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. 2024 Feb 15;31(2):221-233.e14.
doi: 10.1016/j.chembiol.2023.09.014. Epub 2023 Oct 23.

Methotrexate-based PROTACs as DHFR-specific chemical probes

Sandeep Rana  1 , Patricia Dranchak  1 , Jayme L Dahlin  1 , Laurence Lamy  1 , Wenqing Li  2 , Erin Oliphant  1 , Jonathan H Shrimp  1 , Girish H Rajacharya  3 , Ravi Tharakan  1 , David O Holland  1 , Apryl S Whitten  3 , Kelli M Wilson  1 , Pankaj K Singh  4 , Scott K Durum  2 , Dingyin Tao  1 , Ganesha Rai  5 , James Inglese  6
Affiliations

Methotrexate-based PROTACs as DHFR-specific chemical probes

Sandeep Rana et al. Cell Chem Biol. .

Abstract

Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.

Keywords: DHFR-HiBiT; MTX-Cy5; PROTAC; antifolates; dihydrofolate reductase; methotrexate; one-carbon metabolism; targeted protein degradation; thymidylate synthase.

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Conflict of interest statement

Declaration of interests S.R., G.R.B., L.L., and J.I. are listed as inventors for the patent application, WO2021262693A1 (“Methotrexate analogs and methods of use”).

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