Allosteric Targeting of the KEAP1 BTB Homodimerisation Interface: Structure-Based Virtual Screening Identifies Indazole and Benzimidazole Scaffolds

Inside every cell, a delicate balance keeps the body safe from the constant wear and tear of daily life. This balance is managed by a master regulator protein called NRF2, which acts like a switch, turning on the genes that repair damage and fight off toxic stress. However, this switch is usually kept in the "off" position by a watchdog protein named KEAP1. Under normal conditions, KEAP1 grabs NRF2 and marks it for destruction, ensuring the cell does not overreact to minor irritations. When cells face serious threats, such as the buildup of toxic fats or the presence of certain cancer mutations, this system can fail. In some aggressive cancers, the watchdog is either broken or bypassed, leaving the cancer cells protected and resistant to treatment. Scientists have long tried to fix this by designing drugs that either block the grabbing mechanism or chemically jam the watchdog's sensors, but these approaches have limitations and can be outmaneuvered by the cancer.

A new study by independent researcher Cumhur Gokhan Ekmekci explores a different, largely uncharted path to control this system. Instead of trying to stop KEAP1 from grabbing NRF2, the researcher asked if it were possible to stop two KEAP1 proteins from holding hands with each other. KEAP1 works as a pair; two molecules must join together to form a functional unit capable of destroying NRF2. This joining happens at a specific interface, a flat surface where the two proteins touch. The study suggests that if a drug could wedge itself into this meeting place, it would prevent the pair from forming, effectively disabling the watchdog without needing to compete with the natural proteins that usually bind to it. Using a powerful computer simulation, the researcher tested dozens of chemical structures to see which ones might fit into this specific gap, looking for a new way to reactivate the cell's natural defenses.

The investigation began by building a digital model of the KEAP1 protein pair, using a known structure from a database as a blueprint. The researcher focused on the specific surface where the two proteins connect, a region that had been largely ignored by drug developers until now. To ensure the search was thorough, the computer was programmed to test sixty-one different chemical compounds against this interface. These compounds were chosen from a diverse mix, including known drugs that might be repurposed, small fragments of molecules, and new chemical designs based on specific ring-shaped structures known as indazoles and benzimidazoles. The computer simulated how each compound would sit in the gap, calculating how tightly it would bind and whether it would prefer this joining spot over other known sites on the protein.

The results of this virtual screening revealed a promising new direction. Among the many compounds tested, a specific family of molecules built around an indazole core emerged as the strongest candidates. Two of these, named BTBn_Ind_001 and BTBn_Ind_002, showed the highest predicted ability to stick to the protein pair's interface. The simulation suggested they would bind with a strength comparable to the best existing drugs that target other parts of the protein, but with a crucial difference: they were designed to disrupt the partnership between the two KEAP1 molecules rather than block the grabbing site. These top candidates were also checked for safety and drug-like properties, and they passed the initial tests, showing they are small enough and stable enough to potentially become medicines.

Interestingly, the study also found that two compounds, which were originally designed for a different purpose, also fit well into this interface. One of these, JKE-1674, was developed to stop a process called ferroptosis, a type of cell death driven by iron and fat oxidation. The other, PipAryl_005, was designed to block a transporter protein involved in cancer metabolism. The fact that these unrelated drugs also engaged the KEAP1 joining site suggests that a single chemical structure might be able to hit multiple targets in the cell's defense network. This dual action could be particularly useful in treating cancers that have become resistant to standard therapies, as it would attack the problem from two angles at once.

Despite these promising computer results, the researcher is careful to note that these findings are currently just predictions. The study relied entirely on digital models and simulations, which are powerful tools for narrowing down possibilities but cannot prove that a drug will work in a living system. The computer models showed that the interface is a viable target and that specific chemical shapes fit well, but the actual binding strength and biological effects have not yet been measured in a lab. The researcher proposes that the next steps must involve physical experiments, such as testing if these compounds can actually stop the proteins from joining in a test tube and observing if they can turn on the protective genes in living cells.

The study concludes that the KEAP1 joining interface is a real, accessible target for new drugs, offering a fresh strategy to control the cell's antioxidant defenses. The indazole-based molecules identified in this work represent the first computationally discovered candidates for this specific site, providing a new starting point for scientists to build better medicines. While the path from a computer simulation to a real treatment is long and uncertain, this work opens a door to a mechanism that has been overlooked for years. By focusing on how the watchdog proteins connect to each other, rather than just how they grab their targets, researchers may have found a way to outsmart the cancer cells that have learned to evade current treatments. The journey from these digital scores to a working drug will require rigorous testing, but the potential to unlock a new class of therapies makes the effort worthwhile.