Dihydrofolate ReductaseEdit
Dihydrofolate reductase (DHFR) is a small but pivotal enzyme in cellular metabolism. It catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, the latter acting as a carrier of one-carbon units that drive the de novo synthesis of thymidine and purines. Because this folate cycle supplies the precursors for DNA replication and repair, DHFR sits at a bottleneck in cell proliferation. Its activity is essential in all domains of life, from bacteria and archaea to eukaryotes, including humans, and its central role has made it a classic pharmacological target in medicine. In humans, DHFR is exploited by antifolate drugs for cancer and autoimmune diseases, while in bacteria it is a target for antibiotics. Drugs such as methotrexate, trimethoprim, pemetrexed, and raltitrexed illustrate the broad therapeutic reach of DHFR inhibition across disciplines like oncology, infectious disease, and immunology. folate metabolism one-carbon metabolism thymidylate synthase purine biosynthesis methotrexate trimethoprim pemetrexed raltitrexed NADPH dihydrofolate tetrahydrofolate
DHFR operates at the core of the folate cycle, converting dihydrofolate (DHF) into tetrahydrofolate (THF) with the reducing power of NADPH. THF and its one-carbon derivatives act as donors in the synthesis of thymidylate (via thymidylate synthase) and the purine ring, integrating DHFR activity with essential DNA building blocks. In this way, DHFR helps maintain the reduced folate pool required for nucleotide biosynthesis. Inhibition of DHFR disrupts these pathways, depleting nucleotide pools and arresting cell division, a consequence exploited therapeutically in rapidly proliferating cells such as tumor cells or certain pathogenic microbes. thymidylate synthase purine biosynthesis folate cycle NADPH
Biochemical function
The canonical reaction catalyzed by DHFR is the transfer of a hydride from NADPH to DHF, yielding THF and NADP+. The THF pool serves as a versatile one-carbon donor in multiple biosynthetic routes, most notably the synthesis of thymidine and purines. DHFR activity is tightly connected to the cellular demand for DNA precursors and is therefore especially critical in contexts of fast cell growth. DHFR is expressed broadly, with homologous enzymes found in bacteria, yeasts, plants, and animals, yet subtle differences in structure and dynamics between species provide a basis for selective inhibition by drugs. NADPH tetrahydrofolate dihydrofolate enzyme inhibition
Structure and mechanism
DHFR is a relatively small enzyme that adopts a compact fold accommodating both the NADPH cofactor and the dihydrofolate substrate in proximity within the active site. The catalytic cycle involves binding of NADPH and DHF, hydride transfer to DHF to form THF, and product release. Bacterial and human DHFR share the core catalytic chemistry, but differences in active-site geometry and surrounding loops create exploitable selectivity for inhibitors. Structural studies, including X-ray crystallography, have illuminated how inhibitors mimic the substrate or cofactor and how conformational changes regulate ligand binding and release. These structural insights underpin the design of selective antifolates that target microbial DHFR while sparing human DHFR as much as possible. NADPH dihydrofolate tetrahydrofolate X-ray crystallography drug design enzyme inhibition
Clinical significance and therapeutic inhibitors
DHFR has long been a pharmacological target, yielding several important therapeutic classes.
Antifolate chemotherapy: Methotrexate (MTX) is a high-affinity DHFR inhibitor used in a range of cancers and autoimmune diseases. It acts by broadly suppressing nucleotide synthesis and cell proliferation, with toxicity managed in part by dosing strategies and rescue approaches. Other antifolates such as pemetrexed and raltitrexed target DHFR and related folate cycle enzymes, providing therapeutic options for various solid tumors. The clinical use of MTX, pemetrexed, and related drugs is tempered by potential toxicities, requiring careful monitoring and sometimes folinic acid (leucovorin) rescue to mitigate harm to normal tissues. methotrexate pemetrexed raltitrexed folinic acid leucovorin autoimmune disease cancer
Antibiotics: Trimethoprim inhibits bacterial DHFR with relatively selective affinity for prokaryotic enzymes, and is commonly used in combination with sulfamethoxazole (co-trimoxazole) to enhance antibacterial activity. This combination exploits sequential blockade of folate synthesis in bacteria, providing broad-spectrum activity while attempting to minimize host toxicity. trimethoprim sulfamethoxazole antibiotics bacteria
Resistance and toxicity: The clinical and translational challenges include the emergence of drug-resistant DHFR variants in bacteria and parasites, as well as toxicity from antifuppressive therapy in humans. Resistance can arise via point mutations in DHFR that reduce drug binding, gene amplification, or altered substrate/cofactor interactions, as well as via efflux or metabolic bypass mechanisms. In malaria, for example, mutations in parasite DHFR confer resistance to pyrimethamine, illustrating how target evolution can undercut therapy. Strategies to mitigate resistance include combination therapies, dosing regimens, and new inhibitors designed to exploit differences between human and microbial DHFR. drug resistance Plasmodium falciparum pyrimethamine trimethoprim antibiotics
Safety and pharmacology: Because DHFR inhibitors affect nucleotide synthesis, they can cause toxicity in rapidly dividing human tissues (bone marrow, gastrointestinal mucosa, etc.). Clinicians use dosing schedules, rescue agents, and monitoring to balance efficacy against adverse effects. The concept of selective toxicity—treating disease-causing cells while limiting harm to normal cells—drives ongoing research into increasingly selective inhibitors and combination regimens. toxicity bone marrow suppression pharmacology
Drug design and selectivity: The partial divergence between bacterial and human DHFR enables selective targeting in antibiotic development, while recent advances in structural biology inform the design of compounds with improved selectivity, potency, and pharmacokinetic properties. drug design structure-activity relationship NADPH
Evolution and pharmacology
DHFR is a highly conserved enzyme across life, reflecting its central role in one-carbon metabolism. Yet evolutionary divergence between species provides exploitable differences for drug design, especially between bacterial DHFR and human DHFR. The ongoing arms race between drug development and resistance underscores the importance of stewardship and innovation in antifolate chemistry. In clinical and agricultural settings, DHFR-targeting compounds continue to shape treatment paradigms in oncology, infectious disease, and immunology, while researchers pursue inhibitors with better selectivity and fewer adverse effects. evolution phylogeny antibiotic resistance drug development