Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC 50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC 50 values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutic

Fourier Analysis of Gapped Time Series: Improved Estimates of Solar and Stellar Oscillation Parameters

Quantitative helio- and asteroseismology require very precise measurements of the frequencies, amplitudes, and lifetimes of the global modes of stellar oscillation. It is common knowledge that the precision of these measurements depends on the total length (T), quality, and completeness of the observations. Except in a few simple cases, the effect of gaps in the data on measurement precision is poorly understood, in particular in Fourier space where the convolution of the observable with the observation window introduces correlations between different frequencies. Here we describe and implement a rather general method to retrieve maximum likelihood estimates of the oscillation parameters, taking into account the proper statistics of the observations. Our fitting method applies in complex Fourier space and exploits the phase information. We consider both solar-like stochastic oscillations and long-lived harmonic oscillations, plus random noise. Using numerical simulations, we demonstrate the existence of cases for which our improved fitting method is less biased and has a greater precision than when the frequency correlations are ignored. This is especially true of low signal-to-noise solar-like oscillations. For example, we discuss a case where the precision on the mode frequency estimate is increased by a factor of five, for a duty cycle of 15%. In the case of long-lived sinusoidal oscillations, a proper treatment of the frequency correlations does not provide any significant improvement; nevertheless we confirm that the mode frequency can be measured from gapped data at a much better precision than the 1/T Rayleigh resolution.Comment: Accepted for publication in Solar Physics Topical Issue "Helioseismology, Asteroseismology, and MHD Connections

Experimental data on SOA formation from mixtures of anthropogenic and biogenic organic compounds

AbstractSecondary organic aerosols (SOA) constitute a significant fraction of the atmospheric particulate matter. Theses particles are formed as a consequence of the oxidation reaction of certain organic gases that leads to the formation of low-volatility compounds. As for other pollutants, air quality models allow the simulation of particle levels and thus models constitute a powerful tool in air quality management. Nevertheless, the accepted use of models must be based on the validation of its capacity to reproduce observed concentrations. Air monitoring sites provide measured information of a large variety of ambient pollutants. Unfortunately, measurements on SOA are not normally available, as current monitoring networks do not include instrumentation to distinguish primary from secondary sources of organic carbonaceous aerosol. This paper presents a set of photooxidation experiments performed in the European Photorreactor (EUPHORE) smog chamber (CEAM, Spain) under different experimental conditions to investigate SOA formation. The use of chambers allows the isolation of atmospheric chemistry and aerosol formation processes. Thus, although these measurements were obtained at initial precursor concentrations higher than those in atmospheric conditions, they constitute a valuable set of information for SOA model evaluation purposes

Structure–activity relationship study of beta-carboline derivatives as haspin kinase inhibitors

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure–activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented

Structure–activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors

Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC50 <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC50 <400 nM) with a 5.4-fold selectivity over haspin was also identified

Fusion of secretory vesicles isolated from rat liver

Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10–6 m. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells. Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system. The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72384/1/j.1365-2958.2001.02629.x.pd