Structure-activity relationships, and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of ROCK-II

ROCK has been implicated in many diseases ranging from glaucoma to spinal cord injury and is therefore an important target for therapeutic intervention. In this study, we have designed a series of 1-(4-(1H-indazol-5-yl)piperazin-1-yl)-2-hydroxy(or 2-amino) analogs and a series of 1-(4-(1H-indazol-5-yl amino)piperidin-1-yl)-2-hydroxy(or 2-amino) inhibitors of ROCK-II. SR-1459 has IC(50)=13nM versus ROCK-II while the IC(50)s for SR-715 and SR-899 are 80nM and 100nM, respectively. Many of these inhibitors, especially the 2-amino substituted analogs for both series, are modest/potent CYP3A4 inhibitors as well. However, a few of these inhibitors (SR-715 and SR-899) show strong selectivity for ROCK-II over CYP3A4, but the overall potency of the 2-amino analogs (SR-1459) on CYP3A4 and the high clearance and volume of distribution of these compounds makes the in vivo utility of these analogs undesirable.     

Sample entropy analysis of neonatal heart rate variability

Abnormal heart rate characteristics of reduced variability and transient decelerations are present early in the course of neonatal sepsis. To investigate the dynamics, we calculated sample entropy, a similar but less biased measure than the popular approximate entropy. Both calculate the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point. We studied 89 consecutive admissions to a tertiary care neonatal intensive care unit, among whom there were 21 episodes of sepsis, and we performed numerical simulations. We addressed the fundamental issues of optimal selection of m and r and the impact of missing data. The major findings are that entropy falls before clinical signs of neonatal sepsis and that missing points are well tolerated. The major mechanism, surprisingly, is unrelated to the regularity of the data: entropy estimates inevitably fall in any record with spikes. We propose more informed selection of parameters and reexamination of studies where approximate entropy was interpreted solely as a regularity measure.     

Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells

Objectives

Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.

Methods

MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.

Results and Conclusions

We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.     

Effects of DCPA,EPTC, and Chlorpropham on Pathogenicity of Meloidogyne hapla to Alfalfa

Treatments wilh the herbicides chlorpropham (isopropyl m-chlorocarbinilate), DCPA (dimethyl tetrachloroterephthalate), and El''TC (S-ethyl dipropylthiocarbamate), alone or in combination with Meloidogyne hapla Chilwood, significantly reduced the growth of both nemalode-resistant ''Nev Syn XX'' atttt susceptible ''Ranger" alfalfa (Medicago saliva L.) seedlings. M. hapla infection of both alfalfas was reduced by all herbicides because of fewer available infective courts in the treated plants. EPTC, however, reduced resistance to M. hapla, as indicated by increased galling of ''Nev Syn XX'' plants.     

Benzimidazole- and benzoxazole-based inhibitors of Rho kinase

Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral bioavailability.     

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.     

Phototrophs in High-Iron-Concentration Microbial Mats: Physiological Ecology of Phototrophs in an Iron-Depositing Hot Spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [¹⁴C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.