It has been proposed previously that the sustained activation of mitogen-activated protein kinase may be necessary for the differentiation of PC12 cells. Differentiation of PC12 cells is induced by many extracellular agonists including nerve growth factor (NGF) and cyclicAMP analogues, but not epidennal growth factor (EGF), insulin or phorbol esters. Our results demonstrate that: (i) 8-(4-chlorophenylthio)-cyclicAMP (CPT-CAMP) activates MAP kinase; this raises the possibility that the MAP kinase pathway may be activated by agents that act through adenylate cyclase; (ii) NGF and UT-CAMP as well as phorbol esters promote sustained activation of MAP kinase. This suggests that while sustained MAP kinase activation may be associated with differentiation it may not be sufficient, and that other as yet unidentified parallel pathways may be involved.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure−activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (K i = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme’s natural substrate, adenosine 5′-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}­methyl)­benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}­methyl)­benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gα i -coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. (Journal of Biomolecular Screening 2008:737-747)

A case of cortical blindness after cerebral angiography is presented. Serial computerized tomography scans of the brain revealed persistence of contrast medium in occipital visual areas as well as areas that may have been associated with "focal seizures" that occurred after angiography. This case supports the concept that cortical blindness may be secondary to the direct effect of contrast medium on the brain. The persistence of contrast material was in part due to decreased renal function.