Synthesis and biological evaluation of a water-soluble phosphate prodrug salt and structural analogues of KGP94, a lead inhibitor of cathepsin L

The magnitude of expression of cathepsin L, often upregulated in the tumor microenvironment, correlates with the invasive and metastatic nature of certain tumors. Inhibition of cathepsin L represents an emerging strategy for the treatment of metastatic cancer. A potent, small-molecule inhibitor (referred to as KGP94) of cathepsin L, and new KGP94 analogues were synthesized. (3,5-Dibromophenyl)-(3-hydroxyphenyl) ketone thiosemicarbazone (22), with an IC₅₀ value of 202 nM, exhibited similar inhibitory activity against cathepsin L compared to KGP94 (IC₅₀ = 189 nM). Due to limited aqueous solubility of KGP94, a water-soluble phosphate salt (KGP420) was prepared in order to facilitate future in vivo studies. Enzymatic hydrolysis with alkaline phosphatase (ALP) demonstrated that the phosphate prodrug, KGP420, was readily converted to the parent compound, KGP94.     

Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees

Background  

Tropical rain forests are the most diverse terrestrial ecosystems on the planet. How this diversity evolved remains largely unexplained. In Africa, rain forests are situated in two geographically isolated regions: the West-Central Guineo-Congolian region and the coastal and montane regions of East Africa. These regions have strong floristic affinities with each other, suggesting a former connection via an Eocene pan-African rain forest. High levels of endemism observed in both regions have been hypothesized to be the result of either 1) a single break-up followed by a long isolation or 2) multiple fragmentation and reconnection since the Oligocene. To test these hypotheses the evolutionary history of endemic taxa within a rain forest restricted African lineage of the plant family Annonaceae was studied. Molecular phylogenies and divergence dates were estimated using a Bayesian relaxed uncorrelated molecular clock assumption accounting for both calibration and phylogenetic uncertainties.     

Identifying the important HIV-1 recombination breakpoints

Recombinant HIV-1 genomes contribute significantly to the diversity of variants within the HIV/AIDS pandemic. It is assumed that some of these mosaic genomes may have novel properties that have led to their prevalence, particularly in the case of the circulating recombinant forms (CRFs). In regions of the HIV-1 genome where recombination has a tendency to convey a selective advantage to the virus, we predict that the distribution of breakpoints--the identifiable boundaries that delimit the mosaic structure--will deviate from the underlying null distribution. To test this hypothesis, we generate a probabilistic model of HIV-1 copy-choice recombination and compare the predicted breakpoint distribution to the distribution from the HIV/AIDS pandemic. Across much of the HIV-1 genome, we find that the observed frequencies of inter-subtype recombination are predicted accurately by our model. This observation strongly indicates that in these regions a probabilistic model, dependent on local sequence identity, is sufficient to explain breakpoint locations. In regions where there is a significant over- (either side of the env gene) or under- (short regions within gag, pol, and most of env) representation of breakpoints, we infer natural selection to be influencing the recombination pattern. The paucity of recombination breakpoints within most of the envelope gene indicates that recombinants generated in this region are less likely to be successful. The breakpoints at a higher frequency than predicted by our model are approximately at either side of env, indicating increased selection for these recombinants as a consequence of this region, or at least part of it, having a tendency to be recombined as an entire unit. Our findings thus provide the first clear indication of the existence of a specific portion of the genome that deviates from a probabilistic null model for recombination. This suggests that, despite the wide diversity of recombinant forms seen in the viral population, only a minority of recombination events appear to be of significance to the evolution of HIV-1.     

Genomic organization and chromosomal localization of a new repeat MS7, specific for heterochromatin of sex chromosomes in Microtus species voles of the arvalis group

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis [1, 2]. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Synthesis,in vitro,and in vivo evaluation of phosphate ester derivatives of combretastatin A-4

Combretastatin A-4 disodiumphosphate (CA4P), a prodrug formulation of the natural product combretastatin A-4 (CA4), is currently in clinical investigation for the treatment of cancer. In vivo, CA4P is rapidly enzymatically converted to CA4, a potent inhibitor of tubulin polymerization (IC(50)=1-2 microM), and rapidly causes bloodflow shutdown in tumor tissues. A variety of alkyl and aryl di- and triesters of CA4P have been synthesized and evaluated as potential CA4 prodrugs and/or stable CA4P analogues.     

