The design, synthesis, and biological evaluation of potent receptor tyrosine kinase inhibitors

Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.     

Systems Dynamic Modeling of a Guard Cell Cl? Channel Mutant Uncovers an Emergent Homeostatic Network Regulating Stomatal Transpiration

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K⁺ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H⁺ load on the cytosol and promoted Ca²⁺ influx to elevate cytosolic pH (pH_i) and free cytosolic Ca²⁺ concentration ([Ca2+]_i), in turn regulating the K⁺ channels. We have confirmed these predictions, measuring pH_i and [Ca2+]_i in vivo, and report that experimental manipulation of pH_i and [Ca2+]_i is sufficient to recover K⁺ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K⁺ channels. Additionally, these findings underscore the importance of H⁺-coupled anion transport for pH_i homeostasis.Guard cells surround stomatal pores in the epidermis of plant leaves and regulate pore aperture to balance the demands for CO₂ in photosynthesis with the need to conserve water by the plant. Transpiration through stomata accounts for much of the 70% of global water usage associated with agriculture, and it has a profound impact on the water and carbon cycles of the world (Gedney et al., 2006; Betts et al., 2007). Guard cells open the pore by transport and accumulation of osmotically active solutes, mainly K⁺ and Cl⁻ and the organic anion malate²⁻ (Mal), to drive water uptake and cell expansion. They close the pore by coordinating the release of these solutes through K⁺ and anion channels at the plasma membrane. The past half-century has generated a wealth of knowledge on guard cell transport, signaling, and homeostasis, resolving the properties of the major transport processes and metabolic pathways for osmotic solute uptake and accumulation, and many of the signaling pathways that control them (Blatt, 2000; Schroeder et al., 2001; McAinsh and Pittman, 2009; Hills et al., 2012). Even so, much of stomatal dynamics remains unresolved, especially how the entire network of transporters in guard cells works to modulate solute flux and how this network is integrated with organic acid metabolism (Wang and Blatt, 2011) to achieve a dynamic range of stomatal apertures.This gap in understanding is most evident in a number of often unexpected observations, many of which have led necessarily to ad hoc interpretations. Among these, recent studies highlighted a diurnal variation in the free cytosolic Ca²⁺ concentration ([Ca2+]_i), high in the daytime despite the activation of primary ion-exporting ATPases, and have been interpreted to require complex levels of regulation (Dodd et al., 2007). Other findings wholly defy intuitive explanation. For example, the tpk1 mutant of Arabidopsis (Arabidopsis thaliana) removes a major pathway for K⁺ flux across the tonoplast and suppresses stomatal closure, yet the mutant has no significant effect on cellular K⁺ content (Gobert et al., 2007). Similarly, the Arabidopsis clcc mutant eliminates the H⁺-Cl⁻ antiporter at the tonoplast; it affects Cl⁻ uptake, reduces vacuolar Cl⁻ content, and slows stomatal opening; however, counterintuitively, it also suppresses stomatal closure (Jossier et al., 2010). In work leading to this study, we observed that the slac1 anion channel mutant of Arabidopsis paradoxically profoundly alters the activities of the two predominant K⁺ channels at the guard cell plasma membrane. The SLAC1 anion channel is a major pathway for anion loss from the guard cells during stomatal closure (Negi et al., 2008; Vahisalu et al., 2008), and its mutation leads to incomplete and slowed closure of stomata in response to physiologically relevant signals of dark, high CO₂, and the water-stress hormone abscisic acid. Guard cells of the slac1 mutant accumulate substantially higher levels of Cl⁻, Mal, and also K⁺ when compared with guard cells of wild-type Arabidopsis (Negi et al., 2008). The latter observation is consistent with additional impacts on K⁺ transport; however, a straightforward explanation for these findings has not been not forthcoming.Quantitative systems analysis offers one approach to such problems. Efforts to model stomatal function generally have been driven by a “top-down” approach (Farquhar and Wong, 1984; Eamus and Shanahan, 2002) and have not incorporated detail essential to understanding the molecular and cellular mechanics that drive stomatal movement. Only recently we elaborated a quantitative systems dynamic approach to modeling the stomatal guard cell that incorporates all of the fundamental properties of the transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the essential cytosolic pH (pH_i) and [Ca2+]_i buffering characteristics that have been described in the literature (Hills et al., 2012). The model resolved with this approach (Chen et al., 2012b) successfully recapitulated a wide range of known stomatal behaviors, including transport and aperture dependencies on extracellular pH, KCl, and CaCl₂ concentrations, diurnal changes in [Ca2+]_i (Dodd et al., 2007), and oscillations in membrane voltage and [Ca2+]_i thought to facilitate stomatal closure (Blatt, 2000; McAinsh and Pittman, 2009; Chen et al., 2012b). We have used this approach to resolve the mechanism behind the counterintuitive alterations in K⁺ channel activity uncovered in the slac1 mutant of Arabidopsis. Here, we show how anion accumulation in the mutant affects the H⁺ and Ca²⁺ loads on the cytosol, elevating pH_i and [Ca2+]_i, and in turn regulating the K⁺ channels. We have validated the key predictions of the model and, in so doing, have uncovered a previously unrecognized homeostatic network that ameliorates the effects of the slac1 mutant on transpiration from the plant.     

Electrical phenotypes of calcium transport mutant strains of a filamentous fungus, Neurospora crassa

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca(2+)/H(+) exchange protein (cax), and Ca(2+)-ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H(+)-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca(2+) levels, indicative of lesions in Ca(2+) homeostasis. However, the net Ca(2+) effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca(2+)-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca(2+) signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca(2+)] was elevated. Thus, although Ca(2+) homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654-661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H(+)-ATPase activity.     

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

Identification of several new classes of low-molecular-weight wheat gliadin-related proteins and genes

During the initial phases of a wheat endosperm Expressed-Sequence-Tag (EST) project, several clones were determined to be related to wheat gliadin sequences, but not similar enough to be classified into any of the traditional gliadin families [α-, γ-, and ω-gliadins, low-molecular-weight (LMW) glutenins]. Complete sequences of these cDNA clones revealed four new classes of gliadin-related endosperm proteins, but lacking a prominent repeat domain which until now has been characteristic of the gliadins. Two of these classes are related to different minimally described groups of Triticeae endosperm proteins. One class of proteins, which has N-terminal amino-acid sequences matching members of a reported 25-kDa globulin family from wheat, is shown by amino-acid sequencing to match to a family of 25-kDa endosperm proteins, is encoded by a multigene family, and is most similar to the LMW-glutenins. A second new class shows N-terminal homologies to LMW secalins from rye, and has an amino-acid composition similar to wheat and barley LMW proteins with extraction properties similar to prolamins. The third class is most similar to α-gliadins, and the fourth class has no close association to previously described wheat endosperm proteins. Received: 20 October 2000 / Accepted: 20 November 2000     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.