The post-translational sulfation of tyrosine residues occurs in numerous secreted and integral membrane proteins and, in many cases, plays a crucial role in controlling the interactions of these proteins with physiological binding partners as well as invading pathogens. Recent advances in our understanding of protein tyrosine sulfation have come about owing to the cloning of two human tyrosylprotein sulfotransferases (TPST-1 and TPST-2), the development of novel analytical and synthetic methodologies and detailed studies of proteins and peptides containing sulfotyrosine residues. In this article, we describe the TPST enzymes, review the major techniques available for studying the presence, location and function of tyrosine sulfation in proteins and discuss the biological functions and biochemical interactions of several proteins (or protein families) in which tyrosine sulfation influences the protein function. In particular, we describe the detailed evidence supporting the importance of tyrosine sulfation in the cellular adhesion function of P-selectin glycoprotein ligand-1, the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the ligand binding and activation of other G-protein-coupled receptors by complement proteins, phospholipdis and glycoprotein hormones.

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

Substrate specificity in the aldo-keto reductase (AKR) superfamily is determined by three mobile loops positioned at the top of the canonical (α/β)(8)-barrel structure. These loops have previously been demonstrated to be modular in a well-studied class of AKRs, in that exchanging loops between two similar hydroxysteroid dehydrogenases resulted in a complete alteration of substrate specificity (Ma,H. and Penning,T.M. (1999) Proc. Natl Acad. Sci. USA, 96, 11161-11166). Here, we further examine the modularity of these loops by grafting those from human aldose reductase (hAR) into the hyperthermostable AKR, alcohol dehydrogenase D (AdhD), from Pyrococcus furiosus. Replacement of Loops A and B was sufficient to impart hAR activity into AdhD, and the resulting chimera retained the thermostability of the parent enzyme. However, no active chimeras were observed when the hAR loops were grafted into a previously engineered cofactor specificity mutant of AdhD, which displayed similar kinetics to hAR with the model substrate dl-glyceraldehyde. The non-additivity of these mutations suggests that efficient turnover is more dependent on the relative positioning of the cofactor and substrate in the active site than on binding of the individual species. The ability to impart the substrate specificities of mesostable AKRs into a thermostable scaffold will be useful in a variety of applications including immobilized enzyme systems for bioelectrocatalysis and fine chemical synthesis.

Objective Structured Clinical Examinations (OSCEs) are routinely used in healthcare education programs. Traditionally, students undertake OSCEs as face-to-face interactions to assess competency in soft skills. Due to physical distancing restrictions during COVID-19, alternative methods were required. This study utilized a mixed-method design (online survey and interviews) to evaluate second-year pharmacy students’ and examiners’ experiences of their first virtual OSCEs in Australia. A total of 196 students completed their first virtual OSCE in June 2020 of which 190 students completed the online survey. However, out of the 190 students, only 88% (n = 167) consented to the use of the data from their online survey. A further 10 students and 12 examiners were interviewed. Fifty-five students (33%) who participated in the online survey strongly agreed or agreed that they preferred the virtual experience to face-to-face OSCEs while 44% (n = 73) neither agreed nor disagreed. Only 20% (n = 33) felt more anxious with the virtual OSCEs. Additionally, thematic analysis found non-verbal communication as a barrier during the OSCE. Positive aspects about virtual OSCEs included flexibility, decreased levels of anxiety and relevance with emerging telehealth practice. The need for remote online delivery of assessments saw innovative ways of undertaking OSCEs and an opportunity to mimic telehealth. While students and examiners embraced the virtual OSCE process, face-to-face OSCEs were still considered important and irreplaceable. Future opportunities for OSCEs to be delivered both face-to-face and virtually should be considered.

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

No abstract

Pharmacists have a crucial role in the supply of medications and ensuring optimal patient outcomes. However, with the increased use of prescription medications, there is a potential for dispensing errors to occur. Some dispensing errors can result in patient harm, with some leading to death. The development of safe and accurate dispensing skills in pharmacy students is an essential part of the pharmacy curriculum to prevent such dispensing errors from occurring. A retrospective study was conducted on a virtual dispensing assessment completed by first-year pharmacy students using MyDispense at Monash University. Students were assessed on their ability to safely and accurately dispense four prescriptions. The students’ answers in the assessment were then analyzed using qualitative and quantitative methods. Errors in drug quantity, number of repeats, product, patient and prescriber selection were quantitatively analyzed. Through the development of a codebook, frequency of errors was determined for label directions and appropriate use of ancillary labels. In this study, the dispensing errors that were identified depended on the class of medication. Errors in label directions were most common, with the majority of errors displaying incorrect route of administration, drug formulation and/or frequency of dosing. Identified errors were then further categorized into potential severity of harm, ranging from “no harm” to “severe harm”. The findings from this study show the types of errors made by students that are preventable and the potential for first-year pharmacy students to benefit from more comprehensive introductions to dispensing guides and safe environments to practice.