The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase

Abstract

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Small Molecule Screen Identifies Pyrimethamine as an Inhibitor of NRF2-driven Esophageal Hyperplasia

“…Since PYR was recently reported to inhibit DHFR and STAT3 in human cancer cells 29, 30 , we evaluated whether PYR inhibited STAT3 and DHFR activity in human ESCC cells. In KYSE70 cells, PYR (up to 10 µM) did not significantly inhibit pSTAT3 and STAT3 expression (Figure S5A), but did significantly inhibit DHFR activity starting at 5 µM (Figure S5B).…”

“…PYR inhibits plasmodial DHFR 23 and STAT3 in leukemia cells 29 , down-regulates mutant Cu/Zn superoxide dismutase (an NRF2 transcriptional target) in the cerebrospinal fluid of amyotrophic lateral sclerosis patients 35 , and suppresses the growth of various cancer cells [36][37][38][39][40] . In NRF2 Mut -ESCC cells, PYR inhibited NRF2 expression in a dose-and time-dependent manner (Figure 2, S3).…”

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection

“…The reason for this variation in efficacy is unclear. Pyrimethamine is an inhibitor of dihydrofolate receptor (DHFR), involved in the biosynthesis of nucleotides used in viral genome replication [65]. Accordingly, the impact of this drug may depend on the availability of free nucleotides within the infected cell.…”

Discovery of new STAT3 inhibitors as anticancer agents using ligand-receptor contact fingerprints and docking-augmented machine learning

“…A panel of 10 cell lines (Fibroblasts, HEK293, 3T3, PANC1, DU145, U87, MDA-MB-231, A549, doxorubicin resistant and sensitive MCF7) was screened against the selective STAT3 inhibitor pyrimethamine, at 10 mM, to identify cells that rely on STAT3 expression for their livelihood and proliferation. [63][64][65] The cytotoxicity of pyrimethamine against the selected cell lines was assessed using MTT procedure (see ESI Section SM3 † for details). HEK-239, MCF-7, U87, MDA-MB-231 and Fibroblasts were found to be good indicators of STAT3 signicance/redundancy (see results Section 3.7, Table 7).…”