March 2006
Volume 5
|
March 2006
|
|
 |
|
Designing a Chemical Probe to Find a Molecular Target
Figure 1. Chemical structures of flavone-8-acetic acid (FAA) and azido-FAA. The blue circle denotes the locus of chemical substitution that still retains the full activity of the parent FAA molecule. Azido-FAA can undergo efficient coupling via a modified Staudinger ligation (Saxon E and Bertozzi CR. Science 287: 2007–10, 2000) with a FLAG peptide-phosphine tag under mild, biologically relevant conditions (pH 7.0–7.4, 25°C) to generate a peptide-tagged probe for locating and isolating proteins that bind to FAA. The red portions of the peptide-phosphine tag and the azido-FAA conjugate represent the FLAG peptide epitope used for immunorecognition. Numerous studies at the NCI and elsewhere have suggested that the antitumor activity of FAA is the result of indirect effects engaging the immune system and acting as a biological response modifier. NCI researchers found that FAA had very potent immunomodulatory activity against murine kidney tumors when combined with interleukin 2 (IL-2) (Mace KF et al. Cancer Res 50: 1742–7, 1990). These same researchers investigated the effect of FAA in mouse macrophage cell lines and found it to potently induce cytokines and interferons in primary splenocytes, including macrophages and T cells. Cytokines are thought to mediate an increase in natural killer cell activity and, through action on vascular endothelial cells, a reduction of tumor blood flow and the resultant onset of tumor necrosis. Indeed, one of the main effects of FAA was tumor necrosis factor-α (TNF-α)–mediated tumor vascular collapse followed by tumor necrosis. Species differences also apparently existed for the immunomodulatory action of FAA. In particular, FAA did not induce cytokines/interferons in human cells, and the NCI researchers who carried out this study hypothesized that the failed clinical trials were possibly due to the failure of FAA to activate cytokine genes in human cells (Futami H et al. Cancer Res 51: 6596–602, 1991). Since that time, FAA has been found to activate the transcription factor NFκB, which is critical for the transcription of many cytokine genes, and it has recently been confirmed to directly induce interferon. It was against this backdrop of much tantalizing information but no defined and confirmable molecular target for FAA that the interests of investigators in the Laboratory of Experimental Immunology and the Laboratory of Medicinal Chemistry converged. As part of a collaborative effort, we decided to design and synthesize a compound with a close structural resemblance to FAA that would be capable of capturing a protein target. To accomplish this goal, we wanted an FAA-like molecule that was modified with a functional group capable of reacting in a biological milieu with a molecular tag. We were aided in the task by the recent development of a rapid mouse macrophage tissue culture screen with which we could dissect the molecular structure of FAA and determine which chemical variations abrogated, maintained, or enhanced its cytokine-inducing activity. We found that FAA derivatives substituted in the para-position of the phenyl ring (Figure 1, blue circle) retained biological activity. We therefore designed the compound azido-FAA (Figure 1, X = N3), which uses an azide group as an affinity label that can be activated either by conventional chemistry or photochemically. When tested, azido-FAA possessed the same capacity as FAA to induce chemokine expression in a mouse macrophage cell line and induced an identical pattern of chemokine gene expression. This indicated that the azide group did not interfere with the activity of the parent FAA. In view of the technical difficulties expected with photochemical activation of our probe molecule, we investigated chemical approaches to reacting it with a specific peptide tag. Especially attractive was a reaction that allowed us to covalently trap our probe molecule with a reactive tag tethered to the amino terminus of a FLAG octapeptide (Figure 1, FLAG peptide-phosphine); this conjugate would then provide a way to fish out the drug-bound complex. This approach utilizing the Staudinger reaction was developed by Dr. Carolyn R. Bertozzi at University of California, Berkeley, who kindly provided us with the FLAG peptide-phosphine tag (Vocadlo DJ et al. Proc Natl Acad Sci U S A 100: 9116–21, 2003). We were able to demonstrate that azido-FAA could readily and efficiently couple with the FLAG peptide-phosphine tag in a nanoscale reaction under conditions that mimicked those of a potential biological experiment. High-resolution MALDI mass spectrometry was used to track the progress of this reaction and to unambiguously confirm the identity of the coupled product (Figure 1). Western blot analysis or immunoprecipitation with a mouse anti-FLAG antibody are possible strategies for detecting the drug-bound complex in the murine cellular systems of interest. Indeed, in partnership with the Laboratory of Proteomics and Analytical Technology (SAIC-Frederick, Inc.) at NCI-Frederick, we have shown that the conjugate can be mixed with extracts from mouse macrophage cells to interact with potential FAA-binding proteins expressed in these macrophages. The conjugate can also be used as a competitor in incubation with extracts of FAA- or azido-FAA–treated cells. Several FAA-FLAG conjugate–bound protein complexes have been immunoprecipitated using anti-FLAG antibody immobilized on protein G-beads, and the isolated proteins are being characterized with the help of the Laboratory of Proteomics and Analytical Technology in order to identify and validate the FAA molecular target. Our approach thus represents an important model for identifying the molecular targets of novel small molecules whose mechanism of action is unknown. |
 |
wo
decades ago, a synthetic flavonoid known as flavone-8-acetic acid 