hormonal regulator of gastric acid secretion. The effects of gastrin on acid secretion occur both acutely and chronically because gastrin directly stimulates gastric acid secretion and also exerts trophic effects on the enterochromaffin-like and parietal cells that together constitute the acid secretory apparatus of the stomach. Several antagonists that target the CCK2 receptor have been identified and investigated for the treatment of gastroesophageal reflux disease and pancreatic cancer. In this paper, we discuss the contribution of gastrin to these disease pathologies and the data generated to date from clinical studies investigating CCK2 receptor antagonists.

Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1α for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2181-417), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1α, with succinic acid produced as an end product, radiolabeled [5-14C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2181-417 enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The Km value for 2-oxoglutaric acid was determined to be 0.9 µM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1α (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases. (Journal of Biomolecular Screening 2009:627-635)

SNC-80-induced preconditioning: selective activation of the mitochondrial adenosine triphosphate-gated potassium channel.

Pharmacologic preconditioning by &dgr;-opioid agonists occurs via activation of an adenosine triphosphate (ATP)-gated potassium channel (IKATP). Opening of mitochondrial IKATP confers pharmacologic preconditioning whereas opening the sarcolemmal IKATP shortens action potential duration and is proarrhythmic. This study investigated whether SNC-80, a selective &dgr;-opioid agonist, is associated with development of ventricular arrhythmia due to activation of IKATP. Rabbit isolated hearts were subjected to 12 min of hypoxia and 40 min of reoxygenation after pretreatment with SNC-80 (1 &mgr;M, n = 6), pinacidil (1.25 &mgr;M, n = 12), or BMS-191095 (6.0 &mgr;M, n = 4). Nine additional hearts served as controls. The cytoprotective effects of SNC-80 at a concentration of 1 &mgr;M were confirmed using 30 min of regional ischemia followed by 120 min of reperfusion. Ventricular fibrillation (VF) developed in 11 of 12 pinacidil-treated hearts whereas none of the SNC-80-treated (zero of six) hearts developed VF (P < 0.001 compared with pinacidil pretreatment) and zero of four BMS-191095-pretreated hearts developed VF. Similarly, zero of nine control hearts developed VF. SNC-80 reduced infarct size expressed as a percentage of the area at risk from 33 ± 4% to 14 ± 3% (P = 0.004) compared with control. SNC-80, which selectively activates the &dgr;-opioid receptor, provided cytoprotection but did not induce VF after hypoxia reoxygenation. The results indicate that pinacidil-induced nonselective activation of IKATP results in proarrhythmia that is dependent on activation of the sarcolemmal IKATP. Selectivity for the mitochondrial IKATP is necessary to prevent induction of a proarrhythmic state.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: tuning of receptor selectivity and in vivo efficacy.

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.

Role of CCK and potential utility of CCK1 receptor antagonism in the treatment of pancreatitis induced by biliary tract obstruction

Background and purpose: Cholecystokinin (CCK) stimulates the release of amylase and lipase from the normal pancreas. However, it is not clear to what extent this occurs in the early stages of pancreatitis induced by biliary tract obstruction in the rat and whether CCK initiates an inflammatory cascade in this condition.

Inhibitors of HIF Prolyl Hydroxylases

Publisher Summary This chapter broadly characterizes the classes of small-molecule prolyl hydroxylase (PHD) inhibitors and summarizes their biological profiles. The ability of animals to detect, absorb, transport, and enzymatically harness the oxidizing potential of molecular oxygen is a crucial factor in their development and evolution, and represents the fundamental processes of life. The most significant regulator of O 2 sensing and homeostasis is hypoxia inducible factor (HIF)—a transcription factor that has been found to be tightly regulated itself via degradation initiated by PHD enzymes. HIF is directly responsible for the transcription of hundreds of gene products involved in respiration, metabolism, angiogenesis, erythropoiesis, and many other functions at the cellular and organ levels. HIF-α—the regulatory subunit of the HIF dimer—is constitutively produced with a half-life of approximately 5 minutes, being degraded as it is produced by enzymatic hydroxylation on proline residues leading to the recognition of the hydroxyl form by the von Hippel–Lindau tumor suppressor protein (pVHL) and subsequent ubiquitin-mediated proteasomal destruction. The moderation of PHD activity with small-molecule inhibitors has already led to the introduction of four compounds— FG-2216, FG-4592, GSK1278863A, and AKB-6548—into clinical trials for the treatment of various anemias.