IDENTIFICATION OF ENDOGENOUS FUNCTION AND SUBSTRATE-DEPENDENT INTERACTIONS OF ORGANIC CATION TRANSPORTER 1

The human organic cation transporter 1 (hOCT1) is a polyspecific transporter, primarily expressed in the liver, which is known to interact with a large number of structurally dissimilar compounds. Several clinically-relevant drugs, as well as some endogenous compounds and other xenobiotics have been shown to be transported by or inhibit hOCT1. Due to its hepatic expression and general ADME function, hOCT1 has been implicated in adverse drug events (ADEs), including drug-drug interactions. As such, multiple regulatory agencies recommend including hOCT1, in pre-clinical transporter interaction studies. Limited structural information is available for hOCT1, and recently, endogenous functions and substrate-dependent effects have been identified for close relatives of hOCT1. Taken together, these suggest a need for further scrutiny of hOCT1 structure-activity relationships for development of critical drug-transporter interaction studies. The hypothesis was developed that both endogenous and xenobiotic compounds modulate the functional activity of hOCT1 in a substrate-dependent manner through interaction with specific, but perhaps distinct ligand-binding domains within the transporter. The hypothesis was tested via the following specific aims: 1) investigate the effect of xenobiotics on endogenous substrate transport by hOCT1, 2) identify and characterize substrate-dependent interactions with hOCT1, and 3) examine the role of the extracellular loop domain of hOCT1 in substrate affinity and translocation. In the first specific aim, dopamine and serotonin were identified as substrates for hOCT1. Serotonin proved to be a moderate-affinity substrate, while hOCT1 was able to transport it at high capacity. Several clinically-relevant drugs inhibited hOCT1-mediated serotonin transport, and these results were capitulated in primary human hepatocytes. Combined data from this inhibition screen and those previously published by other groups suggested the possibility of substrate-dependent effects. In specific aim two, substrate-dependent effects were screened for in a relatively new assay method, competitive counterflow (CCF). The CCF assay allowed for identification of novel substrates for hOCT1, including negatively-charged bromosulfophthalein (BSP). CCF results also identified numerous substrate-dependent effects which were explored further using computational (homology) modeling and ligand docking. Docking experiments identified three distinct binding sites within the hOCT1 homology model which explain several of the overserved substrate-dependent interactions, and supports previous claims that hOCT1 has a large substrate binding region versus a singular binding site and may be the reason for hOCT1’s polyspecificity. In the final specific aim, an attempt was made to generate human and rat OCT1 chimeric proteins. The goal of this study was to examine the role of the extracellular loop (ECL) domain in the observed differences in substrate affinity between species. Issues during the cloning process prevented the completion of this aim. However, had the chimeras successfully been generated, important information relating hOCT1 structure to its function could have been collected. This dissertation demonstrates that hOCT1 possesses important endogenous function and exhibits substrate-dependent effects, while also revealing important structural information relating to its function. Ultimately, this knowledge will be useful in improving pre-clinical trials for new drugs in the hope of identifying and preventing dangerous adverse drug events

Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children

Introduction: Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Methods: Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Results: Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Conclusions: Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27. The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607

Additional file 4: Table S4. of Differential expression of the Nrf2-linked genes in pediatric septic shock

List of Nrf2-linked gene probes (n =138) corresponding to 92 unique genes differentially regulated between septic shock endotypes A and B subjects. (XLSX 20 kb

Additional file 3: Table S3. of Differential expression of the Nrf2-linked genes in pediatric septic shock

List of network genes shown in Fig. 1. (XLSX 13 kb

Additional file 5: Table S5. of Differential expression of the Nrf2-linked genes in pediatric septic shock

List of network genes shown in Fig. 3. (XLSX 12 kb