Discovery and Efficacy of AZ-PRMT5i-1, a Novel PRMT5 Inhibitor with High MTA Cooperativity.

PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties.

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa.

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study.

A temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of α-maltose-1-phosphate.

Background: The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene.

Structure of the Mycobacterium smegmatis α-maltose-1-phosphate synthase GlgM.

Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M.

Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE.

GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not.

Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants.

Assembly of α-Glucan by GlgE and GlgB in Mycobacteria and Streptomycetes.

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis.

Background: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected.

Results: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M.

Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

GlgE (EC 2.4.99.

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated.

Structure of Streptomyces maltosyltransferase GlgE, a homologue of a genetically validated anti-tuberculosis target.

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis.

Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions.

Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism.

Self-poisoning of Mycobacterium tuberculosis by targeting GlgE in an alpha-glucan pathway.

New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to alpha-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M.

Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy.

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor.

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease.

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis.

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease.

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures.

The denatured state under native conditions: a non-native-like collapsed state of N-PGK.

The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character.

Solution structure of the helicase-interaction domain of the primase DnaG: a model for helicase activation.

The helicase-primase interaction is a critical event in DNA replication and is mediated by a putative helicase-interaction domain within the primase. The solution structure of the helicase-interaction domain of DnaG reveals that it is made up of two independent subdomains: an N-terminal six-helix module and a C-terminal two-helix module that contains the residues of the primase previously identified as important in the interaction with the helicase. We show that the two-helix module alone is sufficient for strong binding between the primase and the helicase but fails to activate the helicase; both subdomains are required for helicase activation.

Enhanced ligand affinity for receptors in which components of the binding site are independently mobile.

Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized.