16 Antimetabolites as cancer drugs

In this chapter, we will talk about anti-metabolites as anti-cancer agents, exemplified by Methotrexate, an anti-folate, and 5-FU as well as Capecitabine as pyrimidine analogues. “Anti-metabolite drugs work by inhibiting essential biosynthetic processes or by incorporating into macromolecules such as DNA and RNA, and inhibiting their normal function.”

16.1 Folate antagonists: Methotrexate

In this chapter we will discuss folate antagonists, in particular Methotrexate. Anti-folates are drugs that block (antagonise) the actions of folic acid. Methotrexate was one of the first chemotherapeutic drugs used in the early 1950’s.

The primary function of folic acid (Figure 14.1), also called vitamin B9 or simply folate, is as a cofactor for various enzymes called methyltransferases. It is involved in the biosynthesis of the amino acids serine and methionine, but also in the synthesis of thymidine and purine. There are two major protein targets for anti-metabolites: The majority of anti-folates work by inhibiting the enzyme Dihydrofolate Reductase (DHFR), but some are inhibitors of Thymidylate Synthase (TS). DHFR catalyses the reduction of dihydrofolate to tetrahydrofolate. Anti-folates act specifically during DNA and RNA syntheses and are thus cytotoxic during the S-phase of the cell cycle. Anti-folates inhibit cell division, DNA or RNA synthesis and repair and protein synthesis. DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor synthesis.

Chemical structure of folic acid, also known as vitamin B9 or folate.

Figure 16.1: Chemical structure of folic acid, also known as vitamin B9 or folate.

16.2 Antimetabolites targeting Dihydrofolate Reductase

By means of reductive methylation, deoxyuridine monophosphate (dUMP) and N5,N10-methylene tetrahydrofolate are together used to form deoxythymidine monophosphate (dTMP) and dihydrofolate as a secondary product (Figure 14.2). DHFR catalyses the conversion of dihydrofolate back to the active tetrahydrofolate. One important drug that blocks regeneration from dihydrofolate to tetrahydrofolate is Methotrexate.

Protein targets for antimetabolites, Dihydrofolate Reductase and Thymidylate Synthase. Both targets are involved in the synthesis of DNA precursers.

Figure 16.2: Protein targets for antimetabolites, Dihydrofolate Reductase and Thymidylate Synthase. Both targets are involved in the synthesis of DNA precursers.

The two chemical structures of dihydrofolate and Methotrexate (MTX) (Figure 14.3) are very similar and differ only in two positions: the hydroxy group in dihydrofolate is substituted with an amino-group in the 4-position. In addition, the N10 position is methylated in methotrexate.

Chemical structures of dihydrofolate and Methotreaxate. Both are very similar differing in only two positions, highlighted in red and blue.

Figure 16.3: Chemical structures of dihydrofolate and Methotreaxate. Both are very similar differing in only two positions, highlighted in red and blue.

Methotrexate competitively inhibits DHFR that participates in tetrahydrofolate synthesis. The affinity of Methotrexate for DHFR is about 1000-fold increased, compared to dihydrofolate. Therefore, Methotrexate is extremely potent, essentially a stoichiometric inhibitor of DHFR. The crystal structure of the DHFR-MTX complex is shown in Figure 14.4. The protein is coloured in pink and blue, whereas the inhibitor is shown in red bound in the inhibitor-binding pocket.

Crystal structure of DHFR in complex with Methotrexate (PDB entry 1DDS); Matthews 1997). α-helices are coloured in blue, β-strands in purple and Methotrexate is shaded in red.

Figure 16.4: Crystal structure of DHFR in complex with Methotrexate (PDB entry 1DDS); Matthews 1997). α-helices are coloured in blue, β-strands in purple and Methotrexate is shaded in red.

16.3 Mechanism of action of Methotrexate

The mechanism of action of Methotrexate is very interesting (Figure 16.5) because it is also the basis for the development of resistance at later stages of tumour treatment (see section 21).

  • Methotrexate enters cells by two different proteins, either the reduced-folate carrier (1) or the membrane folate binding protein (2).
  • Methotrexate is then polyglutamylated by the enzyme folypolyglutamate synthetase (3). Polyglutamylation is a form of reversible posttranslational modification of glutamate residues.
  • Polyglutamylated Methotrexate (glu)~n~ is a potent inhibitor of Dihydrofolate Reductase (DHFR) (4).
  • Methotrexate polyglutamates are hydrolyzed to Methotrexate in the lysosome by γ-Glutamyl Hydrolase (GGH) (5). Hydrolysis of the polyglutamate tails by this enzyme makes (anti)-folates exportable from the cell again.
 Mechanism of action of Methotrexate (MTX). The numbers 1 to 5 refer to the different enzymes involved in uptake or efflux of Methotrexate. CH~2~FH~4~ = N5, N10-methylene tetrahydrofolate (add other abbreviations and ref).

