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PROTACs are heterobifunctional molecules that induce targeted protein degradation via ternary complex formation with E3 ubiquitin ligases. They offer advantages over conventional inhibitors, including high selectivity and access to "undruggable" targets. Over 30 candidates currently in clinical trials. However, their clinical translation is hampered by on-target toxicity in healthy tissues and off-target effects caused by the intrinsic activity of E3 ligase ligands, such as CRBN-based degraders' unintended degradation of neosubstrates like IKZF1/IKZF3.

Hypoxia-Activated Prodrugs (HAPs) represent an effective strategy for targeting therapeutic agents to tumors in vivo. Hypoxia is a hallmark of solid tumors, resulting from rapid tumor growth and a disorganized vasculature leading to insufficient oxygen supply. HAPs exploit the reductive environment of hypoxic conditions: they remain inactive prodrugs in normoxic cells but undergo enzyme-catalyzed reduction under hypoxia, resulting in the release of the active drug. Several HAPs have advanced to clinical stages.

To address these challenges, this study focuses on leveraging the unique hypoxic microenvironment of solid tumors. By employing indolequinone as a bioreductive trigger to mask the E3 ligase ligand, hypoxia-activated PROTACs (HAP-TACs) were designed. These aim to achieve selective activation and degradation of the target protein (e.g., BRD4) within tumors while minimizing impact on normal tissues. This strategy is broadly applicable because it targets commonly used E3 ligases.

Figure 1. (A) A cartoon illustrating the principle of hypoxia-activated PROTACs (HAP-TACs).
(B) The mechanism by which the indolequinone group functions as a bioreductive group.

Molecular Design and Construction

In this study, the authors selected two BRD4 degraders—MZ1 and dBET1, which recruit VHL and CRBN respectively—as the prototype molecules for designing HAP-TACs.

Design of VHL-Recruiting HAP-TACs

The hydroxyproline residue in VHL ligands (e.g., VH032) was identified as the critical binding site, with its modification effectively ablating activity. Initial attempts using traditional bioreductive groups (e.g., 4-nitrobenzyl and 1-methyl-2-nitroimidazole) either failed to efficiently release the active molecule or exhibited chemical instability—as seen with the carbonate-linked nitroimidazole derivative NI-VHL, which underwent hydrolysis under normoxia. Ultimately, incorporation of an indolequinone group yielded IQ-VHL, a compound with superior stability and enhanced selectivity.

Figure 2. Design of VHL-Recruiting HAP-TACs.

Design of CRBN-Recruiting HAP-TACs

The glutarimide nitrogen of CRBN ligands (e.g., pomalidomide) was selected for bioreductive group attachment, as its methylation disrupts a key hydrogen bond with CRBN. NI-CRBN compromised ligand stability, triggering glutarimide ring opening and failing to release active PROTAC. To address this, more stable phenyl glutarimide-based CRBN ligands were adopted, enabling successful synthesis of indolequinone-protected IQ-CRBN.

Figure 3. Design of CRBN-Recruiting HAP-TACs.

Experimental Validation and Characterization

In Vitro Enzymatic Validation

The HAP-TACs were evaluated using purified human CYP450 reductase to simulate bioreductive conditions.

IQ-VHL (7) demonstrated excellent hypoxia selectivity, releasing 41% of active MZ1 under hypoxia (<0.1% O₂) versus only 8% in normoxia (21% O₂), with high stability in enzyme-free controls. 
IQ-CRBN (16) also showed selective release, with ~9% of active PG-4c generated only under hypoxia and negligible release in normoxia.
In contrast, early analogs performed poorly: NI-VHL (6) was highly unstable in normoxia, while NI-CRBN (15) failed to release active PROTAC and instead yielded degradation byproducts.

Figure 4. Enzyme-Based Bioreduction Assays.

Cellular Validation

Experiments in A549 and other cancer cell lines confirmed the functionality of HAP-TACs.

NI-VHL degraded BRD4 under both normoxic and hypoxic conditions, demonstrating no selectivity. In contrast, IQ-VHL efficiently degraded BRD4 (up to 98%) specifically under hypoxia, with minimal degradation in normoxia, showing statistically significant differences. While NI-CRBN exhibited negligible degradation activity, IQ-CRBN achieved potent BRD4 degradation (up to 82%) under hypoxic conditions, with only marginal effects in normoxia.

Figure 5. Hypoxia-Selective Degradation of BRD4 by VHL-Recruiting HAP-TACs in Cells.

Figure 6. Hypoxia-Selective Degradation of BRD4 by CRBN-Recruiting HAP-TACs in Cells.

Treatment with the proteasome inhibitor carfilzomib and the neddylation inhibitor MLN4924 completely blocked BRD4 degradation induced by both IQ-VHL and IQ-CRBN, confirming that their degradation mechanism depends on the E3 ligase pathway.

Figure 7. Validation of HAP-TAC Degradation Mechanism via Inhibitor Assay.

Oxygen Threshold and Degradation Kinetics

In A549 cells treated with IQ-VHL (7) or IQ-CRBN (16) for 24 hours at varying O₂ levels (21%, 2%, 0.5%, <0.1%), BRD4 degradation became apparent at 0.5% O₂ and peaked under <0.1% O₂. This activation profile aligns with severe hypoxia in solid tumors, while sparing normal tissues with higher physiological O₂ (e.g., bone marrow, kidney), indicating a wide therapeutic window.

Kinetic analysis revealed that IQ-VHL acts rapidly, achieving 26% BRD4 degradation within 2 hours and reaching a plateau by 16 hours, whereas IQ-CRBN exhibits a slower onset of action.

Figure 8. Oxygen Dependency and Degradation Kinetics of HAP-TACs

The hypoxia-selective degradation mediated by HAP-TACs demonstrates universality across two distinct cell lines, HCT116 and SF8628 (Figure 9).

Figure 9. Cell Line Validation of HAP-TAC Specificity.

Washout experiments demonstrated that only hypoxia-treated HAP-TACs (IQ-VHL and IQ-CRBN) induced sustained anti-proliferative effects comparable to active PROTACs (MZ1 and PG-4c). Neither HAP-TACs under normoxia nor the BRD4 inhibitor (+)-JQ1 under any conditions produced such persistent responses. Following hypoxia treatment, BRD4 levels remained suppressed for several days, resulting in irreversible suppression of cell proliferation.

Figure 10. Selective antiproliferative effects of HAP-TACs IQ-VHL (7) and IQ-CRBN (16) under hypoxic conditions.

Figure 11. Sustained BRD4 Degradation and Delayed Recovery Following HAP-TAC Treatment and Washout.

Conclusion

Through rigorous chemical, enzymatic, and cell-based assays, the team have conclusively demonstrated that indolequinone-based HAP-TACs—particularly IQ-VHL and IQ-CRBN—enable highly efficient and selective hypoxia-dependent protein degradation with corresponding functional outcomes. This approach presents a promising strategy to address on-target toxicity associated with conventional PROTAC technology.

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