Pharmaceutical drug misuse: are industry of employment and occupation risk factors?

We explore the misuse of pharmaceutical drugs in the Australian workforce, focusing on whether any differences exist between workers in particular industries or occupations. In terms of industry, being employed in hospitality is positively associated with pharmaceutical drug misuse, while being employed in finance, insurance and retail is inversely related. In terms of occupation, we find that being a labourer is positively related to misuse of pharmaceutical drugs, while being employed in managerial, professional, sales, clerical or administrative roles is associated with a lower tendency. Further analysis of occupational effects revealed that being in a blue-collar occupation, as a whole, is positively related to pharmaceutical drug misuse relative to white-collar employment. Moreover, being employed in higher status roles is associated with a lower likelihood of such behaviour. Our findings imply that particular workplace pressures, cultural norms and/or working conditions might be influential factors behind workers' drug misuse

Characterization and Exploitation of Bidirectional Allosteric Coupling in Multi-Domain Tyrosine Kinases using Conformation-Selective ATP-Competitive Inhibitors

Thesis (Ph.D.)--University of Washington, 2016-08Protein kinases are a large family of enzymes that play integral roles in cell signaling networks and are thus critical for effecting appropriate cellular responses to environmental stimuli. Much of kinase biological function has been studied in terms of catalytic activity: phosphorylation of substrate proteins as part of signaling cascades. However, recent evidence has shown that kinases play many important non-catalytic functions such as DNA-binding, scaffolding, and participating in a variety of physiologically relevant protein-protein interactions. While critical, these roles have not been thoroughly explored, in large part due to limited availability of selective ATP-competitive inhibitors. Selectivity for a specific kinase is difficult to achieve due to high structural homology between the ATP-binding sites of the 518 human kinases. Additionally, it has been shown over the past two decades that it is possible to stabilize structurally distinct ATP-binding site conformations using conformation-selective inhibitors, termed Type I and Type II inhibitors, in many kinases. In several cases, inhibition of kinase ATP-binding sites with Type I or Type II inhibitors has been shown to divergently affect cell signaling events as a result of allosteric coupling between important structural features in the ATP-binding site and distal protein-protein interaction sites on the inhibited kinase. Thus, it is important not only to build selective ATP-competitive inhibitors but to understand how their binding affects global kinase conformation through allosteric coupling. This thesis describes my work characterizing allosteric networks in multi-domain tyrosine kinases (Src-Family Kinases (SFKs) and Abl) using conformation-selective inhibitors as well as developing a method for using conformation-selective inhibitors in cells to better understand how non-catalytic function of a kinase of interest determines its role in cell signaling networks

Bioassay Development for Bispecific Antibodies—Challenges and Opportunities

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules

How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture

Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the glob-al conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix aC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix aC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix aC can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.</p