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

Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism

Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismatepyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin. Compounds identified in the screen are high nanomolar to low micromolar inhibitors of the enzyme and produce growth inhibition in PAO1 P. aeruginosa in the millimolar range under iron-limiting conditions. The identified compounds were also tested for enzymatic inhibition of E. coli chorismate mutase, a protein of similar fold and similar chemistry, and of Y. enterocolitica salicylate synthase, a protein of differing fold but catalyzing the same lyase reaction. In both cases, subsets of the inhibitors from the screen were found to be inhibitory to enzymatic activity (mutase or synthase) in the micromolar range and capable of growth inhibition in their respective organisms (E. coli or Y. enterocolitica)

Gluon Polarization from QCD Sum Rules

The gluon polarization $\Delta G$ in a nucleon can be defined in a gauge invariant way as the integral over the Ioffe-time distribution of polarized gluons. We argue that for sufficiently regular polarized gluon distributions $\Delta G$ is dominated by contributions from small and moderate values of the Ioffe-time z < 10. As a consequence $\Delta G$ can be estimated with 20% accuracy from the first two even moments of the polarized gluon distribution, and its behavior at small values of Bjorken x or, equivalently, at large Ioffe-times z. We employ this idea and compute the first two moments of the polarized gluon distribution within the framework of QCD sum rules. Combined with the color coherence hypothesis we obtain an upper limit for $\Delta G \sim 2 \pm 0.5$ at a typical scale $\mu^2 \sim 1 GeV^2$.Comment: 12 pages, Latex, 2 figures include

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening

Phosphatidylcholine transfer protein (PC–TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC–TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC–TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC–TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC–TP activity, with IC50 values that ranged from 4.1 to 95.0 ?M under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC–TP. Because mice with homozygous disruption of the PC–TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases

QCD Sum Rule Calculation of Twist-3 Contributions to Polarized Nucleon Structure Functions

Using the framework of QCD sum rules we predict the twist-3 contribution to the second moment of the polarized nucleon structure function $g_2(x)$. As the relevant local operator depends explicitely on the gluon field, we employ a recently studied interpolating nucleon current which contains three quark field and one gluon field operator. Despite the fact that our calculation is based on the analysis of a completely different correlation function, our estimates are consitent with those of Balitsky, Braun and Kolesnichenko who used a three-quark current.Comment: 16pp. , 2 figures (uuencoded eps-files), LateX. Some misprints corrected, results unchange

The Escape Problem for Irreversible Systems

The problem of noise-induced escape from a metastable state arises in physics, chemistry, biology, systems engineering, and other areas. The problem is well understood when the underlying dynamics of the system obey detailed balance. When this assumption fails many of the results of classical transition-rate theory no longer apply, and no general method exists for computing the weak-noise asymptotics of fundamental quantities such as the mean escape time. In this paper we present a general technique for analysing the weak-noise limit of a wide range of stochastically perturbed continuous-time nonlinear dynamical systems. We simplify the original problem, which involves solving a partial differential equation, into one in which only ordinary differential equations need be solved. This allows us to resolve some old issues for the case when detailed balance holds. When it does not hold, we show how the formula for the mean escape time asymptotics depends on the dynamics of the system along the most probable escape path. We also present new results on short-time behavior and discuss the possibility of focusing along the escape path.Comment: 24 pages, APS revtex macros (version 2.1) now available from PBB via `get oldrevtex.sty

Structure–activity relationship study of EphB3 receptor tyrosine kinase inhibitors

A structure–activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure–activity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also profiled for inhibitory activity against a panel of 288 kinases and found to be quite selective for tyrosine kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and potentially in vivo kinase-dependent function of EphB3 receptor

A pilot Internet "Value of Health" Panel: recruitment, participation and compliance

Objectives To pilot using a panel of members of the public to provide preference data via the Internet Methods A stratified random sample of members of the general public was recruited and familiarised with the standard gamble procedure using an Internet based tool. Health states were perdiodically presented in "sets" corresponding to different conditions, during the study. The following were described: Recruitment (proportion of people approached who were trained); Participation (a) the proportion of people trained who provided any preferences and (b) the proportion of panel members who contributed to each "set" of values; and Compliance (the proportion, per participant, of preference tasks which were completed). The influence of covariates on these outcomes was investigated using univariate and multivariate analyses. Results A panel of 112 people was recruited. 23% of those approached (n = 5,320) responded to the invitation, and 24% of respondents (n = 1,215) were willing to participate (net = 5.5%). However, eventual recruitment rates, following training, were low (2.1% of those approached). Recruitment from areas of high socioeconomic deprivation and among ethnic minority communities was low. Eighteen sets of health state descriptions were considered over 14 months. 74% of panel members carried out at least one valuation task. People from areas of higher socioeconomic deprivation and unmarried people were less likely to participate. An average of 41% of panel members expressed preferences on each set of descriptions. Compliance ranged from 3% to 100%. Conclusion It is feasible to establish a panel of members of the general public to express preferences on a wide range of health state descriptions using the Internet, although differential recruitment and attrition are important challenges. Particular attention to recruitment and retention in areas of high socioeconomic deprivation and among ethnic minority communities is necessary. Nevertheless, the panel approach to preference measurement using the Internet offers the potential to provide specific utility data in a responsive manner for use in economic evaluations and to address some of the outstanding methodological uncertainties in this field

Explaining Evidence Denial as Motivated Pragmatically Rational Epistemic Irrationality

This paper introduces a model for evidence denial that explains this behavior as a manifestation of rationality and it is based on the contention that social values (measurable as utilities) often underwrite these sorts of responses. Moreover, it is contended that the value associated with group membership in particular can override epistemic reason when the expected utility of a belief or belief system is great. However, it is also true that it appears to be the case that it is still possible for such unreasonable believers to reverse this sort of dogmatism and to change their beliefs in a way that is epistemically rational. The conjecture made here is that we should expect this to happen only when the expected utility of the beliefs in question dips below a threshold where the utility value of continued dogmatism and the associated group membership is no longer sufficient to motivate defusing the counter-evidence that tells against such epistemically irrational beliefs

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