Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain Journal Article


Authors: Bezerra, G. A.; Holenstein, A.; Foster, W. R.; Xie, B.; Hicks, K. G.; Bürer, C.; Lutz, S.; Mukherjee, A.; Sarkar, D.; Bhattacharya, D.; Rutter, J.; Talukdar, A.; Brown, P. J.; Luo, M.; Shi, L.; Froese, D. S.; Yue, W. W.
Article Title: Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain
Abstract: The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors. © 2021 The Authors
Keywords: small molecules; one-carbon metabolism; drug-development; 5,10-methylenetetrahydrofolate reductase; enzymatic inhibition
Journal Title: Biochimie
Volume: 183
ISSN: 0300-9084
Publisher: Editions Scientifiques Elsevier  
Date Published: 2021-04-01
Start Page: 100
End Page: 107
Language: English
DOI: 10.1016/j.biochi.2021.01.007
PUBMED: 33476699
PROVIDER: scopus
DOI/URL:
Notes: Article -- Export Date: 1 April 2021 -- Source: Scopus
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MSK Authors
  1. Minkui Luo
    61 Luo