Edward A. O'Neill

A Novel FK506 Binding Protein Can Mediate the Immunosuppressive Effects of FK506 and Is Associated with the Cardiac Ryanodine Receptor

FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12•FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum. Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.

Pyrroles and other heterocycles as inhibitors of P38 kinase

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

Patients with type 2 diabetes mellitus have higher risk for acute pancreatitis compared with those without diabetes.

Aim: The aetiology of acute pancreatitis (AP) is complex, and many risk factors for AP are shared by patients with type 2 diabetes mellitus (T2DM). However, few have assessed risk factors for AP specifically in T2DM patients.

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase.

The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).

Efficacy and safety of initial combination therapy with sitagliptin and pioglitazone in patients with type 2 diabetes: a 54-week study

Aim: To assess the 54‐week efficacy of initial combination therapy with sitagliptin and pioglitazone, compared with pioglitazone monotherapy, and to assess safety in these groups during the 30 weeks after the dosage of pioglitazone was increased from 30 to 45 mg/day, in drug‐naÏve patients with type 2 diabetes mellitus and inadequate glycaemic control [haemoglobin A1c (HbA1c) 8–12%].

p38MAP kinase inhibitors. Part 1: design and development of a new class of potent and highly selective inhibitors based on 3,4-dihydropyrido[3,2-d]pyrimidone scaffold.

A new class of p38 antagonists based on 3,4-dihydropyrido[3,2,-d]pyrimidine scaffold has been developed. These inhibitors exhibit unprecedented selectivity towards p38 over other very closely related kinases. Compounds 25, 33, and 34 were identified as benchmark analogues for follow-up studies. They show good potency for enzyme inhibition and excellent functional activity.

Impact of diabetes duration and chronic pancreatitis on the association between type 2 diabetes and pancreatic cancer risk

To examine the impact of diabetes duration, chronic pancreatitis and other factors on pancreatic cancer risk.

A gene expression signature that classifies human atherosclerotic plaque by relative inflammation status.

Background— Atherosclerosis is a complex disease requiring improvements in diagnostic techniques and therapeutic treatments. Both improvements will be facilitated by greater exploration of the biology of atherosclerotic plaque. To this end, we carried out large-scale gene expression analysis of human atherosclerotic lesions. Methods and Results— Whole genome expression analysis of 101 plaques from patients with peripheral artery disease identified a robust gene signature (1514 genes) that is dominated by processes related to Toll-like receptor signaling, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction, and lysosomal activity. Further analysis of gene expression in microdissected carotid plaque samples revealed that this signature is differentially expressed in macrophage-rich and smooth muscle cell–containing regions. A quantitative PCR gene expression panel and inflammatory composite score were developed on the basis of the atherosclerotic plaque gene signature. When applied to serial sections of carotid plaque, the inflammatory composite score was observed to correlate with histological and morphological features related to plaque vulnerability. Conclusions— The robust mRNA expression signature identified in the present report is associated with pathological features of vulnerable atherosclerotic plaque and may be useful as a source of biomarkers and targets of novel antiatherosclerotic therapies.

The three-dimensional structure of MAP kinase p38beta: different features of the ATP-binding site in p38beta compared with p38alpha.

The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38alpha and p38beta) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38alpha isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38beta may not provide any additional benefit. In order to aid the development of p38alpha-selective compounds, the three-dimensional structure of p38beta was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38beta were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05 A resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38alpha C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38beta. This difference in size between the two pockets could be exploited in order to achieve selectivity.