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Annual canarygrass (Phalaris canariensis L.) has a larger response to chloride (Cl−) fertilizer than other cereal crops. This unexpected response prompted further research for unexpected interactions between Cl− and other nutrients in annual canaryseed. The objective of this study was to compare the interaction of macronutrients and micronutrients with Cl− on the development and grain yield of annual canarygrass. Thirteen fertilizer combinations were applied to determine the effect of macro- and micronutrients on annual canarygrass. A field study was conducted at six locations across Saskatchewan over a 4 yr period using a randomize complete block design. Grain yield had a strong chloride response at 7 of the 21 site years with a 70% increase in grain yield over the 7 site-years. A sideband application or surface application of Cl− were both effective. At the responsive sites without the addition of Cl−, the addition of other nutrients is not effective while at the non-responsive sites, responses to the nutrients can be achieved without the addition of Cl−. Chloride impacted canarygrass during reproductive development. Nitrogen is the major nutrient Cl− interacted with in this study. Canarygrass is not more responsive to phosphorus, potassium, sulfur, zinc, copper, manganese, and boron than other cereals. In conclusion, at Cl−-responsive sites the application of nutrients are ineffective in the absence of Cl− fertilizer. In addition, the sensitivity of annual canarygrass to Cl− indicates that it could be used to investigate the role of Cl− in cereal crop development and grain yield.

Introduction: The aberrant activation of oncogenic signaling pathways can drive tumorigenesis and malignant transformation, where deregulation of the activity of protein kinases or phosphatases can play a critical role. Several protein tyrosine phosphatases (PTPs) that are overexpressed in human cancers have recently been shown to activate signaling pathways and promote tumor development and progression (Sivaganesh et al., 2021. Protein Tyrosine Phosphatases: Mechanisms in Cancer. Int J Mol Sci. 22(23):12865). This reinforces the need for protein phosphatase inhibitors for cancer therapy, which requires robust assays to monitor PTP activity. Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on known physiological substrates. PTP enzyme activity was monitored using the PhosphoSens® platform in kinetic mode (a progress curve in every well) using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode with Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: Using commercially available phosphatases, we demonstrated the ability to rapidly identify novel Sox-based phosphopeptide substrates for each phosphatase. Performance measures included initial reaction rates, Km's, signal/background, linear reaction kinetics, absence of initial lags, assay sensitivity and specificity. Final optimized assay conditions were established for 28 different phosphatases. Each phosphatase was titrated to determine the lowest optimal assay concentration, demonstrating linearity and detection of low nM enzyme. Potency assessments using a set of compounds revealed sub nM inhibitor IC50 values and this included using sodium orthovanadate as a positive control. Finally, we demonstrated the ability to profile a range of novel phosphatase inhibitors to determine selectivity. Conclusions: The generation of robust activity-based Sox-sensor phosphopeptide substrates opens new areas for effective drug discovery with protein phosphatases as an emerging target class. The PhosphoSens-Kinetic assay format is ideal for assessing enzyme regulation and elucidating drug mechanism of action, potency, and profiling. The PhosphoSens-Red endpoint format is ideal for HTS and high-throughput SAR. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a dramatic improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Susan Cornell-Kennon, Erik Schaefer, Earl May, Matthew Hakar. Sox-based sensor phosphopeptides for continuous, homogeneous and quantitative monitoring of protein phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1426.

We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)

A fluorescence polarization assay is described that measures the binding of fluorescently labeled erythromycin to 70S ribosomes from Escherichia coli and the displacement of the erythromycin from these ribosomes. The assay has been validated with several macrolide derivatives and other known antibiotics. We demonstrate that this assay is suitable for determining the dissociation constants of novel compounds that have binding sites overlapping those of macrolides. This homogeneous binding assay provides a valuable tool for defining structure-activity relationships among compounds during the discovery and development of new ribosome-targeting drugs.

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.