Synthesis and biological evaluation of 2-(4-fluorophenoxy)-2-phenyl-ethyl piperazines as serotonin-selective reuptake inhibitors with a potentially improved adverse reaction profile

Three new 2-(4-fluorophenoxy)-2-phenyl-ethyl piperazines, 1-(3-chlorophenyl)-4-[2-(4-fluorophenoxy)-2-phenylethyl]-piperazine 7, 1-[2-(4-fluorophenoxy)-2-phenylethyl]-4-(2-methoxyphenyl)-piperazine 8, and 1-[2-(4-fluorophenoxy)-2-phenylethyl]-4-(3-trifluoromethylphenyl)-piperazine 9, modeled after the potent antidepressant fluoxetine and coupled with several functionalized piperazines, have been prepared by chemical synthesis as selective serotonin reuptake inhibitors (SSRIs) with a potentially improved adverse reaction profile. Typical SSRIs, although very effective in the treatment of depression, still face the troublesome side effect of sexual dysfunction. A number of pharmacological agents-notably, drugs in the piperazine class-have been used to reverse SSRI-induced sexual dysfunction, and evidence for developing an improved SSRI by coupling a fluoxetine congener with the pharmacophore of a reversal agent holds promise. Preliminary data indicates that the hydrochloride (HCl) salts 10, 11, and 12 each exhibit single-site binding at the site of the serotonin reuptake transporter (SERT). However, each of the three compounds are much less potent than typical SSRIs, showing micromolar (microM) affinity for the SERT with IC(50) values of 1.45 microM, 3.27 microM, and 9.56 microM, respectively. Further biological evaluation of compounds 10, 11, and 12 is needed before definitive conclusions can be made with regard to each compound's potential for use as an SSRI-type candidate which is devoid of sexual side effects. Nevertheless, the initial findings are quite encouraging, thus lending credence to the idea of hybridizing an SSRI congener with that of the pharmacophore of an agent known to reverse or treat SSRI-induced sexual dysfunction.     

Individual and geographical variation in display behaviour of male harbour seals in Scotland

Studying variations in behaviour at the individual or population level enables insight into the reproductive strategies within a species. We examined individual and geographical variation in the vocal and dive behaviour of male harbour seals, Phoca vitulina, which is associated with aquatic mating. This display behaviour was recorded in the Moray Firth, Scotland, from July 1994 to 1997, and in Orkney, Scotland, during July 1998. One vocalization type was apparent in the Moray Firth and two in Orkney. Time parameters (total and pulse duration) varied between males in the population in the Moray Firth. We used both frequency and time parameters in a discriminant analysis, which showed that 73.2% of individual male vocalizations could be correctly classified; 94.6% of male vocalizations from the Moray Firth and Orkney could be correctly classified according to their geographical areas. Therefore, vocal variation was greater between geographical areas than between individuals. No individual variation was apparent between dive and surface interval durations. However, individuals varied significantly in the percentage of short surface intervals. Male harbour seals showed substantial variability in the parameters affecting their vocal and dive behaviour during the mating season. We suggest that these variations may be indicative of adaptations to varying environmental challenges influencing the reproductive strategies of discrete populations. Copyright 2000 The Association for the Study of Animal Behaviour.     

Metabolic reconstruction and analysis for parasite genomes

With the completion of sequencing projects for several parasite genomes, efforts are ongoing to make sense of this mass of information in terms of the gene products encoded and their interactions in the growth, development and survival of parasites. The emerging science of systems biology aims to explain the complex relationship between genotype and phenotype by using network models. One area in which this approach has been particularly successful is in the modeling of metabolism. With an accurate picture of the set of metabolic reactions encoded in a genome, it is now possible to identify enzymes or transporters that might be viable targets for new drugs. Because these predictions greatly depend on the quality and completeness of the genome annotation, there are substantial efforts in the scientific community to increase the numbers of metabolic enzymes identified. In this review, we discuss the opportunities for using metabolic reconstruction and analysis tools in parasitology research, and their applications to protozoan parasites.     

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fcγ receptors (FcγR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil FcγRs were examined with a specific FcγR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of FcγRIIa and FcγRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative FcγR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.