Targeted protein degradation (TPD) is an attractive strategy for drug discovery by eliminating a protein of interest (Zhao et al., 2022). Proteolysis-targeting chimeras (PROTAC) (Ruffilli et al., 2022) and molecular-glues (Sasso et al., 2023) are two major TPD technologies to induce E3-ligase-dependent degradation of protein targets in proteasomes. While PROTACs are heterobifunctional containing ligands for both E3 ligase and protein target with a flexible linker, molecular glues are simple chemicals without a linker but also interact with E3 ligase or protein targets to induce or enhance their binding and degradation of the target. In essence, their mechanisms of action are similar.

TPD is gaining rapid traction since its discovery with 48 TPD agents in clinical testing (Fang et al., 2023, Ruffilli et al., 2022). However, most of these studies were on intracellular/soluble proteins and little progress has been made on membrane proteins although technologies including LYTAC by taking advantage of lysosome receptor and targeting a membrane protein with a small molecule−antibody conjugate and AbTAC that uses a bivalent antibody for membrane protein targets and the membrane resident E3 ligase have recently been developed to induce membrane protein degradation in lysosomes (Ruffilli et al., 2022, Sasso et al., 2023). Of 48 TPD agents in clinical trials, only two target membrane proteins including EGFR and HER2 (Fang et al., 2023, Ruffilli et al., 2022). Furthermore, the majority of these agents in clinical trials target classically drugged proteins such as androgen/estrogen receptors with readily available chemical matters (Bekes et al., 2022).

ABCG2 is a member of the ATP-binding cassette membrane transporter superfamily and its over-expression causes multidrug resistance (MDR) in cancer chemotherapy by actively exporting anticancer drugs, effectively reducing their intracellular accumulation (Mo and Zhang, 2012, Robey et al., 2011). ABCG2 has also been thought to protect cancer stem cells (CSCs) (Mo and Zhang, 2012, Stacy et al., 2013). Interestingly, ABCG2 knockout had no apparent adverse effect on the development, biochemistry, and life of mice (Zhou et al., 2002). Thus, ABCG2 is an interesting and perhaps an ideal target for developing chemo-sensitizing agents to overcome MDR and eliminate CSCs.

Indeed, many ABCG2 inhibitors have been identified, including known receptor tyrosine kinase inhibitors (TKIs) (Houghton et al., 2004, Ozvegy-Laczka et al., 2004), natural products (Rabindran et al., 1998), and chemical compounds (Henrich et al., 2007). However, few inhibitors were tested clinically, and none has been approved although some approved TKIs inhibit ABCG2 due to off-target effects. Recently, a group of inhibitors with dynamic properties in inhibiting the function and inducing degradation of ABCG2 have been discovered (Peng et al., 2009, Peng et al., 2010, Wei et al., 2012). While the static inhibitors have been well studied, the detailed mechanism of dynamic inhibitor action in inducing ABCG2 degradation remains largely elusive.

One of these dynamic inhibitors is PZ-39 (Peng et al., 2009). Several active analogues of PZ-39 including PZ-39C8 have also been identified. Both PZ-39 and PZ-39C8 have the core benzothiazole linked to a triazine ring backbone with different side groups (Fig. 1A). It has been shown that PZ-39C8 is less potent and slower in inducing ABCG2 degradation than PZ-39, which is known to reverse ABCG2-mediated MDR without cytotoxicity (Peng et al., 2009). Thus, PZ-39C8 is a better probe for studying the mechanism of dynamic inhibitor-induced ABCG2 degradation for convenience of observing ABCG2 degradation process while PZ-39 a better probe for in-vivo studies.

In the present study, we investigated the mechanism of dynamic inhibitor action in inducing ABCG2 degradation and its in-vivo activity using PZ-39C8 and PZ-39 as representative dynamic inhibitor probes. We showed that the inhibitor-induced ABCG2 degradation is due to its direct binding to the third extracellular loop, inducing clathrin-dependent endocytosis and trafficking of ABCG2 into lysosome for degradation. The newly synthesized ABCG2 was hijacked from ER by the inhibitor and targeted to lysosome for degradation via autophagy. PZ-39 causes ABCG2 degradation and sensitizes ABCG2-mediated doxorubicin resistance in xenograft tumors. Hence, the dynamic inhibitors represent the next generation ABCG2 inhibitors that may function as a new class of TPD agents with a potential to be developed to overcome ABCG2-mediated MDR in cancer chemotherapy.