Pathway assays

This chapter focuses on a novel antibiotic discovery paradigm. Metallo-hydrolases and Mur ligases are used to illustrate this approach. New methods to identify and prioritize targets, develop screens, and evaluate new inhibitors are discussed. New developments in enzyme-based assays, such as pathway assays, are also presented. This new approach is opening new venues for screening targets that are difficult to screen because substrates are not easily available. 

Several hits with IC5o less than 10 p,g/ml were identified using this DASA-pathway assay, including one compound with an IC50 of 0.08 dg/ml [38,40], To deconvolute the effects of potential inhibitors in the pathway, inhibition of individual enzymes is tested for hits identified in the primary screening. For ex-... 

Pathway assays have several advantages (1) individual assays can be combined into one, saving labor and reagents (2) the reaction product of a previous step is the substrate for the subsequent reaction and avoids the need to obtain the individual substrates (3) a pathway assay can detect dead-end substrates, compounds that are capable of being incorporated into the product of previous reaction but prevent the further elongation of the pathway [37],... 

Oxidation of DBT using the recombinant DszC enzyme produced DBTO2 in 79% yield no DBTO was observed. In the absence of reductase, there was no detectable conversion. When DBTO was used as a substrate, it was quantitatively converted to DBTO2, indicating that DBTO is indeed an intermediate in the desulfurization pathway. Assays using benzyl sulfide as the substrate produced 71% benzyl sulfoxide, 6% benzyl sulfone, and the remainder unreacted substrate, demonstrating that sulfides are also substrates for the enzyme. Under identical conditions, using benzyl sulfoxide as the substrate, 11% was converted to the sulfone. 

AMPK can also be activated by a Ca2+-mediated pathway involving phosphorylation at Thr-172 by the Ca2+/calmodulin-dependent protein kinase, CaMKK 3. CaMKKa and CaMKK 3 were discovered as the upstream kinase for the calmodulin-dependent protein kinases-1 and -IV they both activate AMPK in a Ca2+/ calmodulin-dependent manner in cell-free assays, although CaMKK 3 appears to much more active against AMPK in intact cells. Expression of CaMKKa and CaMKK(3 primarily occurs in neural tissues, but CaMKKp is also expressed in some other cell types. Thus, the Ca2+-mediated pathway for AMPK activation has now been shown to occur in response to depolarization in rat neuronal tissue, in response to thrombin (acting via a Gq-coupled receptor) in endothelial cells, and in response to activation of the T cell receptor in T cells. 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

The HPLC assay is fully coupled to the impurities A-D on the assumption that there is a direct competition between the major component and some impurity-producing reaction pathways. The basis-value 99 was introduced to simulate other concentration losses that were not accounted for by impurities A-D. 

Metabolic disorders of urea biosynthesis, while extremely rare, illustrate four important principles (1) Defects in any of several enzymes of a metabolic pathway enzyme can result in similar clinical signs and symptoms. (2) The identification of intermediates and of ancillary products that accumulate prior to a metabolic block provides insight into the reaction that is impaired. (3) Precise diagnosis requires quantitative assay of the activity of the enzyme thought to be defective. (4) Rational therapy must be based on an understanding of the underlying biochemical reactions in normal and impaired individuals. 

Wildlife toxicologists should be attuned to developments in human health mercury, as assays that have been used successfully on humans may be suitable or adaptable for other vertebrate species. Echeverria and co-workers (Echeverria et al. 2005, 2006 Heyer et al. 2006) have characterized a gene encoding coproporphyrinogen oxidase, a gene in the heme biosynthetic pathway. Polymorphism in this gene predicts differential response to elemental mercury exposure in human subjects. Plans to modify this assay for other mercury species in matrices from wildlife are under way. 

Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

Generally, all of the assays used for characterization of hits in terms of potency, selectivity, function, and or cellular activity will continue to be used to characterize new compounds synthesized to identify leads. However, additional assays will normally be added to characterize compound selectivity more fully, to provide additional evidence that compounds are acting on the desired pathway and by the desired mechanism. 

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