Fabiana Lo Mascolo
| Prodrug strategies to improve the drug-like properties of fragment-derived Keap1-Nrf2 Inhibitors |
| Fabiana Lo Mascolo1, Paola Barraja1, Anders Bach2 1 Department of Sciences and Chemical Biology and Pharmaceutical Technology (STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy; 2 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark; |
| Abstract Oxidative stress is a key driver of neurodegenerative, inflammatory and renal diseases [1]. Pharmacological activation of the Nrf2 pathway via disruption of the Keap1-Nrf2 protein-protein interaction is a promising therapeutic strategy; however, most non-covalent Keap1 inhibitors rely on carboxylic acid moieties required for binding to the arginine-rich pocket, often resulting in poor membrane permeability and suboptimal drug-like properties [2,3]. To overcome this limitation, we investigated a prodrug approach to transiently mask the carboxylic acid functionality of fragment-derived Keap1 inhibitors identified through fragment-based drug discovery. A focused library of 26 prodrugs was synthesized and evaluated for aqueous solubility (PBS, pH 7.4, 0.5-4% DMSO; 30-200 µM) and passive permeability using PAMPA. Quantitative LC-MS methods based on selective ion monitoring (SIM) were developed for sensitive compound detection. Initial screening identified three methyl ester derivatives with markedly improved permeability, reaching Pe (cm/s) values up to 1.16 ×10⁻⁵ cm/s versus ≤ 8.62 ×10⁻⁷ cm/s for parent acids (~13-fold increase), including complete rescue from undetectable to high permeability. Building on these results, corresponding thiazolidinone-based prodrugs were designed and synthesized to further optimize physicochemical properties and enable ROS-responsive activation. These analogues retained favorable solubility and permeability profiles and were subjected to oxidative activation studies. LC-MS kinetic analysis confirmed concentration-dependent oxidative cleavage. Under mild oxidative conditions (1 mM H₂O₂), prodrugs showed gradual and controlled degradation, with 56-68% remaining after 180 min, whereas faster conversion occurred at higher ROS levels, indicating a tunable balance between stability and activation. Overall, this study demonstrates that prodrug strategies can effectively address permeability limitations of fragment-derived Keap1 inhibitors while enabling ROS-triggered activation. Selected compounds are currently under biological evaluation in cellular models of Nrf2 activation in collaboration with Prof. Olagnier’s laboratory (Aarhus University). Supporting further development of drug-like modulators of the Keap1-Nrf2 pathway. |
| References [1] Cuadrado, A.; Rojo, A.I.; Wells, G.; Hayes, J.D.; Cousin, S.P.; Rumsey, W.L.; Attucks, O.C.; Franklin, S.; Levonen, A.L.; Kensler, T.W.; Dinkova-Kostova, A.T. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat. Rev. Drug Discov. 2019, 18, 295–317. DOI: 10.1038/s41573-018-0008-x. [2] Barreca, M.; Qin, Y.; Cadot, M.E.H.; Barraja, P.; Bach, A. Advances in developing noncovalent small molecules targeting Keap1. Drug Discov. Today 2023, 28, 103800. DOI: 10.1016/j.drudis.2023.103800. [3] Pallesen, J.S.; Tran, K.T.; Bach, A. Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Inhibitors and Their Potential for Targeting Central Nervous System Diseases. J. Med. Chem. 2018, 61, 8088–8103. DOI: 10.1021/acs.jmedchem.8b00358. |