Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis.

The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF.

Exploring the potential of adjunct therapy in tuberculosis.

A critical unmet need for treatment of drug-resistant tuberculosis (TB) is to find novel therapies that are efficacious, safe, and shorten the duration of treatment. Drug discovery approaches for TB primarily target essential genes of the pathogen Mycobacterium tuberculosis (Mtb) but novel strategies such as host-directed therapies and nonmicrobicidal targets are necessary to bring about a paradigm shift in treatment. Drugs targeting the host pathways and nonmicrobicidal proteins can be used only in conjunction with existing drugs as adjunct therapies. Significantly, host-directed adjunct therapies have the potential to decrease duration of treatment, as they are less prone to drug resistance, target the immune responses, and act via novel mechanism of action. Recent advances in targeting host-pathogen interactions have implicated pathways such as eicosanoid regulation and angiogenesis. Furthermore, several approved drugs such as metformin and verapamil have been identified that appear suitable for repurposing for the treatment of TB. These findings and the challenges in the area of host- and/or pathogen-directed adjunct therapies and their implications for TB therapy are discussed.

MmpL3 a potential new target for development of novel anti-tuberculosis drugs.

INTRODUCTION: Tuberculosis (TB) is still a leading cause of mortality in the developing world and there is an unmet clinical need for new drugs with novel mechanism of action. Targeting the complex and unique cell wall of TB-causing pathogen Mycobacterium tuberculosis (Mtb) has been a mainstay of TB drug discovery. Though, the composition of the cell wall of Mtb is well understood, little is known about the assembly process of the cell wall such as the transport of mycolic acids across the cell wall. AREAS COVERED: Recent research demonstrating MmpL3 protein as a transmembrane transporter of mycolic acids is discussed. In addition, MmpL3 has also been implicated in heme transport. Research describing several diverse chemical inhibitors that inhibit MmpL3 is reviewed. EXPERT OPINION: Evidence so far suggests MmpL3 is a transporter of mycolic acids. It has emerged as a novel therapeutic target for Mtb that is essential and for which several small molecule inhibitors have been identified. Identifying the interacting partners, understanding the substrate specificity and the mechanism of transport by MmpL3 are some of the gaps in knowledge that need to be addressed.

Identification of berberine as a novel agonist of fatty acid receptor GPR40.

Several herbal plants such as Chinese herb Rhizoma Coptidis have been reported to possess antidiabetic activity. Berberine is its major active constituent and functions as an insulin sensitizer and insulin secretagogue. It has been reported to modulate several signaling pathways and targets. The objective of the current study is to investigate if berberine can function as a ligand of fatty acid receptor GPR40, which stimulates glucose dependent insulin secretion. Towards this objective, a mammalian cell line with stable overexpression of GPR40 was generated and characterized. Berberine stimulated calcium mobilization with an EC(50) of 0.76 microM in this GPR40 overexpressing cell line. Further, berberine stimulated glucose dependent insulin secretion from rat pancreatic beta cell line. This suggests that berberine functions as an agonist of fatty acid receptor GPR40 and might be a novel antidiabetic mechanism of action for berberine.

Glycogen synthase kinase 3: more than a namesake.

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

Fatty acid receptors as new therapeutic targets for diabetes.

G-protein-coupled receptors (GPCRs) are key regulators of several physiological functions. Their roles in cellular signal transduction have made them the target for majority of all currently prescribed drugs. Additionally, there are many orphan GPCRs that provide potential novel therapeutic targets. Several GPCRs are involved in metabolic regulation and glucose homeostasis such as GLP-1 receptor, glucagon receptor, adiponectin receptor and so on. Recently, free fatty acids (FFAs) have been demonstrated as ligands for orphan GPCRs and have been proposed to play a critical role in physiological glucose homeostasis. GPR40 and GPR120 are activated by medium and long-chain FFAs, whereas GPR41 and GPR43 can be activated by short-chain FFAs. GPR40, which is preferentially expressed in pancreatic beta-cells, mediates the majority of the effects of FFAs on insulin secretion. In this review, these findings and also critical analysis of these GPCRs as novel targets for diabetes are discussed.

NSD1 is essential for early post-implantation development and has a catalytically active SET domain.

The nuclear receptor-binding SET domain-containing protein (NSD1) belongs to an emerging family of proteins, which have all been implicated in human malignancy. To gain insight into the biological functions of NSD1, we have generated NSD1-deficient mice by gene disruption. Homozygous mutant NSD1 embryos, which initiate mesoderm formation, display a high incidence of apoptosis and fail to complete gastrulation, indicating that NSD1 is a developmental regulatory protein that exerts function(s) essential for early post-implantation development. We have also examined the enzymatic potential of NSD1 and found that its SET domain possesses intrinsic histone methyltransferase activity with specificity for Lys36 of histone H3 (H3-K36) and Lys20 of histone H4 (H4-K20).