Abstract: The antifolate, methotrexate (MTX) is a potent dihydrofolate reductase (DHFR) inhibitor. MTX, like folate undergoes poly-gamma-glutamylation by folylpolyglutamate synthetase (FPGS). This modification affects the cellular retention and target specificity of MTX. MTX has been used for decades as an anti-cancer agent and immune system suppressant despite its significant toxic side effects. MTX resistance in cancers can be mediated by a decrease in FPGS and an upregulation of DHFR. Efforts to improve on its medicinal properties have been hampered by a complex and poorly understood polypharmacology. The PROTAC strategy involves the degradation of a target protein by hijacking cellular quality control machinery. We have leveraged the PROTAC modality to develop a first-in-class selective DHFR degrader, versortrexate (VSTX). To facilitate this work, we developed several novel assays, including a bioluminescence cell-based assay to monitor DHFR abundance. Our studies revealed key pharmacological differences between MTX and VSTX. MTX treatment upregulates DHFR levels whereas VSTX potently degrades DHFR in a dose- and time-dependent manner across multiple cell lines. In addition, VSTX showed a strong folate-dependent cellular toxicity, significantly greater than observed for MTX. Furthermore, we demonstrate that VSTX-mediated DHFR degradation is unaffected by the mechanisms giving rise to MTX resistance. We believe VSTX and its analogs will be useful chemical probes to help dissect the biology of one-carbon metabolic pathways and might have therapeutic potential to treat autoimmune and neoplastic disease.
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