Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.     

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.     

S-100-Mediated Inhibition of Brain Protein Phosphorylation

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.     

Repurposing Hsp90 inhibitors as antibiotics targeting histidine kinases

To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.     

Cryptic structure and niche divergence within threatened Galápagos giant tortoises from southern Isabela Island

Although Galápagos giant tortoises are an icon for both human-mediated biodiversity losses and conservation management successes, populations of two species on southern Isabela Island (Chelonoidis guntheri, and C. vicina) remain threatened by hunting and persistence of feral animals. Conservation management of these tortoises has been hampered by lack of clarity regarding their taxonomy, ecological and morphological diversity, and the spatial distribution of evolutionarily significant units that may exist. Analyses of 16 microsatellite loci did not group samples according to current taxonomy. Instead, three (rather than two) genetic clusters were revealed. We show that the three regions of southern Isabela associated with these genetic clusters are significantly different in their ecological niches, which could suggest that ecological divergence may have shaped patterns of genetic differentiation in these tortoises. Furthermore, results suggest limited recent gene flow among sampled localities and between each of the three regions associated with genetic clusters. We discuss the need for further research on the ecological factors shaping the genetic and morphological diversity of southern Isabela tortoises. We suggest that current strategies whereby populations are managed separately are warranted pending further study, but due to mixed ancestry we recommend that Cerro Paloma tortoises be excluded from management programs.     

Nonrecombining genes in a recombination environment: the Drosophila "dot" chromosome

Rate of recombination is a powerful variable affecting several aspects of molecular variation and evolution. A nonrecombining portion of the genome of most Drosophila species, the "dot" chromosome or F element, exhibits very low levels of variation and unusual codon usage. One lineage of Drosophila, the willistoni/saltans groups, has the F element fused to a normally recombining E element. Here, we present polymorphism data for genes on the F element in two Drosophila willistoni and one D. insularis populations, genes previously studied in D. melanogaster. The D. willistoni populations were known to be very low in inversion polymorphism, thus minimizing the recombination suppression effect of inversions. We first confirmed, by in situ hybridization, that D. insularis has the same E + F fusion as D. willistoni, implying this was a monophyletic event. A clear gradient in codon usage exists along the willistoni F element, from the centromere distally to the fusion with E; estimates of recombination rates parallel this gradient and also indicate D. insularis has greater recombination than D. willistoni. In contrast to D. melanogaster, genes on the F element exhibit moderate levels of nucleotide polymorphism not distinguishable from two genes elsewhere in the genome. Although some linkage disequilibrium (LD) was detected between polymorphic sites within genes (generally <500 bp apart), no long-range LD between F element loci exists in the two willistoni group species. In general, the distribution of allele frequencies of F element genes display the typical pattern of expectations of neutral variation at equilibrium. These results are consistent with the hypothesis that recombination allows the accumulation of nucleotide variation as well as allows selection to act on synonymous codon usage. It is estimated that the fusion occurred ～20 Mya and while the F element in the willistoni lineage has evolved "normal" levels and patterns of nucleotide variation, equilibrium may not have been reached for codon usage.     

The synthesis and SAR of 2-arylsulfanylphenyl-1-oxyalkylamino acids as GlyT-1 inhibitors

Elevation of glycine levels by inhibition of the glycine transporter-1 (GlyT-1) and activation of the NMDA receptor is a potential strategy for the treatment of schizophrenia. A novel series of 2-arylsulfanylphenyl-1-oxyalkyl amino acids have been identified. The most prominent member of this series S-1-{2-[3-(3-fluoro-phenylsulfanyl)biphenyl-4-yloxy]ethyl}pyrrolidine-2-carboxylic acid (38) is a potent GlyT-1 inhibitor (IC50=59 nM). In vitro and in vivo assessment of CNS exposure indicates this compound is a likely substrate for active efflux transporters.     

Volatile fatty and dissolved free amino acids in Organic Lake,Vestfold Hills,East Antarctica

The distributions of volatile fatty acids (C1–C5) (VFAs) and dissolved free amino acids (DFAAs) in Organic Lake, a shallow hypersaline meromictic lake in the Vestfold Hills, East Antarctica, were determined during the winter and spring of 1987. The concentrations of the two VFAs detected, formic and acetic acid, were low (<10 M) in the oxic upper waters of the lake, but increased dramatically (up to 250M) beneath the oxycline. The distribution of DFAAs was similar, with a total concentration of 280M in the anoxic water early in the study. The amino acids occurring at the highest concentrations were alanine, glutamic acid, leucine and valine. Total VFA concentrations in the anoxic zone increased during the study period, but the total amino acid concentration dropped significantly to 64 M by the end of the study. The high concentrations beneath the oxycline were probably the result of bacterial utilisation of these substrates that was slow due to the high salinity and low temperature of the lake water.     

Naturally rare versus newly rare: demographic inferences on two timescales inform conservation of Galápagos giant tortoises

Long‐term population history can influence the genetic effects of recent bottlenecks. Therefore, for threatened or endangered species, an understanding of the past is relevant when formulating conservation strategies. Levels of variation at neutral markers have been useful for estimating local effective population sizes (N_e) and inferring whether population sizes increased or decreased over time. Furthermore, analyses of genotypic, allelic frequency, and phylogenetic information can potentially be used to separate historical from recent demographic changes. For 15 populations of Galápagos giant tortoises (Chelonoidis sp.), we used 12 microsatellite loci and DNA sequences from the mitochondrial control region and a nuclear intron, to reconstruct demographic history on shallow (past ~100 generations, ~2500 years) and deep (pre‐Holocene, >10 thousand years ago) timescales. At the deep timescale, three populations showed strong signals of growth, but with different magnitudes and timing, indicating different underlying causes. Furthermore, estimated historical N_e of populations across the archipelago showed no correlation with island age or size, underscoring the complexity of predicting demographic history a priori. At the shallow timescale, all populations carried some signature of a genetic bottleneck, and for 12 populations, point estimates of contemporary N_e were very small (i.e., < 50). On the basis of the comparison of these genetic estimates with published census size data, N_e generally represented ~0.16 of the census size. However, the variance in this ratio across populations was considerable. Overall, our data suggest that idiosyncratic and geographically localized forces shaped the demographic history of tortoise populations. Furthermore, from a conservation perspective, the separation of demographic events occurring on shallow versus deep timescales permits the identification of naturally rare versus newly rare populations; this distinction should facilitate prioritization of management action.     

Divalent metal transporter-1 decreases metal-related injury in the lung

Exposure to airborne particulates makes the detoxification of metals a continuous challenge for the lungs. Based on the fate of iron in airway epithelial cells, we postulated that divalent metal transporter-1 (DMT1) participates in detoxification of metal associated with air pollution particles. Homozygous Belgrade rats, which are functionally deficient in DMT1, exhibited diminished metal transport from the lower respiratory tract and greater lung injury than control littermates when exposed to oil fly ash. Preexposure of normal rats to iron in vivo increased expression of the isoform of DMT1 protein that lacked an iron-response element (-IRE), accelerated metal transport out of the lung, and decreased injury after particle exposure. In contrast, normal rats preexposed to vanadium showed less expression of the -IRE isoform of DMT1, decreased metal transport, and greater pulmonary injury after particle instillation. Respiratory epithelial cells in culture gave similar results. Also, DMT1 mRNA and protein expression for the -IRE isoform increased or decreased in these cells when exposed to iron or vanadium, respectively. These results thus demonstrate for the first time a primary role for DMT1 in lung metal transport and detoxification.