Dihydrofolate reductase (DHFR) is responsible for catalyzing the reduction of folate and dihydrofolate (DHF) into tetrahydrofolate (THF) using nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor [1]. This reaction is an essential step in one-carbon metabolism, which cycles dietary folate as a precursor for purine synthesis and for maintenance of cellular methionine levels [1]. Antifolates, or drugs which disrupt the folate cycle via DHFR inhibition, are clinically used to treat several conditions including cancer, inflammatory conditions, autoimmune diseases and parasitic infections [[2], [3], [4], [5]].

DHFR is also one of the earliest therapeutic targets for human cancers. Inhibiting human (h) DHFR disrupts folate metabolism, thereby inhibiting nucleic acid synthesis, DNA replication and cell division. More recently, it was discovered that DHFR inhibition can regulate the activity of pro-oncogenic transcription factors, such as signal transducer and activator of transcription 3 (STAT3) [6]. Indirect inhibition of STAT3 results in prevention of transcription of pro-tumorigenic genes, such as those responsible for cell proliferation and metastasis [7]. In many types of cancer, including triple negative breast cancer [8], STAT3 is constitutively active, leading to a sustained oncogenic effect. Therefore, effective DHFR inhibition, particularly in cancers with increased STAT3 activity, represents a promising therapeutic approach.

Several classic inhibitors of hDHFR are clinically used in anti-cancer therapy. Classic DHFR inhibitors, such as methotrexate (MTX), mimic folic acid and compete for substrate binding in the active site to inhibit DHFR's enzymatic activity. However, the therapeutic use of MTX has some notable disadvantages. First, due to its similarity to folate, cellular uptake of MTX requires functional reduced folate carrier, which can lead to diminished inhibitor uptake and resistance if this active transport mechanism is compromised [9]. Clinically, MTX has also been shown to cause disease to the lungs [10], damage to the gastrointestinal tract [11], and epithelial-mesenchymal transition of epithelial cells, thus enhancing cell migration [12,13]. Treatment with DHFR inhibitors also commonly results in a transient accumulation of DHFR protein, due to disruption of the DHFR's autoregulatory functions [6,14,15].

Pyrimethamine (Pyr) is a clinically approved antifolate used to treat toxoplasmosis and parasitic malaria. Despite reported selectivity for parasitic and bacterial DHFR, recent evidence has demonstrated that Pyr has notable activity towards hDHFR and has promising anti-cancer activity in several in vivo model systems [6,[16], [17], [18]]. Pyr has also recently undergone clinical trials as an anti-cancer drug for the treatment of chronic lymphocytic leukemia and small lymphocytic leukemia (CLL and SLL, respectively) following promising pre-clinical results [19]. Notably, this study reports a relatively high number of serious adverse events at the highest testing dose (50 mg, daily, oral), and several other non-serious adverse events across all tested doses (12.5, 25 and 50 mg), with anemia, fatigue and decreased platelet count being most common [19]. A final publication based on this completed study was not available at the time of this manuscript's submission.

Compounds related to Pyr have been thoroughly studied for their ability to inhibit DHFR and elicit anti-cancer and antibiotic activity. Notably, in silico modeling has been previously used to explore how modifications of the Pyr scaffold could be used to overcome resistance-mediating mutations in parasitic DHFR [20,21]. Additionally, further efforts to investigate the anti-cancer activity of Pyr demonstrated nearly identical predicted binding orientations between Pyr and human and Plasmodium falciparum (pf) DHFR [16].

By applying state-of-the-art techniques to investigate Pyr analogues, this current study seeks to examine how modifications on the Pyr scaffold impact its drug-like properties and biological activity. A series of Pyr analogues were synthesized and evaluated for their ability to bind DHFR in biochemical and cellular settings. In silico docking and physicochemical properties analyses provided solid rationales for the observed trends. Building on recent evidence that DHFR inhibition may be a promising therapeutic strategy for breast cancer [23], cellular assays in this study were performed in breast cancer cell lines, including MDA-MB-231 and MDA-MB-468 which have constitutively active STAT3 [8], and are susceptible to DHFR inhibition. This study identifies analogues of Pyr that are highly potent and cell-active inhibitors of DHFR.