Structure-Based Design and Optimization of Methionine Adenosyltransferase 2A (MAT2A) Inhibitors with High Selectivity, Brain Penetration, and In Vivo Efficacy.

Synthetic lethality has recently emerged as a new approach for the treatment of mutated genes that were previously considered undruggable. Targeting methionine adenosyltransferase 2A (MAT2A) in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene leads to synthetic lethality and thus has attracted significant interest in the field of precise anticancer drug development. Herein, we report the discovery of a series of novel MAT2A inhibitors featuring a pyrazolo[3,4-]quinolin-4-one skeleton based on structure-based drug design.

Leveraging Structure-Based Drug Design to Identify Next-Generation MAT2A Inhibitors, Including Brain-Penetrant and Peripherally Efficacious Leads.

Inhibition of the -adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, , to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, , and a potent, but limited brain-penetrant compound, .

Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production.

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure.

Discovery of Potent and Selective Covalent Protein Arginine Methyltransferase 5 (PRMT5) Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is known to symmetrically dimethylate numerous cytosolic and nuclear proteins that are involved in a variety of cellular processes. Recent findings have revealed its potential as a cancer therapeutic target. PRMT5 possesses a cysteine (C449) in the active site, unique to PRMT5.

Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown.

Structural Insight into Substrate Selection and Catalysis of Lipid Phosphate Phosphatase PgpB in the Cell Membrane.

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro Here we report the crystal structure of the lipid-bound form of PgpB.

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli.

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E.

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation.

Ca(2+) efflux by Ca(2+) cation antiporter (CaCA) proteins is important for maintenance of Ca(2+) homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na(+)/Ca(2+) exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca(2+) efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca(2+)/H(+) antiporter protein YfkE from Bacillus subtilis at 3.

Structural studies of the Ca(2+) regulatory domain of Drosophila Na(+)/Ca (2+) exchanger CALX.

CALX, the NCX homolog in Drosophila, involves in light-mediated Ca(2+) homeostasis in sensory neuronal cells. CALX exhibits a unique negative Ca(2+) regulatory property mediated by Ca2+ binding at its intracellular regulatory domain. Our structural studies of individual CBD1 or CBD2 domain reveal that CBD1 is the only Ca(2+) binding domain in CALX.

Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing.

The Na(+)/Ca(2+) exchanger CALX promotes Ca(2+) efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca(2+) homeostasis. CALX activity is negatively regulated by specific Ca(2+) interaction within its two intracellular Ca(2+) regulatory domains CBD1 and CBD2, yet how the Ca(2+) binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca(2+) regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex.

The cleavage factor I(m) (CF I(m)), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I(m), comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I(m)-RNA complex.

Crystallization and preliminary X-ray diffraction analysis of ARO9, an aromatic aminotransferase from Saccharomyces cerevisiae.

Saccharomyces cerevisae ARO9 protein, an aromatic aminotransferase II, catalyzes the transamination step of the catabolism of aromatic amino acids, mainly tryptophan. ARO9 also belongs to a novel subfamily of enzymes within the aminotransferase subgroup I. Crystals of ARO9 protein have been grown using the hanging-drop vapour-diffusion method.

Structure of the second PDZ domain from human zonula occludens 2.

Human zonula occludens 2 (ZO-2) protein is a multi-domain protein that consists of an SH3 domain, a GK domain and three copies of a PDZ domain with slight divergence. The three PDZ domains act as protein-recognition modules that may mediate protein assembly and subunit localization. The crystal structure of the second PDZ domain of ZO-2 (ZO-2 PDZ2) was determined by molecular replacement at 1.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human phosphoribosyl pyrophosphate synthetase 1 (PRS1).

Phosphoribosyl pyrophosphate synthetase (PRS; EC 2.7.6.