Design and synthesis of Hsp90 inhibitors: Exploring the SAR of Sansalvamide A derivatives

Utilizing the structure–activity relationship we have developed during the synthesis of the first two generations and mechanism of action studies that point to the interaction of these molecules with the key oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their synthesis. Our new structures, designed from previously reported potent compounds, were tested for cytotoxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed using computational studies involving blind docking of derivatives using Autodock. Further, we show new evidence that our molecules bind directly to Hsp90 and modulate Hsp90’s binding with client proteins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

Insights from Characterizing Extinct Human Gut Microbiomes

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (∼8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.     

Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes

The AddAB and RecBCD helicase-nucleases are related enzymes prevalent among bacteria but not eukaryotes and are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although these enzymes have been extensively studied both genetically and biochemically, inhibitors specific for this class of enzymes have not been reported. We developed a high-throughput screen based on the ability of phage T4 gene 2 mutants to grow in Escherichia coli only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized this screen for use in 1536-well plates and screened 326,100 small molecules in the NIH molecular libraries sample collection for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli recBCD deletion strain. Secondary screening used assays with cells expressing AddAB or RecBCD and a viability assay that measured the effect of compounds on cell growth without phage infection. From this screening campaign, 12 compounds exhibiting efficacy and selectivity were tested for inhibition of purified AddAB and RecBCD helicase and nuclease activities and in cell-based assays for recombination; seven were active in the 0.1-50 μM range in one or another assay. Compounds structurally related to two of these were similarly tested, and three were active in the 0.1-50 μM range. These compounds should be useful in further enzymatic, genetic, and physiological studies of these enzymes, both purified and in cells. They may also lead to useful antibacterial agents, since this class of enzymes is needed for successful bacterial infection of mammals.     

Natural recolonisation of seagrasses at a disused sewage sludge outfall

Digested sludge from a sewage outfall that operated for 15 years adjacent to Adelaide in South Australia caused total seagrass loss in a 365 ha area around the outfall. Eight years after its closure, an underwater survey was conducted to determine the extent of seagrass recovery in a 2 ha area immediately adjacent to the disused outfall. Total seagrass cover was estimated to be 28% comprising 23% Halophila australis, 3% Posidonia angustifolia, 1% Posidonia sinuosa, and less than 1% each for Zostera tasmanica and Amphibolis antarctica. The recovery of seagrasses at the outfall site is probably due to recolonisation by propagules from a distant source. While results from this study suggest that seagrasses can return to a severely polluted site if the pollution source is removed and that Posidonia can be a primary coloniser of disturbed sites, they also suggest that it will take many decades for the seagrass community to recover to its former state.     

The effects of cambial age and position within the stem on specific conductivity in Douglas-fir (Pseudotsuga menziesii) sapwood

Specific conductivity (k_s, m²s^-1MPa^-1) describes the permeability of xylem and is determined by all aspects of xylem anatomy that create resistance to the flow of water. Here we test the hypothesis that k_s is a function of radial and vertical position within the stem, rather than solely a function of cambial age (ring number from the pith), by measuring k_s on samples excised from 35-year-old Douglas-fir [Pseudotsuga menziesii var. menziesii (Mirb.) Franco] trees at six heights and two or three radial positions. Sapwood k_s decreased from the cambium to the heartwood boundary, and the difference between outer and inner sapwood increased with height in the tree. Beneath the live crown, inner sapwood had 80-90% the k_s of outer sapwood, but only 55% just 10 m higher in the stem (about 10 nodes down from the tree top). Outer sapwood k_s peaked near the base of the crown and declined toward both the base and top of the stem. These patterns can be explained by two superimposed effects: the effect of cambial age on the dimensions of tracheids as they are produced, and the effect of xylem aging, which may include accumulation of emboli and aspiration of bordered pits. Tracheid lumen diameter and earlywood and latewood density and width, all factors known to vary with cambial age, were measured on different trees of the same age and from the same stand. Lumen diameter increased with cambial age, whereas the proportion of latewood and growth ring density increased after an initial decrease in the first 5 years. Our results suggest that the effect of cambial age on xylem anatomy is not sufficient to explain variation in k_s. Instead, physical position (both vertical and radial) in the stem and cambial age must be considered as determinants of conductivity.     

Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database

Abstract The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (Ptsl), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.