Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and ß-Tubulin.

Photoaffinity labeling approaches have historically been used in pharmacology to identify molecular targets. This methodology has played a pivotal role in identifying drug-binding domains and searching for novel compounds that may interact at these domains. In this review we focus on studies of microtubule stabilizing agents of natural product origin, specifically taxol (paclitaxel). Taxol and other microtubule interacting agents bind to both P-glycoprotein (ABCB1), a drug efflux pump that reduces intracellular drug accumulation, and the tubulin/microtubule system. Both binding relationships modulate drug efficacy and are of immense interest to basic and translational scientists, primarily because of their association with drug resistance for this class of molecules. We present this body of work and acknowledge its value as fundamental to understanding the mechanisms of taxol and elucidation of the taxol pharmacophore. Furthermore, we highlight the ability to multiplex photoaffinity approaches with other technologies to further enhance our understanding of pharmacologic interactions at an atomic level. Thus, photoaffinity approaches offer a relatively inexpensive and robust technique that will continue to play an important role in drug discovery for the foreseeable future.

A label-free mass spectrometry method for relative quantitation of ß-tubulin isotype expression in human tumor tissue.

PURPOSE: Quantitation of ß-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of ß-tubulin isotypes. EXPERIMENTAL DESIGN: Using isotype-specific reporter peptides, we determined relative ß-tubulin isotype expression levels in human lung tumor tissue. RESULTS: Four reporter peptides were chosen to quantitate the ßI/ßII, ßIV, ßIII, and ßV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine ß-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low ßIII-tubulin expressing using immunohistochemistry. It was found that ßI/ßII (accounting for 18.7-65.7% of total ß-tubulin) and ßIVa/ßIVb (26.3-79.1%) were the most abundant isotypes and that the ßIII (0-8.9%) and ßV (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low ßIII-tubulin expressing. CONCLUSION AND CLINICAL RELEVANCE: With this method we can determine the relative expression levels of ß-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.