Preliminary X-ray crystallographic data on mouse submaxillary gland renin and renin-inhibitor complexes

We have crystallized renin from the submaxillary gland of male mice, both in its native state, and in binary complex with transition-state analog inhibitors. The best of the many crystal forms examined consisted of tetragonal bipyramids with space group symmetry P41 21 2 (or its enantiomorph) and unit cell dimensions a = b = 91.4 A, c = 211.6 A; alpha = beta = gamma = 90 degrees. This tetragonal form was compatible with both inhibited and uninhibited renin. Two of the inhibitors used were synthesized as iodinated analogs; their binary complexes with renin may serve as single-site rational heavy atom derivatives. X-ray data beyond 2.8-A resolution have been collected by oscillation photography using the Cornell High Energy Synchrotron Source in Ithaca, NY.     

Heterocyclic sulfoxide and sulfone inhibitors of fatty acid amide hydrolase

A novel series of heterocyclic sulfoxides and sulfones was prepared and examined as potential inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for inactivation of neuromodulating fatty acid amides including anandamide and oleamide.     

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.     

Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents

The solution-phase synthesis of two 1000-membered positional scanning libraries of distamycin A analogues is described enlisting acid/base liquid-liquid extractions for isolation and purification of all intermediates and final products. The results of their screening for functional activity (L1210 cytotoxic potency) and DNA binding affinity were compared with those derived from libraries containing the same compound members but prepared in a smaller 10-compound mixture format. The positional scanning libraries, which are substantially less demanding to prepare, allowed the accurate detection of the global observations and the clearly more potent activities, but more subtle discoveries and less distinguishable activities were not detected. This is a natural consequence of testing the larger 100-compound mixtures and the relative insensitivity of the assays to the contribution of any single, uniquely acting compound in the mixture. Thus, the disadvantages associated with the loss of some information contained within the library must be balanced against the advantages of the ease of library synthesis and judged in light of the library screening objectives.     

Stereocilia: the long and the short of it

Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness and short cochlear hair cell stereocilia in whirler mice and recessive deafness (DFNB31) in humans. Through its PDZ domains, whirlin might organize a group of proteins into a functional complex required for stereocilia elongation. Identifying these protein partners will advance our understanding of the development of stereocilia and their function as mechanosensory organelles indispensable for normal hearing.     

Thiazole orange as the fluorescent intercalator in a high resolution fid assay for determining DNA binding affinity and sequence selectivity of small molecules.

The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.     

N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases

A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide?>?carbamate?>?urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme inhibitory activity displayed pronounced effects with the activity improving substantially as one alters both the nature of the reacting carbonyl group (urea?>?carbamate?>?amide) and the pyrazole C4 substituent (CN?>?H?>?Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase. Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent K_i?=?100–200?pM), dual inhibitors of FAAH and monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC₅₀?=?10–20?nM) while simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).     

Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress

The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophi-cally grown, suspension-cultured soybean celts ( Glycine max L.). Within two days, 50 to 500 n M oxyfluorfen. a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants ascorbate and glutathione showed different responses. Under stress conditions with more than 250 n M oxyfluorfen, the cellular ascorbate- concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of ascorbate in untreated control cells) increased nearly 3-fold in the presence of 250 n M oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely ascor-bate peroxidase, glutathione reductase, rnonodehydroascorbate reductase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 n M , while dehydroascorbate reductase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.