Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Bioenergetic analysis of peroxisome proliferator-activated receptor gamma coactivators 1alpha and 1beta (PGC-1alpha and PGC-1beta) in muscle cells.

Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha is a coactivator of nuclear receptors and other transcription factors that regulates several components of energy metabolism, particularly certain aspects of adaptive thermogenesis in brown fat and skeletal muscle, hepatic gluconeogenesis, and fiber type switching in skeletal muscle. PGC-1alpha has been shown to induce mitochondrial biogenesis when expressed in muscle cells, and preliminary analysis has suggested that this molecule may specifically increase the fraction of uncoupled versus coupled respiration. In this paper, we have performed detailed bioenergetic analyses of the function of PGC-1alpha and its homolog PGC-1beta in muscle cells by monitoring simultaneously oxygen consumption and membrane potential. Cells expressing PGC-1alpha or PGC-1beta display higher proton leak rates at any given membrane potential than control cells. However, cells expressing PGC-1alpha have a higher proportion of their mitochondrial respiration linked to proton leak than cells expressing PGC-1beta. Although these two proteins cause a similar increase in the expression of many mitochondrial genes, PGC-1beta preferentially induces certain genes involved in the removal of reactive oxygen species, recently recognized as activators of uncoupling proteins. Together, these data indicate that PGC-1alpha and PGC-1beta profoundly alter mitochondrial metabolism and suggest that these proteins are likely to play different physiological functions.