The biotin-tagged electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) have been used as model electrophile probes in complex proteomes to identify protein targets associated with chemical toxicity. Whereas IAB activates stress signaling and apoptosis in HEK293 cells, BMCC does not. Cysteine Michael adducts formed from BMCC and non-biotinylated analogs rapidly disappeared in the intact cells, whereas the adducts were stable in BMCC-treated subcellular fractions, even in the presence of the cellular reductants reduced glutathione, NADH and NADPH. In contrast, cysteine thioether adducts formed from IAB and its non-biotinylated analogs were stable in intact cells. Loss of the BMCC adduct in cells was reduced at 4 °C, which suggests the involvement of a metabolic process in adduct removal. Model studies with a glutathione-BMCC conjugate indicated rapid hydrolysis of the adducted imide ring, but neither the conjugate nor its hydrolysis product dissociated to release the electrophile in neutral aqueous buffer at significant rates. The results suggest that low BMCC toxicity reflects facile repair that results in transient adduction, which fails to trigger damage signaling pathways.

BackgroundNon-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research.Methods and Findings[18F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [18F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [18F]-FLT-PET data from standard estimates of proliferative index.ConclusionsOur findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [18F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [18F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [18F]-FLT-PET correlative results previously reported and suggest future best-practices when [18F]-FLT-PET is employed in oncology.

CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR5 subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an EC50 value of 77 +/- 15 nM in potentiating mGluR5-mediated responses in cortical astrocytes and a Ki value of 3760 +/- 430 nM in displacing [3H]methoxyPEPy binding in membranes of cultured HEK-293 cells expressing rat mGluR5. The structure-activity relationships showed that electronegative aromatic substituents in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing Ki = 156 +/- 29 nM and EC50 = 9.6 +/- 1.9 nM in the binding and functional assays, respectively.

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323:147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[ 4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G␣ 15 . Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative cooperativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G i/o -coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G i/o signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.The G protein-coupled receptors (GPCRs) play critical roles in regulating a broad range of signaling pathways within virtually every organ system (George et al., 2002) and are the targets of intense discovery efforts for new drug candidates. Despite this concentrated focus, selective ligands do not exist for the majority of these receptors (Howard et al., 2001). Many GPCRs interact with natural ligands based on complex or highly restricted chemical scaffolds (for example, amino acids, peptides, lipids, etc.) that are not well suited to optimization of selective small-molecule reagents. In addition, high conservation of the orthosteric sites of GPCR subfamilies presents challenges in developing high selectivity for a given receptor subtype. For these reasons, increasing interest has been placed on identifying compounds that interact with GPCRs at allosteric, rather than orthosteric, binding