Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Silencing of microRNA-708 promotes cell growth and epithelial-to-mesenchymal transition by activating the SPHK2/AKT/ß-catenin pathway in glioma.

Aberrant microRNA-708 (miR-708) expression is frequently reported in cancer studies; however, its role in glioma has not been examined in detail. We investigated miR-708 function in glioma and revealed that miR-708 expression was significantly down-regulated in glioma tissues and cell lines. Restoration of miR-708 inhibited glioma cell growth and invasion both in vitro and in vivo. The oncogene SPHK2 (sphingosine kinase 2) was identified as a downstream target of miR-708 using luciferase and western blot assays. miR-708 inhibited AKT/ß-catenin signaling, which is activated by SPHK2. In addition, we revealed that miR-708 was transcriptionally repressed by EZH2 (enhancer of zeste homolog 2)-induced histone H3 lysine 27 trimethylation and promoter methylation. In summary, our findings revealed that miR-708 is a glioma tumor suppressor and suggest that miR-708 is a potential therapeutic target for glioma patients.

Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase is an archetypal multifunctional moonlighting protein involved in diverse cellular processes including metabolism, insulin signaling, proliferation, differentiation, apoptosis, neuronal function and embryonic development. The two known isoforms, GSK-3α and GSK-3ß that undergo activation/inactivation by post-translational, site-specific phosphorylation incorporate a vast number of substrates in their repertoire. Dysregulation of GSK-3 has been linked to diverse disease entities including cancer. The role of GSK-3 in cancer is paradoxical and enigmatic. The enzyme functions as a tumour promoter or suppressor based on the context, cell type and phosphorylation status. GSK-3 is the central hub that orchestrates signals from the Wnt/ß-catenin, PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, hedgehog, Notch and TP53 pathways to elicit regulatory influences on cancer initiation, epithelial-mesenchymal transition, and resistance to therapy. As a direct target of several microRNAs, GSK-3 influences hallmark attributes of cancer, cancer stemness and treatment resistance. There is overwhelming evidence to indicate that GSK-3 is aberrantly regulated in different cancer types. Consequently, GSK-3 has emerged as a potential therapeutic target in cancer. A plethora of natural and synthetic GSK-3 modulators have been discovered and the number of patents published for GSK-3 inhibitors has also been steadily increasing in recent years. This review focuses on the intricate interactions between GSK-3 and oncogenic signalling circuits as well as the feasibility of targeting GSK-3 for the treatment of cancer.

Novel 8-arylated purines as inhibitors of glycogen synthase kinase.

A series of 8-arylated purine derivatives bearing either an aniline or an alkyl amide at position 6 were found to inhibit glycogen synthase kinase-3, with good selectivity over ten kinases. Molecular modeling studies indicated that the most active compounds (8a and 8e), adopt a planar conformation, close to the shape of AMPPNP in the crystal structure of GSK-3. These compounds are stabilized by hydrophobic contacts between the 8-aromatic group and the protein adenine pocket and by electrostatic contacts.

T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses.

T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (Oryza sativa glycogen synthase kinase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis brassinosteroid insensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes.

Fragment screening: an introduction.

There are clearly many different philosophies associated with adapting fragment screening into mainstream Drug Discovery Lead Generation strategies. Scientists at Astex, for instance, focus entirely on strategies involving use of X-ray crystallography and NMR. However, AstraZeneca uses a number of different fragment screening strategies. One approach is to screen a 2000 compound fragment set (with close to "lead-like" complexity) at 100 microM in parallel with every HTS such that the data are obtained on the entire screening collection at 10 microM plus the extra samples at 100 microM; this provides valuable compound potency data in a concentration range that is usually unexplored. The fragments are then screen-specific "privileged structures" that can be searched for in the rest of the HTS output and other databases as well as having synthesis follow-up. A typical workflow for a fragment screen within AstraZeneca is shown below (Figure 24) and highlights the desirability (particularly when screening >100 microM) for NMR and X-ray information to validate weak hits and give information on how to optimise them. In this chapter, we have provided an introduction to the theoretical and practical issues associated with the use of fragment methods and lead-likeness. Fragment-based approaches are still in an early stage of development and are just one of many interrelated techniques that are now used to identify novel lead compounds for drug development. Fragment based screening has some advantages, but like every other drug hunting strategy will not be universally applicable. There are in particular some practical challenges associated with fragment screening that relate to the generally lower level of potency that such compounds initially possess. Considerable synthetic effort has to be applied for post-fragment screening to build the sort of potency that would be expected to be found from a traditional HTS. However, if there are no low-hanging fruit in a screening collection to be found by HTS then the use of fragment screening can help find novelty that may lead to a target not being discarded as intractable. As such, the approach offers some significant advantages by providing less complex molecules, which may have better potential for novel drug optimisation and by enabling new chemical space to be more effectively explored. Many literature examples that cover examples of fragment screening approaches are still at the "proof of concept" stage and although delivering inhibitors or ligands, may still prove to be unsuitable when further ADMET and toxicity profiling is done. The next few years should see a maturing of the area, and as our understanding of how the concepts can be best applied, there are likely to be many more examples of attractive, small molecule hits, leads and candidate drugs derived from the approaches described.

Incorporation of a nanosplitter interface into an LC-MS-RD system to facilitate drug metabolism studies.

In the work reported here, a novel interface, the nanosplitter, is incorporated into the drug metabolism laboratory in order to enhance the analytical capabilities of detecting and identifying drug-related metabolites to support drug metabolism studies during the drug development process. When an existing LC-MS-radiometric detector (RD) system is coupled with this nanosplitter, the system becomes capable of performing dynamic microspray under a typical analytical LC method. With the superior MS sensitivity offered by this system, most of the analytical LC methods developed for metabolite profiling can then be easily adopted for metabolite identification work. The improvement of these analytical capabilities can streamline the entire process of the drug metabolism study. In the experiments presented here, the nanosplitter interface coupled with analytical HPLC systems (e.g. 4.6 x 250 mm column @ 1 ml/min) demonstrated significant increases in MS signal (2x to 40x peak area) when compared to the standard LC-MS interface for both in vitro and in vivo metabolism studies. Furthermore, this signal gain facilitated the MS detection of additional metabolites (observed in the radiometric trace) that were below the MS level of detection when using the standard interface.