ADP-Glo is a novel bioluminescent, homogeneous assay for monitoring ADP producing biochemical reactions and thus it is an ideal assay for detecting enzyme activity using a wide variety of substrates. It is a universal assay that can be used with protein kinases, lipid kinases, sugar kinases, and many more kinases as well as ATPases. Because of its high sensitivity, it is suitable for monitoring enzyme activities at very early substrate conversions requiring very low amount of enzymes. Furthermore, as the assay is applicable to a broad range of ATP and substrate concentrations, it is optimal for enzymes that require high ATP and substrate concentrations. This is critical since inhibitor potency has to be demonstrated at the cellular level where ATP is present at millimolar concentrations. ADP-Glo is performed in 2 steps upon completion of kinase reaction: a combined termination of kinase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity. Due to its high signal to background and luminescent readout, this assay is less susceptible to generation of false hits and thus it is applicable to not only primary and secondary screening but also kinase profiling.
Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.Clostridial proteins, such as tetanus toxin and seven serologically distinct botulinum neurotoxins (botulinum neurotoxin serotype A [BoNT/A], BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G) that are produced by Clostridium tetani, Clostridium botulinum, Clostridium argentiensis, and Clostridium baratti, are powerful tools for studying the mechanisms of synaptic vesicle exocytosis (3,(20)(21)(22)(23). These toxins have been also used for therapeutic purposes, such as the treatment of strabismus, blepharospasms (24,25), and many other neurological conditions, as well as in clinical dermatology (4).Currently, BoNT/A and other clostridial neurotoxins and their fragments are purified from native Clostridium strains by using traditional purification protocols. Because these microorganisms are anaerobes, they pose technical problems. In addition, gene manipulation methods have not been developed for these microorganisms. Therefore, it has been difficult to construct Clostridium strains that produce derivatives of neurotoxins and other proteins. Genes for all eight clostridial neurotoxins have been cloned, and their sequences have been identified (2,6,9,18,30,31). Many attempts to express fragments of clostridial neurotoxins in Escherichia coli have failed because of the unusually high AT content of clostridial DNA. Makoff et al. successfully expressed a tetanus toxin fragment in E. coli (12) by optimizing sequences for codon usage in E. coli by complete synthesis of these sequences de novo. This approach, however, is very laborious and expensive.Recently, several groups of workers have demonstrated that rarely used codons can...
Clostridial neurotoxins are the most powerful toxins known. There are no available antidotes to neutralize neurotoxins after they have been internalized by neuronal cells. Enzymatic domains of clostridial neurotoxins are zinc-endopeptidases specific for protein components of the neuroexocytosis apparatus. Thus, attempts were made to find such antidotes among molecules possessing chelating properties. Subsequently, it was proposed that the process of interaction between clostridial neurotoxins and their substrates might be more complex than viewed previously and may include several separate regions of interaction. Phage display technology is free from bias toward any particular model. This technology in combination with recombinantly produced light chains of botulinum neurotoxins serotypes A, B, and C was used to identify potential inhibitors of clostridial neurotoxins. Identified sequences did not show substantial similarity with substrate proteins of clostridial neurotoxins. Nevertheless, three peptides chosen for further analysis were able to inhibit enzymatic activity of all clostridial neurotoxins tested. This work demonstrates that at least one of these peptides could not be cleaved by clostridial neurotoxin. Attempts to delete amino acid residues from this peptide resulted in dramatic loss of its inhibitory activity. Finally, this work presents a novel approach to searching for inhibitors of clostridial neurotoxins.
Pseudomonas exotoxin (PE) binds the heavy chain of the ␣ 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. Nterminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.
Clostridial neurotoxins are the most powerful toxins known. Nevertheless, derivatives of these toxins may find broad applications both in science and medicine because of their unique abilities to recognize neurons and deliver small and large molecules into them. In this paper we describe the construction of two types of such derivatives. Proteins belonging to the first class were designed to allow direct conjugation with one or few molecules of interest. Proteins belonging to the second class contain biotin residue and therefore could be easily connected to streptavidin loaded with multiple molecules of interest. Only C-terminal regions of neurotoxin heavy chains were incorporated in the structure of recombinant proteins. Nevertheless, recombinant proteins were found to be able to recognize specific neuronal receptors and target model molecules to rat synaptosomes and human neuroblastoma cells.
G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.
Protein Kinases are implicated in metabolic and cellular disorders including cancer and thus they represent a very important target for the development of novel therapeutics. The process of developing new drug requires an innovative and low cost strategy to screen and identify compounds that can be used for lead optimization that incorporate the required desirable features. It will be idea to have a universal technology that can be used for the various stages of drug discovery such as assay development, compound screening, lead optimization, and enzyme profiling using the same technology platform. The technology should provide high sensitivity, robustness, and low cost. Towards that goal, we have expanded our luminescent platform for monitoring kinase activity to enable us to detect enzyme activity using low amount of enzymes, measure initial rates at very early substrate conversion, and generate reliable and robust data. This luminescent assay monitors enzyme activity by measuring ADP production using a homogenous and high through put formatted assay. The assay is also ideal for profiling compounds against various kinases since it detects ADP, the universal product of all kinases. Thus, the new homogenous assay is most suitable for drug discovery targeting kinases since it can be used in all various stages of drug development leading to animal or cellular studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2011-5445
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