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First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival
Maria V. Guijarro, … , Frederic J. Kaye, Maria Zajac-Kaye
Maria V. Guijarro, … , Frederic J. Kaye, Maria Zajac-Kaye
Published April 25, 2023
Citation Information: JCI Insight. 2023;8(10):e158798. https://doi.org/10.1172/jci.insight.158798.
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Research Article Therapeutics

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival

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Abstract

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

Authors

Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Michael Feely, Elham Nasri, Robert P. Seifert, Xiaoping Luo, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens III, Frederic J. Kaye, Maria Zajac-Kaye

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Figure 1

Compound 19-S inhibits TYMS catalytic activity, shows cytotoxicity in vitro, and does not increase TYMS levels.

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Compound 19-S inhibits TYMS catalytic activity, shows cytotoxicity in vi...
(A) Diagram illustrating the TYMS (TS) tritium assay and conversion of tritiated dUMP to dTMP, generating tritiated water for quantification of TS activity. (B) TS tritium assay showing TS activity for control reactions and reactions performed in the presence of 5-fluorouridine (FUrd), 5-fluoro-2′-deoxyuridine (FdUrd), and compound 19-S. Drug concentrations were 250 μM using 10 μg/mL bacterially expressed TS protein. Mean ± SD of 4 data points from 2 independent experiments shown. The DMSO control represents 10 data points. Compound 19-S treatment represents 6 data points. (C) Viability assays comparing known TS inhibitor 5-FU with compound 19-S in the indicated cell lines following a 72-hour drug incubation. Data are expressed as mean ± SD of 2 independent experiments; n = 4 to 5 technical replicates. (D) Immunoblot analysis showing TS overexpression following 5-FU treatment (TS5FU) and stable TS expression levels following compound 19-S treatment. PANC-1 cells were treated for 72 hours with the indicated 5-FU and 19-S concentrations. Experiment was repeated independently twice with similar results.

Copyright © 2023 American Society for Clinical Investigation
ISSN 2379-3708

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