5Flurouracil

1
5-Flurouracil

• Chirag Bhagat

2
Todays Talk

• General info on 5-FU
• Metabolism of 5-FU
• Mechanism of TS inhibition
• Other drug modulation
• Recent drugs
• Future

3
5-FU

• 5-Florouracil (5-FU) is an anti-metabolite drug
  that is widely used for the treatment of cancer,
  particularly for colorectal cancer.
• 5-FU was first introduced in 1957 and since then
  its popularity has not diminished.
• It is marketed under the name Adrucil.
• In 2005 it was given to 2 mil people worldwide
  making it one of the most widely prescrbed
  anti-cancer drugs.

4
5-FU

• 5-FU based chemotherapy improves overall and
  disease-free survival of patients with stage III
  colorectal cancer.
• Only used 1015 of time by itself.
• The combination of 5-FU with newer chemotherapies
  such as OXALIPLATIN has improved the response
  rates for advanced colorectal cancer to 4050

5
5-FU

• 5-FU exerts its anticancer effects through
  inhibition of thymidylate synthase (TS) and
  incorporation of its metabolites into RNA and
  DNA.
• Modulation strategies, such as co-treatment with
  leucovorin and methotrexate, have been developed
  to increase the anticancer activity of 5-FU.
• DNA microarray analysis of 5-FU-responsive genes
  will greatly facilitate the identification of new
  biomarkers, novel therapeutic targets and the
  development of rational drug combinations.

6
5-FU

• 5-FU is an analogue of uracil with a fluorine
  atom at the C-5 position in place of hydrogen
• As a pyramidine analogue, it is transformed
  inside the cell into different cytotoxic
  metabolites which are then incorporated into DNA
  and RNA, finally inducing cell cycle arrest and
  apoptosis by inhibiting the cell's ability to
  synthesize DNA.
• It is an S-phase specific drug and only active
  during certain cell cycles.

7
Metabolism

• 5-FU is converted to 3 main active
  metabolitesfluorodeoxyuridine monophosphate
  (FdUMP), fluorodeoxyuridine triphosphate (FdUTP)
  and fluorouridine triphosphate (FUTP).

8
Metabolism

• The main mechanism of 5-FU activation is
  conversion to fluorouridine monophosphate (FUMP),
  either directly by orotate phosphoribosyltransfers
  e (OPRT) with phosphoribosyl pyrophosphate (PRPP)
  as the cofactor, or indirectly via fluorouridine
  (FUR) through the sequential action of uridine
  phosphorylase (UP) and uridine kinase (UK).

9
Metabolism

• FUMP is then phosphorylated to fluorouridine
  diphosphate (FUDP), which can be either further
  phosphorylated to the active metabolite
  fluorouridine triphosphate (FUTP), or converted
  to fluorodeoxyuridine diphosphate (FdUDP) by
  ribonucleotide reductase (RR). In turn, FdUDP can
  either be phosphorylated or dephosphorylated to
  generate the active metabolites FdUTP and FdUMP,
  respectively.

10
Metabolism

• An alternative activation pathway involves the
  thymidine phosphorylase catalysed conversion of
  5-FU to florodeoxyuridine (FUDR), which is then
  phosphorylated by thymidine kinase (TK) to FdUMP.
  Dihydropyrimidine dehydrogenase (DPD)-mediated
  conversion of 5-FU to dihydrofluorouracil (DHFU)
  is the rate-limiting step of 5-FU catabolism in
  normal and tumour cells. Up to 80 of
  administered 5-FU is broken down by DPD in the
  liver.

11
TS Inhibition

• Thymidylate synthase (TS) catalyses the
  conversion of deoxyuridine monophosphate (dUMP)
  to deoxythymidine monophosphate (dTMP) with
  5,10-methylene tetrahydrofolate (CH2THF) as the
  methyl donor.
• The 5-fluorouracil (5-FU) active metabolite
  fluorodeoxyuridine monophosphate (FdUMP) binds to
  the nucleotide-binding site of TS and forms a
  stable ternary complex with TS and CH2THF,
  blocking access of dUMP to the nucleotide-binding
  site and inhibiting dTMP synthesis.

