By Andres Hernandez Maduro
It is rare for coincidental discoveries to lead to viable prophylactic treatments. This was most probably the case when, in the 1920s, Lucy Wills found that an undiscovered nutrient in yeast extract could be used to treat patients with macrocytic anaemia.1 Soon thereafter, Wills’ results were both corroborated and expanded upon by her peers in research, leading to the identification of folic acid and its metabolism in cells. The result: the Folate Cycle.
Beginning with the ingestion of dietary folic acid – otherwise known as Vitamin B9 – the Folate Cycle has been found to be crucial in a variety of cellular mechanisms. Here, folic acid is converted into a folate (belonging to a family of molecules made of a pteridine ring, a p-aminobenzoyl moiety and a variably long polyglutamate group), which in turn is reduced into dihydrofolate (DHF) and finally into tetrahydrofolate (THF). THF is then used to carry single-carbon groups for the synthesis of functionally significant biomolecules, including purines and thymidine monophosphate for nucleic acid synthesis, as well as methionine for both proteostatic regulation and gene methylation.1 All in all, it is apparent why cells are so dependent upon the regular function of folate metabolism – and why it is so attractive a subject for medical investigations around the globe.
Before we look at applications, however, we should first understand the key molecular mechanisms of DHF action. An enzyme known as dihydrofolate reductase (DHFR) utilises reduced nicotinamide adenine dinucleotide phosphate (NADPH) to reduce DHF into THF in a sequential reaction. The gene coding for this enzyme – DHFR in human and mouse nuclei – is also expressed via the DNA-binding of cell cycle regulatory proteins (such as E2F family transcription factors2), hence making it one of the more prominent players in both cell proliferation and survival.1 Structurally, most species of DHFR contain a backbone of central anti-parallel beta sheets, with NADPH bound by its nicotinamide ring and with the active site typically present at the N-terminus.3 This is surrounded by a series of around fifteen residues that are widely referred to as the Met20 loop, further encompassing a highly conserved Pro-Trp dipeptide that increases the affinity of DHFR for DHF. Structural protein images show that conformational changes triggered by substrate and coenzyme binding take place mostly in the Met20 loop, altering from ‘closed’ to ‘occluded’ states via an ‘open’ intermediate phase during the catalytic cycle.3
Along with its structure, the general mechanism behind DHFR catalysis has already been resolved to the atomic level – enough that one might be led to believe that there is no more to find out about it. However, much remains to discover. Recent studies, for example, have demonstrated (with neutron and X-ray crystallography) that protonation of the reactionary pteridine nitrogen in DHF (necessary for reduction to occur) takes place through its charge stabilisation and by the subsequent dissociation of a hydrogen-bonded water molecule.4 This counters the original view that it was the pteridine ring’s tautomerisation that led to its protonation – which is now known to be incorrect. In addition, DHFR catalysis is now even believed to be an example of quantum biology in nature. After protonation, hydride ion transfer from NADPH to a proximally oriented carbon atom in DHF apparently happens via quantum tunnelling, wherein the electrostatic energy barrier between the molecules is bypassed through the quantum superposition of the ion.5
Indisputably, future research is bound to discover further biochemical properties in DHFR, offering us a better insight of how it can be manipulated. Already, a multitude of DHFR inhibitors (called antifolates) have been applied for both tumour chemotherapeutics and pathogenic infection.6 Methotrexate (a DHF analogue first created in the 1960s) was the start of these, giving doctors an alternative way to prevent cell hyperproliferation. Being in the early stages of development, however, this drug displayed poor solubility and was too non-specific and general to most cell types to be used without side-effects – meaning that people were keen on obtaining replacements. In came non-classical antifolates, lipid-soluble molecules that are able to enter cells quicker than their classical counterparts.
Now, non-classical antifolates like piritrexim are used clinically to combat conditions like urothelial and colorectal carcinomas (particularly in tumours that have developed resistance towards methotrexate), with pralatrexate being approved by the FDA in 2009 for its rapid cell internalisation/retention and high affinity for DHFR.6 Furthermore, whereas cancer-fighting antifolates are still rather non-specific to human cells, several species-selective DHFR inhibitors have been discovered. Trimethoprim (TMP) and sulfamethoxazole, for instance, serve as effective antibiotics against many respiratory, intestinal and urinary bacterial infections;7 and pyrimethamine is one of the few therapeutic drugs that can eliminate Plasmodium falciparum (i.e., malaria) from our system. The growing concern over multi-drug resistant superbugs makes this field of study particularly relevant nowadays, as shown by the myriad of attempts at finding more potent TMP analogues.7
Needless to say, the Folate Cycle and subtle mechanisms of DHFR show great potential for further research. And DHFR inhibition, given its near-ubiquitous application in living organisms, could very well be crucial in preventing the looming superbug catastrophe. An interesting prospect to investigate, at the very least.
- Zheng Y, & Cantley LC. Toward a better understanding of folate metabolism in health and disease. The Journal of Experimental Medicine 2019;216(2):253–266. Available from: https://doi.org/10.1084/jem.20181965
- Blake MC & Azizkhan JC. Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Molecular and cellular biology 1989;9(11):4994–5002. Available from: https://doi.org/10.1128/mcb.9.11.4994-5002.1989
- Arora K, Brooks Iii CL 3rd. Functionally important conformations of the Met20 loop in dihydrofolate reductase are populated by rapid thermal fluctuations. J Am Chem Soc 2009;131(15):5642-5647. Available from: https://dx.doi.org/10.1021%2Fja9000135
- Wan Q, et al. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. PNAS 2014;111(51):18225-18230. Available from: https://doi.org/10.1073/pnas.1415856111
- Stojkovic V, Perissinotti LL, Willmer D, Benkovic SJ, Kohen A. Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction. J Am Chem Soc. 2012 Jan 25;134(3):1738-45. Available from: http://dx.doi.org/10.1021/ja209425w
- Raimondi MV, Randazzo O, La Franca M, et al. DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents. Molecules 2019;24(6):1140. Available from: https://dx.doi.org/10.3390%2Fmolecules24061140
- Wróbel A, et al. Trimethoprim and other nonclassical antifolates an excellent template for searching modifications of dihydrofolate reductase enzyme inhibitors. J Antibiot 2020;73:5–27. Available from: https://doi.org/10.1038/s41429-019-0240-6