Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.     

Kinesin spindle protein (KSP) inhibitors. Part 3: synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles with reduced hERG binding and employment of a phosphate prodrug strategy for aqueous solubility

2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.     

Kinesin spindle protein (KSP) inhibitors. Part 2: the design, synthesis, and characterization of 2,4-diaryl-2,5-dihydropyrrole inhibitors of the mitotic kinesin KSP

The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.     

APRIN is a unique Pds5 paralog with features of a chromatin regulator in hormonal differentiation

Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in the Pds5 family). Our results suggest that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. The results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.     

Wirkung von Hypophysenhormonen auf den Farbwechsel einiger Adriafische

Zusammenfassung Bei einer Reihe von Adriafischen (Serranus scriba, Maena zebra, Tripterygium nasus, Crenilabrus pavo und Coris julis) ruft Injektion von H.H.L.-Hormonpräparaten lebhaftere Färbung hervor, die durch Expansion der Erythrophoren, Xanthophoren und Kyanophoren bedingt ist. Die Farbeffekte, die dabei auftreten, gleichen den zur Laichzeit spontan auftretenden. Es dürfte sich dabei um den bisher nur an Plioxinus laevis beobachteten, als Intermedinreaktion bekannten, Effekt handeln.     

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.     

Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.     

Diagnostic Accuracy of Recombinant Immunoglobulin-like Protein A-Based IgM ELISA for the Early Diagnosis of Leptospirosis in the Philippines

BackgroundLeptospirosis is an important but largely under-recognized public health problem in the tropics. Establishment of highly sensitive and specific laboratory diagnosis is essential to reveal the magnitude of problem and to improve treatment. This study aimed to evaluate the diagnostic accuracy of a recombinant LigA protein based IgM ELISA during outbreaks in the clinical-setting of a highly endemic country.Conclusions/SignificanceThe newly developed LigA-IgM ELISA is more sensitive than the whole cell-based IgM based ELISA. Although the final diagnosis must be validated by more specific tests, LigA-IgM ELISA could be a useful diagnostic test in a real clinical-setting, where diagnosis is needed in the early phase of infection.     

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.