422738 Systems-Level Assay of Phosphoproteins Using the Microwestern Array Elucidates the Mechanism of Action for Bioactive Natural Products

Wednesday, November 11, 2015: 10:00 AM
151D/E (Salt Palace Convention Center)
Mark Ciaccio and Neda Bagheri, Chemical & Biological Engineering, Northwestern University, Evanston, IL

Systems-level assay of phosphoproteins using the microwestern array elucidates the mechanism of action for bioactive natural products.  

Mark F. Ciaccio, Neda Bagheri. Department of Chemical & Biological Engineering, McCormick School of Engineering, Northwestern University.

 

Introduction: Technological advances in the collection and isolation of bioactive natural products have provided a rich treasure-trove of potential drug candidates. Compounds derived from natural sources such as aspirin, morphine, and penicillin have brought invaluable improvement to longevity and quality of life. While the untapped potential of natural products seems limitless, the number of compounds that successfully pass clinical trials remains prohibitively low. An efficient pipeline to identify the mechanism of action of drug candidates can (i) identify new purposes, (ii) mitigate potential side effects, and (iii) suggest chemical combinations to improve therapeutic potential. Here we take the interdisciplinary approach of combining a systems-level biological assay, termed the microwestern array, coupled with sparse regression, to discover the mechanism of action of naturally-derived small molecules in a cancer signaling network. Background: New technology that allows for direct quantification of protein abundance and phosphorylation states provides a particularly useful source of data as proteins are often the functional entities of cellular decision-making processes. Because the microwestern array incorporates the separation of proteins using electrophoresis, the sizes of proteins can be cross-referenced against molecular standards, eliminating much of the uncertainty that convolutes the quantification of proteins due to non-specific antibody-antigen binding common in antibody-based assays such as ELISA or reverse phase lysate arrays. Materials and Methods: A non-contact arrayer was used to spot 2 nL of cell lysate from drug-treated and control stimulations on to an acrylamide gel. Each sample was printed 96 times in the configuration of a 96-well plate. After electrophoresis, the samples were transferred on to a nitrocellose membrane, clamped in a multiwell gasket, and probed with 96 distinct antibodies. Fluorescence of the signals, corresponding to phosphorylation abundance, was quantified using a 700-nm infrared scanner. From the cue-signal response matrix, directed relationships between bioactive compounds and phosphorylation sites were inferred using partial least squares regression. Results and Discussion: We identified PTEN as a target of the common antioxidant, n-acetylcysteine. This suggests that this commonly-mutated protein may function as a sensor for the redox state of the cell and may be used to modulate cellular response to oxidative stress. In addition we identified 4EBP1 as a target negatively regulated by wortmannin suggesting that this natural product has pleiotropic effects beyond its use as a canonical PI3K inhibitor. Conclusions:  Use of the microwestern array technology to assay changes in phosphorylation levels after small-molecule perturbation proves to be a powerful new method to harvest the potential of bioactive natural products in drug discovery.

 

References:

[1] Harvey, Alan L., et al. "The re-emergence of natural products for drug discovery in the genomics era." Nat. Rev. Drug Discov. 14. (2015): 111–129.

[2] Ciaccio, Mark F., et al. "Systems analysis of EGF receptor signaling dynamics with microwestern arrays." Nat meth 7.2 (2010): 148-155.


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See more of this Session: High Throughput Technologies
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