A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels.

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance.

Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound.

Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP.

Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive.

Discovery of (S)-6-(3-cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid as a hepatoselective glucokinase activator clinical candidate for treating type 2 diabetes mellitus.

Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk.

Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis.

GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact.

Structure-guided inhibitor design for human acetyl-coenzyme A carboxylase by interspecies active site conversion.

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system.

Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome.

High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha.

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA).

High-throughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase.

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries.

A high-throughput competitive scintillation proximity aminoacyl-tRNA synthetase charging assay to measure amino acid concentration.

An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of scintillation proximity assay (SPA) technology to measure aminoacyl-tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive scintillation proximity assay (cSPA).

A High-throughput two-phase partition assay to measure the activity of lipid-metabolizing enzymes.

A new method to measure the activity of lipid-metabolizing enzymes is described. Subsequent to an enzymatic reaction, a two-phase system (organic/aqueous) is established by the addition of a phase partition scintillation fluid (PPSF). The PPSF serves as a scintillation fluid, a phase partition agent, and a carrier/separator of an organic-soluble radiolabeled reaction substrate or product.

Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants.

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead.

HTS in the new millennium: the role of pharmacology and flexibility.

Over the past decade, high throughput screening (HTS) has become the focal point for discovery programs within the pharmaceutical industry. The role of this discipline has been and remains the rapid and efficient identification of lead chemical matter within chemical libraries for therapeutics development. Recent advances in molecular and computational biology, i.

Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus: elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping.

Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J.

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain.

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design.

C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase.

Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases. The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix. The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide.

Thiol ligation of two zinc atoms to a class I tRNA synthetase: evidence for unshared thiols and role in amino acid binding and utilization.

Class I tRNA synthetases generally contain a characteristic N-terminal catalytic core joined to a C-terminal domain that is idiosyncratic to the enzyme. The closely related class I Escherichia coli methionyl- and isoleucyl-tRNA synthetases each have a single zinc atom coordinated to ligands contained in the catalytic domain. Isoleucyl-tRNA synthetase has a second, functionally essential, zinc bound to ligands at the C-terminal end of the 939 amino acid polypeptide.

Zinc-dependent cell growth conferred by mutant tRNA synthetase.

We present evidence that zinc bound near the C terminus of a long tRNA synthetase polypeptide, and at a location far in the sequence from the catalytic domain, is needed to sustain cell growth and is, therefore, essential for enzyme function. Several class I and class II tRNA synthetases contain bound zinc, including the 939-amino acid class I Escherichia coli isoleucyl-tRNA synthetase, which has two zinc atoms coordinated to cysteine sulfhydryls. The functional significance of these bound zinc atoms has been unclear.

[Treatment of chronic myeloid leukemia using alpha-interferon and hydroxyurea. Study of 30 cases].

Purpose: To evaluate the efficacy of the combination, alpha-interferon (IFN)-hydroxyurea (HU) in the treatment of patients with Philadelphia positive chronic myelogenous leukaemia (Ph'-CML).

Patients And Methods: A prospective study was started in 1988 in which 30 patients with chronic phase, low-risk Ph'-CML, according to Kantarjian's staging system, were included. They were treated with IFN at a dose of 5 MU/m2 subcutaneously twice a week plus HU in doses between 0.

The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate.

The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR.

C-terminal peptide appendix in a class I tRNA synthetase needed for acceptor-helix contacts and microhelix aminoacylation.

The 10 class I tRNA synthetases have an N-terminal nucleotide-binding fold which contains the catalytic center. Insertions into the nucleotide-binding fold provide contacts for acceptor-helix interactions, which stabilize the amino acid acceptor end of the tRNA substrate in the active site. A separate and largely nonconserved C-terminal domain provides contacts with distal parts of the tRNA, including the anticodon.

Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases.

Of the ten class I tRNA synthetases, those for methionine and isoleucine are among the most closely related. In recent work we showed that the 676 amino acid E. coli methionine tRNA synthetase has one zinc bound per polypeptide.

Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold.

The 10 class I aminoacyl-tRNA synthetases share a common N-terminal nucleotide-binding fold. Idiosyncratic polypeptide insertions into this fold introduce residues important for activity, including those that interact with the tRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNA(Met) ligase, EC 6.