Figure 16.5: Mechanism of action of Methotrexate (MTX). The numbers 1 to 5 refer to the different enzymes involved in uptake or efflux of Methotrexate. CH2FH4 = N5, N10-methylene tetrahydrofolate (add other abbreviations and ref).

In summary, Dihydrofolate Reductase catalyses the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is needed in purine and pyrimidine synthesis, important for cell proliferation and cell growth. Inhibiting DHFR by Methotrexate causes depletion of tetrahydrofolate leading to a deficiency of thymidylate. Since DNA synthesis is dependent on thymidylate supply, cessation of DNA synthesis can lead to growth arrest in rapidly proliferating cells. Therefore, Methotrexate interferes with DNA synthesis in S-phase.

16.4 Route of administration, clinical uses and toxicities of methotrexate

Various routes of administration for Methotrexate are available including orally, intravenously and several additional routes. Methotrexate can be used either alone or in combination with other drugs to treat:

  • Breast

  • Head and neck

  • Acute lymphoblastic leukemia (ALL)

  • Lymphoma

  • Osteosarcoma

  • Choricarcinoma

Methotrexate can also be used using lower doses to treat other diseases such as rheumatoid arthritis, psoriasis and graft-versus-host disease.

The most common adverse effects include bone marrow suppression, in particular leukopenia and oral mucositis, which is a painful inflammation that occurs in the mouth and which is a common complication of cancer treatment.

Methotrexate can also affect how the kidneys work but only usually when it’s given in high doses. Patients will have blood tests before and during treatment to monitor this. Patients will have extra fluids through a drip before and after chemotherapy to protect the kidneys.

Routine monitoring is recommended including Methotrexate levels, complete blood count and creatinine at least every two months.

Leucovorin is usually given 24 h after starting Methotrexate treatment to reduce the side effects. Leucovorin is administered at the appropriate time following Methotrexate as part of a total chemotherapeutic plan, where it may "rescue“ bone marrow cells from Methotrexate treatment. Although it will alleviate gut and bone marrow toxicity, it does not alleviate kidney problems.

Leucovorin may also be useful in the treatment of an acute Methotrexate overdose and it should be redosed until the Methotrexate is back to normal levels.

16.5 Antimetabolites targeting thymidylate synthase

We will know look at pyrimidine anti-metabolites. Anti-metabolites masquerade for example as a pyrimidine, the building blocks of DNA. They prevent pyrimidine becoming incorporated into DNA during S phase of the cell cycle.

We have discussed the mechanism of action of Methotrexate and will now look at the mechanism of action of a drug called 5-FU, which targets Thymidylate Synthase (Figure 14.2). By reductive methylation, Thymidylate synthase catalyses the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (Figure 14.2). This is coupled to the transformation of methylene tetrahydrofolate to dihydrofolate.

16.6 Function of Thymidylate Synthase

We want to focus on the reaction catalysed by Thymidylate Synthase in more detail. The reaction catalysed by thymidylate synthase involves dUMP, N5, N10-methylene-tetrahydrofolate and the enzyme (Figure 14.6). Thymidylate Synthase forms a covalent bond to the substrate dUMP, through a cysteine side chain from the enzyme. The cofactor N5,N10-methylene tetrahydrofolate donates a methyl group to the α carbon atom, resulting in a so-called transition state. The generated products are dTMP, which now carries an additional methyl group, and dihydrofolate. In summary, a methyl group has been transferred from the cofactor to the nucleotide during hte reaction.

a) The synthesis of dTMP involves the enzyme thymidylate synthase, dUMP and N5,N10-methylen-tetrahydrofolate. b) The transition state is formed between the three reaction partners. c) dTMP is generated restoring dihydrofolate and thymidylate synthease.

Figure 16.6: a) The synthesis of dTMP involves the enzyme thymidylate synthase, dUMP and N5,N10-methylen-tetrahydrofolate. b) The transition state is formed between the three reaction partners. c) dTMP is generated restoring dihydrofolate and thymidylate synthease.

16.7 Poisoning Thymidylate Synthase: mechanism of inhibition by 5-FU

Having understood function of thymidylate Synthase we can now appreciate how this enzyme is inhibited. What is the mechanism of action of the anti-metabolite drug 5- Fluorouracil (5-FU)? 5-FU is an analogue of uracil with a fluorine atom at the C-5 position in place of hydrogen (Figure 14.7). Upon entering the cell, 5-FU is converted to a variety of active metabolites. One such metabolite is FdUMP. FdUMP differs from dUMP by a fluorine in place of a hydrogen on the α atom where the compound - compared to dUMP- possesses an additional flourine substituent.