12
TS Inhibition

• This results in deoxynucleotide (dNTP) pool
  imbalances and increased levels of deoxyuridine
  triphosphate (dUTP), both of which cause DNA
  damage.
• The extent of DNA damage caused by dUTP is
  dependent on the levels of the pyrophosphatase
  dUTPase and uracil-DNA glycosylase (UDG). dTMP
  can be salvaged from thymidine through the action
  of thymidine kinase (TK).

13
TS Crystal Structure

• C-6 of dUMP is covalently bound to gamma S of
  Cys-198(146) during catalysis, and the reactants
  are surrounded by specific hydrogen bonds and
  hydrophobic interactions from conserved residues.

14
Other Drug Modulation

• Leucovorin (LV) ncreases the intracellular pool
  of 5,10-methylene tetrahydrofolate (CH2THF),
  thereby enhancing thymidylate synthase (TS)
  inhibition by fluorodeoxyuridine monophosphate
  (FdUMP).

15
Other Drug Modulation

• Eniluracil and uracil inhibit DPD-mediated
  degradation of 5-FU. Methotrexate (MTX) is
  thought to increase 5-FU activation by increasing
  phosphoribosyl pyrophosphate (PRPP) levels.

16
Other Drug Modulation

• Interferons (IFNs) have been reported to enhance
  thymidine phosphorylase (TP) activity, abrogate
  acute TS induction caused by 5-FU treatment and
  enhance 5-FU-mediated DNA damage.

17
Other Drug Modulation

• Capecitabine is a 5-FU pro-drug that is converted
  to 5'-deoxy-5-fluorouridine (5'DFUR) in the liver
  by the sequential action of carboxylesterase and
  cytidine deaminase. 5'DFUR is converted to 5-FU
  by TP.

18
Activation of p53

• A tumor supressing agent called p53
  transcriptionally activates CDKN1A GADD45alpha
  whose products induce cell-cycle arrest in
  response to DNA damage
• It can also trigget the elimation of damaged
  cells by inducing apoptosis
• This is done eyther by inducing FAS and BAX
  (pro-apoptotic genes) or by down regulation of
  BCL2 (anti-apoptotic gene)

19
Recent Drugs

• Oxaliplatin - Platin based DNA damaging agent.
• Active as a single agent, but more effective but
  more effective if combined with 5-FU. (more than
  doubles the response rate)
• Irinotecan - Topoisomerase I inhibitor
• Active in the first and second line of treatment.
  
• Used as a substitute for 5-FU.

20
Future Work

• DNA microarray technology can aid in the
  identification of downstream signaling pathways
  involved in tumor cell response to 5-FU
• The molecular determination of chemotherapudic
  drugs can help in rationally determining useful
  drug combinations
• DNA microarray technology can one day help design
  individualized patient treatment.

21
Summary (Take Home Message)

• 5-FU is used in the treatment of colorectal
  cancer.
• It causes DNA and RNA damage.
• Inhibits TS.
• Usually given with other drugs.

22
References

• Longley, D. B., Harkin, P. D. and Johnson, P. G.
  (2003) 5-Florouracil Mechanisms of action and
  clinical strategies. Nature, 3, 330-338.
• Rich,T.A., Shepard,R.C. and Mosley,S.T. (2004)
  Four decades of continuing innovation with
  fluorouracil Current and future approaches to
  fluorouracil chemoradiation therapy. J. Clin.
  Oncol, 22, 22142232.
• Malet-Martino,M. and Martino,R. (2002) Clinical
  studies of three oral prodrugs of 5-fluorouracil
  (capecitabine, UFT, S-1) a review. Oncologist,
  7, 288323.
• Montfort, W.R., Perry, K.M., Fauman, E.B.,
  Finer-Moore, J.S., Maley, G.F., Hardy, L., Maley,
  F., Stroud, R.M. Structure, multiple site
  binding, and segmental accommodation in
  thymidylate synthase on binding dUMP and an
  anti-folate. (1990) Biochemistry, 29, 6964-6977.
• http//en.wikipedia.org/wiki/Fluorouracil

23
Questions

• ?