Anti-folate chemotype protein degraders

National Center For Advancing Translational Sciences

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Abstract

Targeting enzymes in one carbon metabolism. Dihydrofolate reductase (DHFR) is an essential enzyme in the folate pathway that catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid (THF), a cofactor important in several one-carbon transfer reactions which participate in amino acid and nucleobase synthesis. Methotrexate (MTX), a potent DHFR inhibitor, has been used for decades as a potent anti-cancer agent, as well as to treat autoimmune diseases like rheumatoid arthritis. MTX is a prodrug with a complex polypharmacology, leading to reduced intracellular levels of THF cofactors, which results in inhibition of thymidylate, purine, and DNA biosynthesis. Mechanistically, MTX undergoes an enzyme catalyzed polyglutamylation which leads to irreversible intracellular accumulation of these analogs. Reduced THF cofactor concentrations result in DNA replication arrest. Unfortunately, therapeutic resistance to MTX is a significant clinical barrier. Mechanisms of resistance include DHFR amplification, decreased intracellular transport of MTX, decreased retention of MTX (due to lack of polyglutamylation), increased export of MTX (due to overexpression of some members of the ATP binding cassette transporters), mutated DHFR (resulting in weak binding to MTX), increased levels of lysosomal gamma-glutamyl hydrolase (leading to hydrolysis of MTX polyglutamates), and others. While many MTX analogs have been made over the years to improve its specificity and side-effect profile significantly improved versions have been elusive. New insights and technology are being employed to develop novel modulators of one carbon metabolism. We have synthesized a series of MTX-based PROteolysis TArgeting Chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. Several of these MTX-PROTAC chemotypes, we’ve named versortrexates (VSTX) display on-target, selective degradation of DHFR in multiple cancer cell lines, as opposed to the increased cellular DHFR protein expression induced by MTX and related antifolates. Importantly, VSTX and its analogs produced distinct, less-lethal phenotypes compared to MTX. VSTX and its analogs may also serve as leads for potential autoimmune and anti-neoplastic therapeutics.