Glycosylation of Aromatic Amines I: Characterization of Reaction Products and Kinetic Scheme

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D ¹H–¹H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of α and β glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1–6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

Substantivity of zinc salts used as rinsing solutions and their effect on the inhibition of Streptococcus mutans

The antimicrobial efficacy of zinc (Zn) salts (sulfate and acetate) against Streptococcus mutans (S. mutans) present in the oral cavity was tested in this study. The substantivity of Zn salts was assessed by determining the concentration of Zn in whole, unstimulated saliva and by measuring the magnitude of suppression of salivary S. mutans, 2h after rinsing. The concentration of Zn was measured by atomic absorption spectrometry (AAS) with electrothermal atomization (ET AAS) in saliva sampled before (basal) and 24h after mouth rinsing with different concentrations of Zn (0.1%, 0.5% and 1%) administrated as sulfate and acetate. The estimation of Zn levels in samples collected 30, 60, 90 and 120 min after rinsing was carried out by AAS with flame atomization (FAAS). Immediately after rinsing, the concentration of Zn in saliva sharply increased with respect to the baseline values (0.055+/-0.017 mg/L), followed by a sustained decrease, probably due to clearance of salivary flow or swallowing during sampling. A significant reduction (>87%) in the total mean S. mutans counts was found 2h after rinsing either with sulfate or acetate solutions, as evidence of the high substantivity and effectiveness of the Zn salts tested. A statistically significant inverse relationship (p<0.001 and the Pearson correlation coefficients between -34% and -50%) was found between Zn levels and the respective pH values measured in the samples collected 60 and 120 min after rinsing, sustaining the theory of bacterial glycolysis inhibition.     

The discovery of diazetidinyl diamides as potent and reversible inhibitors of monoacylglycerol lipase (MAGL)

Monoacylglycerol lipase (MAGL) has emerged as an attractive drug target because of its important role in regulating the endocannabinoid 2-arachidonoylglycerol (2-AG) and its hydrolysis product arachidonic acid (AA) in the brain. Herein, we report the discovery of a novel series of diazetidinyl diamide compounds 6 and 10 as potent reversible MAGL inhibitors. In addition to demonstrating potent MAGL inhibitory activity in the enzyme assay, the thiazole substituted diazetidinyl diamides 6d–l and compounds 10 were also effective at increasing 2-AG levels in a brain 2-AG accumulation assay in homogenized rat brain. Furthermore, selected compounds have been shown to achieve good brain penetration after oral administration in an animal study.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

Allogibberic acid: An inhibitor of flowering in Lemna perpusilla

Allogibberic acid (I) has been identified as the compound responsible for the inhibition of flowering, increase in frond multiplication rate and decrease in frond size produced in Lemna perpusilla 6746 by autoclaved, unbuffered aqueous solutions of gibberellic acid (VII). 13-Deoxyallogibberic acid (IV), a product of autoclaving aq. GA₇ (VIII) solutions, also inhibits flowering in L. perpusilla and is about 10 times more active than allogibberic acid.     

The Lys 280 → Gln mutation mimicking disease‐linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post‐translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau‐K18 K280Q, in which the Lys 280 → Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX‐MS measurements, to form fibrils with a structural core that is longer than that of tau‐K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau‐K18 for its fibrillar counterpart is only marginally more than that of monomeric tau‐K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis–Menten‐like model, in which the monomer first binds and then undergoes conformational conversion to β‐strand, shows that the fibrils of tau‐K18 K280Q convert monomeric protein more slowly than do fibrils of tau‐K18. In contrast, monomeric tau‐K18 K280Q is converted faster to fibrils than is monomeric tau‐K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not.     

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.