Carol Ann Homon

Three Mechanistically Distinct Kinase Assays Compared: Measurement of Intrinsic ATPase Activity Identified the Most Comprehensive Set of ITK Inhibitors:

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)–compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.

Hit to lead account of the discovery of a new class of inhibitors of Pim kinases and crystallographic studies revealing an unusual kinase binding mode.

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.

The role of 5-lipoxygenase products in preclinical models of asthma

BACKGROUND The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. METHODS Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. RESULTS In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. CONCLUSIONS In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

Relationships Among Cytokines (IL-1, TNF, and IL-8) and Histologic Markers of Acute Ascending Intrauterine Infection

Amniotic fluid levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) were determined in 13 pregnancies presenting with preterm spontaneous membrane rupture in which there were no clinical indications of intraamniotic infection. These levels were compared to the placental histology. All 8 cases in which elevated cytokine levels were identified also had histologic evidence of both maternal and fetal acute inflammation in choriodecidua and chorionic plate, and umbilical and chorionic vessels, respectively. Cytokine concentrations correlated with the severity of maternal inflammation, but not the severity of fetal inflammation as assessed histologically. Elevated levels of both IL-1β and TNF-α were not uniformly observed in amniotic fluid; all cases with either elevated IL-1β of TNF-α had elevated levels of IL-8. Microbial studies of the 8 patients with histologic acute inflammation showed 5 with positive amniotic fluid cultures and/or gram stains. One had positive gram s...

Assay Optimization: A Statistical Design of Experiments Approach

With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. With the use of an integrated approach called automated assay optimization developed in collaboration with Beckman Coulter (Fullerton, CA), the process of conducting these experiments has been greatly facilitated. This approach imports an experimental design from a commercial statistical package and converts it into robotic methods. The data from these experiments are fed back into the statistical package and analyzed, resulting in empirical models for determining optimum assay conditions. The optimized assays are then progressed into HTS. This tutorial will focus on the use of statistically designed experiments in assay optimization.

Radioimmunoassay for clonidine in human plasma and urine using a solid phase second antibody separation.

A reliable, sensitive, and specific radioimmunoassay (RIA) procedure for the quantitation of clonidine in plasma and other biological fluids was developed. The detection limit of the assay is 2 pg based on a 200 microliters sample. Nine commonly used drugs were found not to interfere with the RIA. The utility of the assay was demonstrated in a bioavailability study of clonidine conducted with 24 healthy subjects. Clonidine was readily quantitated in plasma over 4 half-lives. This assay is suitable for pharmacokinetic and bioavailability studies as well as therapeutic drug monitoring of patients.

Glossary of terms used in biomolecular screening (IUPAC Recommendations 2011)

Biomolecular screening is now a crucial component of the drug discovery process, and this glossary will be of use to practitioners in the field of screening and to those who interact with the screening community. The glossary contains definitions related to various aspects of the screening process such as assay types, data handling, and relevant technologies. Many of the terms used in this discipline are not covered by existing glossaries, and where they are, the definitions are often not appropriate for this field. Where appropriate, this document provides new or modified definitions to better reflect the new context. The field of biomolecular screening is multidisciplinary in nature, and this glossary, containing authoritative definitions, will be useful not only for regular practitioners, but also for those who make use of data generated during the screening process.

A selective radioimmunoassay for the determination of pirenzepine in plasma and urine.

A radioimmunoassay procedure for the determination of pirenzepine in either plasma or urine was demonstrated to be both sensitive and specific as well as highly reproducible. The assay could detect levels as low as 1.25 ng/ml. The sensitivity was sufficient to allow the analysis of biological samples from both pharmacokinetic and clinical studies. The two metabolites of pirenzepine, LS 75 and LS 822, did not cross-react with the antiserum. The assay was not affected by a change in anticoagulant or by the presence of several over-the-counter or prescription drugs, even at very high levels. Samples could be frozen and stored for at least a year without affecting the analysis. Repeat analysis could be performed on samples that had been re-frozen. Several thousand plasma and urine samples, including plasma samples from severely renally impaired patients, have been analyzed for pirenzepine by the RIA with no interferences having been detected.

Generation of leukotrienes by antigen and calcium lonophore A23187 stimuli from a mouse mastocytoma cell line, MMC-16

The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.

Discovery of BIRM 270: A New Class of Leukotriene Biosynthesis Inhibitors

Several events in the late 1970s and early 1980s helped to shape the research agenda for pharmaceutical companies engaged in the search for a new generation of anti-inflammatory agents. The structural identity of the arachi- donic acid (AA) metabolite leukotriene B4 (LTB4) (Borgeat and Samuels- son, 1979) and the discovery that it was a potent neutrophil chemoattractant (Ford-Hutchinson et al., 1980) helped to elucidate how leukocytes migrate to a site of inflammation. In addition, the identification of leukotriene C4 (LTC4) as a component of slow reacting substance of anaphylaxis (SRS- A) demonstrated that arachidonic acid-derived substances were also potent bronchoconstrictors (Murphy and Hammarstrom, 1979; Corey et al., 1980; Sirois and Borgeat, 1980). Common to both classes of leukotrienes is the enzyme 5-lipoxygenase (5-LO) which introduces a molecule of oxygen at the C-5 position of AA followed by cyclization to the epoxide intermediate ITA4. Further reaction with a glutathione S-transferase yields the peptidyl LTs, LTC4 and its metabolites LTD4 and LTE4. Conversion of the epoxide by LTA4 hydrolase produces LTB4. Given that AA also serves as a substrate for the cyclooxygenase (CO) pathway to produce pro-inflammatory prostaglandins and thromboxane, interest was certainly reinforced in inhibiting one or both arms of what is often called the arachidonic acid cascade.