Herein, we tested this hypothesis with multiple compound sets and in different cell types. We, therefore, hypothesized that F ic might be a practical metric for predicting compound access to intracellular targets. In a preliminary study with a single cytosolic target (thymidylate synthase), we showed that intracellular concentrations of active hits and reference drugs correlate with intracellular target engagement ( 31). Intracellular fraction of unbound compound (f u,cell) measured after dialysis and intracellular compound accumulation (Kp) are quantified using LC-MS/MS and combined to give F ic. ( B) General protocol for measurement of intracellular bioavailability (F ic). After engaging the target, the compound elicits a functional response, leading, in turn, to a phenotypic response. In cellular assays, only the fraction of compound that is not bound to proteins in cell culture medium (f u,medium) or cellular components (f u,cell) is available for binding to an intracellular target this fraction is the intracellularly bioavailable fraction (F ic). ( A) In biochemical assays, the compound is directly available to engage the target. 1 A).Ī comparison of biochemical and cellular assays used in drug discovery and measurement of intracellular bioavailability (F ic). Importantly, it is this unbound fraction of the dosed drug that is available for interactions in the intracellular environment ( Fig. Our method does not require chemical labeling, has a high sensitivity, and measures the unbound drug concentration. Therefore, we developed a cell-based methodology for determination of intracellular drug concentrations in a high-throughput format, which is applicable to a wide variety of cell systems, not only those directly relevant for the target pharmacology ( 28 – 30). Tissue-based methodologies allow accurate determinations of intracellular drug concentrations using unlabeled compounds, but they are based on animal tissues and have low throughput ( 26, 27). Labeling often alters the molecular properties of the compound, thereby perturbing its distribution and target affinity. Qualitative measurements of the cellular distribution of compounds can be obtained with various imaging modalities ( 19 – 25) however, most of these techniques require labeling of the compound of interest. Furthermore, exposure can only be detected in cell types that express the target, limiting their use when studying off-target effects and drug toxicity. Some of these approaches are limited to certain target classes, because they require probes that are known to bind the target ( 13 – 16, 18) or target properties to allow detection. Importantly, routine measurement of compound levels at intracellular sites in target tissues is hindered by sampling constraints in human subjects ( 5).Ĭurrently, indirect estimates of intracellular drug levels are extrapolated from transcellular permeability experiments ( 7) or cellular target engagement data ( 8 – 17). In a recent analysis of the drug candidate pipeline of a major drug company, all programs in which target exposure was uncertain (18 of 44 programs) resulted in failure of progression to phase III clinical trials ( 4). Furthermore, insufficient exposure at the target is an important contributor to failure in clinical drug development and the high attrition rate in drug discovery programs ( 4 – 6). Inadequate cellular drug exposure is hypothesized to lead to a lower “biochemical efficiency” ( 3), typified by compounds that bind to the isolated target protein with high affinity but fail to perform in cellular assays, a phenomenon termed “cell drop off” ( 2). Most known drug targets are located in the cell interior ( 1, 2).
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