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Targeted Degradation of the Aryl Hydrocarbon Receptor by the PROTAC Approach: A Useful Chemical Genetic Tool

In recent years, small molecules have been a major contributor to the investigation of many biological processes. Given that biologically active small molecules often exert their activities by inhibition of specific proteins, they provide valuable molecular probes for studying the role of targeted proteins in complex signaling pathways. Thus, this small-molecule or “chemical-genetics” approach, which easily affords more temporal and spatial control of targeted biological events than the classical genetic approach, is complementary to the conventional genetic approach. Recently, an exciting chemical genetic approach, which was designed to induce degradation of targeted proteins, was developed by Deshaies, Crews, and colleagues, and has been appropriately named PROTAC (PROteolysis TArgeting Chimera). PROTACs are chimeric small molecules that target proteins of interest for degradation by the intracellular ubiquitin–proteasome pathway (Figure 1). The PROTAC molecule is comprised of a specific E3 ubiquitin-ligase recognition motif linked to a ligand that binds to the targeted protein, and recruits it to the specific E3-ligase complex for multiubiquitination and subsequent degradation by the 26S proteasome. Unlike conventional bioactive small molecules that inhibit protein function by direct or indirect interactions with the targeted protein, PROTAC molecules inhibit protein function by elimination (rather than by simple “inhibition” of protein function) ; thereby they provide an alternative strategy for protein modulation. Thus far, several PROTACs, mostly aimed at nuclear receptors, have been successfully developed. However, despite its great potential, the widespread applications of PROTACs are yet to be fully realized, due largely to a limited number of protein–ligand pairs. Recently, a similar small-molecule approach based on the ability of the natural product geldanamycin to inhibit heat shock protein (HSP)-90 has been reported. This approach induces degradation of nuclear receptors that interact with HSP-90 (e.g. , ER and AR). However, this geldanamycin-based protein degradation system appears to be limited to proteins associated with HSP-90. Although the details specific to the mechanism of action of these chimeric molecules are still inconspicuous, this work can offer an option to specifically degrade certain HSP-associated proteins. Biologically active natural products have been increasingly employed as molecular probes in the investigation of biological pathways. 14, 15] Most of these biologically active natural products exert their action by direct interaction with specific protein targets. Thus, they provide valuable tools for the design of novel PROTAC molecules, as they present biologically prevalidated protein–ligand pairs. This natural product-based PROTAC design approach has been elegantly illustrated by successful development of antiangiogenic natural product fumagillin-based PROTAC that induces degradation of a major intracellular target of fumagillin, methionine aminopeptidase (MetAP)-2, which had not previously been characterized as a natural substrate of the 26S proteasome. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic and carcinogenic effects of environmental carcinogens, such as polyACHTUNGTRENNUNGcyclic and halogenated aromatic hydrocarbons. 17] The role of AHR in chemical carcinogenesis involves binding of the ligand (i.e. , a polycyclic aromatic hydrocarbon) to the receptor and initiation of events that include induction of xenobiotic-metabolizing enzymes, such as cytochrome P450 isoform 1A1, and subsequent generation of DNA adducts. In addition to playing an important role in the initiation stage of carcinogenesis, increasing evidence supports the idea that ligand activation of AHR facilitates tumor development and progression. However, despite increasing attention to AHR-associated physiological disorders, detailed description of the role of AHR in these important pathological processes is yet to be clearly investigated. Currently, the major problem that limits further understanding of the functions of the AHR is the lack of appropriate molecular probes that modulate AHR function in cultured cells and in in vivo models. Figure 1. PROTAC recruits the target protein to a specific E3 ubiquitin ligase for ubiquitination; this is followed by degradation by the 26S proteasome.