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Development of KEAP1-targeting PROTAC and its antioxidant properties: In vitro and in vivo
Se Yong Park1, Raju Gurung2, Jung Ho Hwang2
1College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
Abstract:
Oxidative stress due to abnormal accumulation of reactive oxygen species (ROS) is an initiator of a large number of human diseases, and thus, the elimination and prevention of excessive ROS are important aspects of preventing the development of such diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is an essential transcription factor that defends against oxidative stress, and its function is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1). Therefore, activating NRF2 by inhibiting KEAP1 is viewed as a strategy for combating oxidative stress-related diseases. Here, we generated a cereblon (CRBN)-based proteolysis-targeting chimera (PROTAC), which we named SD2267, that induces the proteasomal degradation of KEAP1 and leads to NRF2 activation. As was intended, SD2267 bound to KEAP1, recruited CRBN, and induced the degradation of KEAP1. Furthermore, the KEAP1 degradation efficacy of SD2267 was diminished by MG132 (a proteasomal degradation inhibitor) but not by chloroquine (an autophagy inhibitor), which suggested that KEAP1 degradation by SD2267 was proteasomal degradation-dependent and autophagy-independent. Following KEAP1 degradation, SD2267 induced the nuclear translocation of NRF2, which led to the expression of NRF2 target genes and attenuated ROS accumulation induced by acetaminophen (APAP) in hepatocytes. Based on in vivo pharmacokinetic study, SD2267 was injected intraperitoneally at 1 or 3 mg/kg in APAP-induced liver injury mouse model. We observed that SD2267 degraded hepatic KEAP1 and attenuated APAP-induced liver damage. Summarizing, we described the synthesis of a KEAP1-targeting PROTAC (SD2267) and its efficacy and mode of action in vitro and in vivo. The results obtained suggest that SD2267 could be used to treat hepatic diseases related to oxidative stress.
Insights
A novel PROTAC molecule, SD2267, effectively degrades KEAP1, activating NRF2 to combat oxidative stress and liver damage. This targeted approach shows promise for treating diseases linked to reactive oxygen species.
Area of Science:
- Biochemistry and Molecular Biology
- Pharmacology and Toxicology
- Drug Discovery
Background:
- Oxidative stress from reactive oxygen species (ROS) contributes to numerous human diseases.
- Nuclear factor erythroid 2-related factor 2 (NRF2) protects against oxidative stress but is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1).
- Inhibiting KEAP1 to activate NRF2 is a therapeutic strategy for oxidative stress-related conditions.
Purpose of the Study:
- To develop and characterize a cereblon (CRBN)-based proteolysis-targeting chimera (PROTAC) targeting KEAP1 for degradation.
- To evaluate the efficacy of the PROTAC, named SD2267, in activating NRF2 and mitigating oxidative stress in vitro and in vivo.
- To investigate the mechanism of KEAP1 degradation induced by SD2267.
Main Methods:
- Synthesis of a CRBN-based PROTAC (SD2267) designed to induce KEAP1 degradation.
- In vitro experiments assessing SD2267's binding to KEAP1, recruitment of CRBN, and induction of KEAP1 proteasomal degradation using inhibitors like MG132.
- In vitro assessment of NRF2 nuclear translocation, target gene expression, and ROS attenuation in hepatocytes. In vivo studies in a mouse model of acetaminophen-induced liver injury.
Main Results:
- SD2267 successfully bound KEAP1, recruited CRBN, and induced KEAP1 degradation via the proteasome.
- KEAP1 degradation by SD2267 led to NRF2 nuclear translocation and activation of NRF2 target genes.
- SD2267 treatment attenuated acetaminophen-induced ROS accumulation in hepatocytes and reduced liver damage in a mouse model.
Conclusions:
- SD2267 is an effective KEAP1-targeting PROTAC that activates NRF2 through proteasomal degradation.
- The compound demonstrates therapeutic potential for hepatic diseases associated with oxidative stress.
- SD2267 represents a promising therapeutic strategy for managing oxidative stress-related pathologies.