The structure of 5-FU is shown on the left, the active metabolite is shown in the middle and dUMP is shown on the right. The effect changes dramatically when FdUMP enters into the same reaction.

Figure 16.7: The structure of 5-FU is shown on the left, the active metabolite is shown in the middle and dUMP is shown on the right. The effect changes dramatically when FdUMP enters into the same reaction.

The cofactor, Thymidylate Synthase and FdUMP come together.

Figure 16.8: The cofactor, Thymidylate Synthase and FdUMP come together.

FdUMP is also able to bind to the nucleotide-binding site of the enzyme. Again, thymidylate synthase forms a covalent bond to the substrate dUMP, through a cysteine residue from the enzyme. The cofactor N5,N10-methylene tetrahydrofolate donates a methyl group to the a carbon atom. However, the FdUMP is unable to have an elimination reaction and complete the methyl donation from methylene-tetrahydrofolate. 5-FU forms a stable ternary complex with the enzyme and tertrahydrofolate, therefore blocking binding of the normal substrate dUMP and inhibiting dTMP synthesis. F+ is highly unlikely as a leaving group, it cannot have an equivalent reaction to the hydrogen.

Thymidylate, FdUMP and N5,N10-methylene-tetrahydrofolate form a frozen transition state, and the reaction cannot continue. The binding of normal substrate dUMP is blocked and dTMP synthesis is inhibited.

Figure 16.9: Thymidylate, FdUMP and N5,N10-methylene-tetrahydrofolate form a frozen transition state, and the reaction cannot continue. The binding of normal substrate dUMP is blocked and dTMP synthesis is inhibited.

Expert box: 5-FU principally acts as a Thymidylate Synthase inhibitor. Another mechanism of cytotoxicity of 5-FU is mis-incorporation of fluoronucleotides into RNA and DNA. 5-FU exerts its anticancer effects through incorporation of its metabolites into RNA and DNA. Again, the molecular basis is not fully understood. The importance of this mechanism may be cell dependent. The 5-FU metabolite FUTP is incorporated into RNA, disrupting normal RNA processing and function.

16.8 Route of administration, clinical uses and toxicities of 5-FU

5-FU is injected intravenously. Oral administration is not advised because the absorption from the GI-tract is unpredictable. The 5-Fu concentration peaks shortly after injection. The elimination half-life from plasma is extremely fast with 10 to 20 min, requiring continuous infusion to maintain 5-FU levels in the plasma. We will talk about this inconveniences in a moment, when we discuss the inhibitor Capecitabine.

5-FU is used to treat

  • Head and neck cancer,

  • Colon cancer,

  • breast cancer.

5-FU has the greatest impact in colon cancer.

5-FU induces a large range of adverse effects, the main ones being nausea and vomiting, stomatitis and diarrhea, which may be dose-limiting. It should be noted that toxicities differ depending on how 5-FU is administered: if 5-FU is administered IV as a bolus (‘administration of a discrete amount of drug within a specific time, generally within 1 -30 min”), its main toxicity is bone marrow depression. If it is administered as a continuous infusion main toxicities include stomatitis and diarrhea.

As we have just heard, 5-FU is very swiftly metabolised with half-lives of 10 to 20 min, involving the enzyme Dihydropyrimidine Dehydrogenase (DPD). About 8% of the population have a DPD deficiency, which leads to slower metabolism and therefore significantly longer half-lives of 5-FU. If undetected this may lead to life threatening toxicity, because 5-FU is continuously administered to the patient but not eliminated by the expected metabolisation.

16.9 Capecitabine, a new 5-FU prodrug

Capecitabine is an orally administered prodrug of 5-FU, in contrast to 5-FU, which his administered via IV. Capecitabine is converted by the liver enzyme carboxyl esterase to 5-DFCR (5-deoxy-5-fluorocytidine) and then to 5-DFUR (5-deoxy-5-fluorouridine) by cytidine deaminase, an enzyme expressed in both the liver and in the tumour. 5-DFUR is then converted intracellularly to the antimetabolite 5-FU by thymidine phosphorylase. Tumour specificity is being achieved due to the high level of expression of this enzyme in malignant relative to normal tissue.

Capecetabine is used in the treatment of breast and colorectal cancers. Clinically, it is more important than 5-FU due to its oral administration and tumour specificity.

16.10 Bibliography

Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M., Hoogsteen, K. (1977). Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate. Science 197, 452-455.

Carreras, C.W. and Santi, D.V. (1995). The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem. 64, 721-762.

Longley, D.B., Harkin, D.P. and Johnston, P.G. (2003). 5-Flyorouracil: mechanism of action and clinical strategies. Nature Reviews Cancer 3, 330-338.

Midgley, R. and Kerr, D.J. (2009). Capecitabine: have we got the dose right? Nature Clinical Practice 6, 17-24.